Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation

Single cell RNA-sequencing of sternal bone marrow reciding Hematopoietic Stem Cells (HSCs) and Megakaryocytes (MKs) from individuals undergoing elective open heart valve replacement. HSCs were defined as Lineage-, CD34+, CD38-, CD45RA-, CD90+, CD49f+ cells. MKs where CD41a+, CD42b+ and ploidy was determined with Hoechst. A sternal bone marrow scraping was taken directly following median sternotomy using a Volkmann’s spoon. The sample was collected into an EDTA Vacutainer tube containing 1.8mg/ml EDTA. 4mL of Dulbecco’s phosphate buffered saline (PBS, Sigma) containing 10% human serum albumin (HSA, Gemini Bio Products) was added and the whole volume was resuspended by pipetting 2-3 times. The sample was then put on metallic thermal beads (ThermoFisher Scientific) at a temperature between 0-4°C and transported to the University of Cambridge for further processing. For HSC isolation the cells were stained with the following antibody cocktail: PECy5 conjugated anti-lineage specific antibodies: CD2 (BD), CD3 (BD), CD10 (BD), CD11b (BD), CD11c (BD), CD19 (BD), CD20 (BD), CD56 (BD), biotinylated CD42b (Pab5, NHS Blood and Transplant, International Blood Group Reference Laboratory [IBGRL]), biotinylated GP6 (Pab5, NHS Blood and Transplant, International Blood Group Reference Laboratory [IBGRL]) used in combination with PECy5 conjugated streptavidin (Biolegend). Alexa Fluor 700 conjugated anti-CD34 (BD), PerCP-Cy5.5 conjugated anti-CD38 (BD), Pacific Blue conjugated anti-CD45RA (Invitrogen), PECy7 conjugated anti-CD90 (BD),PE conjugated anti-CD49f (BD). After staining cells were kept at 4°C before sorting using a FACS Aria Fusion flow sorter (BD). Single HSCs defined as Lineage-, CD34+, CD38-, CD45RA-, CD90+, CD49f+ cells were sorted by FACS directly into individual wells of a 96-well plate. Index sort data was collected for each single cell. For MK isolation the cells were stained for surface MK markers with mouse anti-human CD41a APC conjugated antibody (BD) and mouse anti-human CD42b PE conjugated antibody (BD) and for ploidy analysis with 1ug/ml Hoechst 33342 (Invitrogen). After incubation at 37°C for 30 minutes, the cells were kept at 4°C before sorting using a FACS Aria Fusion flow sorter (BD). Single cells and MK pools of 20 cells were sorted by FACS according to ploidy level using a 100uM nozzle directly into individual wells of a 96-well plate. cDNA synthesis and poly(A) enrichment was performed following the G&T-seq protocol (Macaulay et al. 2015), a variation of the Smart-seq2 protocol1. ERCC spike-in RNA (Ambion) was added to the lysis buffer in a dilution of 1:4,000,000.

07/12/2020
2383 samples
DAC: EGAC00001001850
Technology: Illumina HiSeq 4000

Access Policy1 Study Files

Metadata

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The BPS Consortium anticipates that data generated from the project will be used by others, such as required for developing new analytical methods, in understanding epigenetics, for integrative analysis and for other uses. BPS Consortium “Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation”. Funding for the project was provided by UK Medical Research Council (MR/K024043/1).

THE BAMI PLATELET SUB-STUDY (BPS) CONSORTIUM DATA ACCESS AGREEMENT This agreement governs the terms on which access will be granted through the BAMI Platelet Sub-study Data Access Committee (DAC) to BAMI Platelet Sub-study data available in the European Genome- phenome Archive (EGA) by the European Bioinformatics Institute, the UK Outstation of the European Molecular Biology Laboratory for and mandated by the members of the BAMI Platelet Sub-study Consortium. In signing this agreement, You are agreeing to be bound by the terms and conditions of access set out in this agreement. For the sake of clarity, the terms of access set out in this agreement apply both to the User and the User’s Institution (as defined below). User Institution and User are referred to within the agreement as “You” and “Your” shall be construed accordingly. Definitions: BPS DAC means the BAMI Platelet Sub-study Data Access Committee appointed by the BAMI Platelet Sub-study Consortium to manage access to all data submitted by the BAMI Platelet Sub-study Consortium to the EGA. BAMI Platelet Sub-study Consortium means the consortium of researchers involved in the generation and analysis of the BAMI Platelet Sub-study dataset described in the manuscript “Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation”. EGA means the European Genome-phenome Archive, a permanent archive of controlled access data generated for biomedical research and deposited to enable data sharing. The EGA is described on the website http://www.ebi.ac.ukl/ega. Data means all and any human genetic or phenotype data obtained from the EGA. Data Subject means a person, who has been informed of the purpose for which the Data is held and has given his/her informed consent thereto. User means a researcher whose User Institution has previously completed this Data Access Agreement and has received acknowledgement of its acceptance. Publications means, without limitation, articles published in print journals, electronic journals, reviews, books, posters and other written and verbal presentations of research, including social media. User Institution means the organisation at which the User is employed, affiliated or enrolled. Terms and Conditions: In signing this Agreement: 1. You agree to use the Data only for the advancement of medical research and according to the consent obtained from sample donors.   2. You agree to preserve, at all times, the confidentiality of information and Data pertaining to Data Subjects. In particular, You undertake not to identify or attempt to identify any Data Subject.   3. You agree not to attempt to link the data provided under this agreement to other information or archive data available for the data sets provided, even if access to that data has been formally granted to you, or it is freely available without restriction, without specific permission being sought from the relevant access committees. 4. You agree not to transfer or disclose the Data, in whole or part, or any identifiable material derived from the Data, to others, except as necessary for data/safety monitoring or programme management. Should You wish to share the Data with a collaborator outwith the same Institution, the third party must make a separate application for access to the Data.   5. You agree to use the data for the approved purpose and project described in your application; use of the data for a new purpose or project will require a new application and approval.   6. You accept that Data will be updated from time to time, with suitable versioning. If the reissue is at the request of sample donors and/or other ethical scrutiny, You will destroy earlier versions of the Data.   7. You accept that the BPS DAC, the BPS Consortium, or the funders of the Data or any part of the Data supplied:  a) bear no legal responsibility for the accuracy or comprehensiveness of the Data; and b) accept no liability for indirect, consequential, or incidental, damages or losses arising from use of the Data, or from the unavailability of, or break in access to, the Data for whatever reason.   8. You understand and acknowledge that the Data may be protected by copyright and other intellectual property rights, and that duplication, except as reasonably required to carry out Your research with the Data, or sale of all or part of the Data on any media may not be permitted. Users of the data are solely responsible for establishing the nature of and complying with any such intellectual property restrictions.   9. You recognise that nothing in this agreement shall operate to transfer to the User Institution any intellectual property rights relating to the Data. The User Institution has the right to develop intellectual property based on comparisons with their own data.   10. You accept that this agreement will terminate immediately upon any breach of this agreement by You and You will be required to destroy any Data held.   11. You accept that it may be necessary for the BPS Consortium or its appointed agent to alter the terms of this agreement from time to time in order to address new concerns. In this event, the EGA or its appointed agent will contact You to inform You of any changes and You agree that Your continued use of the Data shall be dependent on the parties entering into a new version of the Agreement.   12. You agree that you will submit a report to the BPS DAC, if requested, on completion of the agreed purpose. The BPS DAC agrees to treat the report and all information, data, results, and conclusions contained within such report as confidential information belonging to the User Institution.   13. You accept that the Data may be protected by and subject to international laws, including but not limited to applicable laws on the protection of personal data, and that You are responsible for ensuring compliance with any such applicable law. You undertake to implement or have implemented adequate technical and organisational security measures to protect Data. The BPS DAC reserves the right to request and inspect data security and management documentation to ensure the adequacy of data protection measures, particularly in countries that have no national laws comparable to those which pertain in the EEA.   BPS DAA ver1, un 2019 APPENDIX 1 BPS Publications Policy The BPS Consortium anticipates that data generated from the project will be used by others, such as required for developing new analytical methods, in understanding epigenetics, for integrative analysis and for other uses. Authors who use data from the project must acknowledge the BPS Consortium using the following wording "This study makes use of data generated by the BPS Consortium. A full list of the investigators who contributed to the generation of the data is available in “Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation”. Funding for the project was provided by UK Medical Research Council (MR/K024043/1). Users must also cite the primary BPS publication mentioned above. Users should note that the BPS Consortium bears no responsibility for the further analysis or interpretation of these data, over and above that published by the Consortium. BPS DAA ver1, un 2019

Studies are experimental investigations of a particular phenomenon, e.g., case-control studies on a particular trait or cancer research projects reporting matching cancer normal genomes from patients.

Study ID	Study Title	Study Type
EGAS00001004844	Single Cell MK and HSC sequencing	Other

This table displays only public information pertaining to the files in the dataset. If you wish to access this dataset, please submit a request. If you already have access to these data files, please consult the download documentation.

ID	File Type	Size	Quality Report
EGAF00004897967	fq.gz	309.7 MB	Report
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EGAF00004897972	fq.gz	153.9 MB	Report
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EGAF00004898034	fq.gz	600.3 MB	Report
EGAF00004898035	fq.gz	160.2 MB	Report
EGAF00004898036	fq.gz	194.1 MB	Report
EGAF00004898037	fq.gz	344.5 MB	Report
EGAF00004898038	fq.gz	366.7 MB	Report
EGAF00004898039	fq.gz	809.5 MB	Report
EGAF00004898040	fq.gz	890.7 MB	Report
EGAF00004898041	fq.gz	319.8 MB	Report
EGAF00004898042	fq.gz	326.8 MB	Report
EGAF00004898043	fq.gz	464.7 MB	Report
EGAF00004898044	fq.gz	481.3 MB	Report
EGAF00004898045	fq.gz	452.9 MB	Report
EGAF00004898046	fq.gz	463.8 MB	Report
EGAF00004898047	fq.gz	671.7 MB	Report
EGAF00004898048	fq.gz	666.7 MB	Report
EGAF00004898049	fq.gz	148.0 MB	Report
EGAF00004898050	fq.gz	144.3 MB	Report
EGAF00004898051	fq.gz	849.5 MB	Report
EGAF00004898052	fq.gz	860.0 MB	Report
EGAF00004898053	fq.gz	214.8 MB	Report
EGAF00004898054	fq.gz	216.7 MB	Report
EGAF00004898055	fq.gz	204.8 MB	Report
EGAF00004898056	fq.gz	196.1 MB	Report
EGAF00004898057	fq.gz	403.1 MB	Report
EGAF00004898058	fq.gz	459.0 MB	Report
EGAF00004898059	fq.gz	197.5 MB	Report
EGAF00004898060	fq.gz	202.1 MB	Report
EGAF00004898061	fq.gz	525.4 MB	Report
EGAF00004898062	fq.gz	640.3 MB	Report
EGAF00004898063	fq.gz	772.5 MB	Report
EGAF00004898064	fq.gz	746.8 MB	Report
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EGAF00004898066	fq.gz	296.5 MB	Report
EGAF00004898067	fq.gz	530.6 MB	Report
EGAF00004898068	fq.gz	591.6 MB	Report
EGAF00004898069	fq.gz	620.2 MB	Report
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EGAF00004898071	fq.gz	609.2 MB	Report
EGAF00004898072	fq.gz	613.0 MB	Report
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EGAF00004898074	fq.gz	289.3 MB	Report
EGAF00004898075	fq.gz	374.1 MB	Report
EGAF00004898076	fq.gz	434.5 MB	Report
EGAF00004898077	fq.gz	261.2 MB	Report
EGAF00004898078	fq.gz	297.3 MB	Report
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EGAF00004898081	fq.gz	450.4 MB	Report
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EGAF00004898083	fq.gz	116.8 MB	Report
EGAF00004898084	fq.gz	140.7 MB	Report
EGAF00004898085	fq.gz	485.0 MB	Report
EGAF00004898086	fq.gz	499.3 MB	Report
EGAF00004898087	fq.gz	313.4 MB	Report
EGAF00004898088	fq.gz	370.4 MB	Report
EGAF00004898089	fq.gz	110.3 MB	Report
EGAF00004898090	fq.gz	106.1 MB	Report
EGAF00004898091	fq.gz	197.7 MB	Report
EGAF00004898092	fq.gz	207.1 MB	Report
EGAF00004898093	fq.gz	214.3 MB	Report
EGAF00004898094	fq.gz	253.8 MB	Report
EGAF00004898095	fq.gz	587.5 MB	Report
EGAF00004898096	fq.gz	567.3 MB	Report
EGAF00004898097	fq.gz	335.0 MB	Report
EGAF00004898098	fq.gz	345.4 MB	Report
EGAF00004898099	fq.gz	186.1 MB	Report
EGAF00004898100	fq.gz	230.6 MB	Report
EGAF00004898101	fq.gz	676.4 MB	Report
EGAF00004898102	fq.gz	695.1 MB	Report
EGAF00004898103	fq.gz	409.3 MB	Report
EGAF00004898104	fq.gz	407.8 MB	Report
EGAF00004898105	fq.gz	201.9 MB	Report
EGAF00004898106	fq.gz	197.1 MB	Report
EGAF00004898107	fq.gz	117.5 MB	Report
EGAF00004898108	fq.gz	114.6 MB	Report
EGAF00004898109	fq.gz	457.4 MB	Report
EGAF00004898110	fq.gz	544.2 MB	Report
EGAF00004898111	fq.gz	295.5 MB	Report
EGAF00004898112	fq.gz	290.5 MB	Report
EGAF00004898113	fq.gz	384.2 MB	Report
EGAF00004898114	fq.gz	381.5 MB	Report
EGAF00004898115	fq.gz	581.8 MB	Report
EGAF00004898116	fq.gz	589.9 MB	Report
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EGAF00004898120	fq.gz	128.7 MB	Report
EGAF00004898121	fq.gz	455.4 MB	Report
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EGAF00004898124	fq.gz	170.7 MB	Report
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EGAF00004898126	fq.gz	162.8 MB	Report
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EGAF00004898132	fq.gz	327.1 MB	Report
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EGAF00004898136	fq.gz	403.5 MB	Report
EGAF00004898137	fq.gz	166.3 MB	Report
EGAF00004898138	fq.gz	165.1 MB	Report
EGAF00004898139	fq.gz	8.2 MB	Report
EGAF00004898140	fq.gz	9.8 MB	Report
EGAF00004898141	fq.gz	123.5 MB	Report
EGAF00004898142	fq.gz	147.2 MB	Report
EGAF00004898143	fq.gz	545.8 MB	Report
EGAF00004898144	fq.gz	555.1 MB	Report
EGAF00004898145	fq.gz	249.1 MB	Report
EGAF00004898146	fq.gz	290.4 MB	Report
EGAF00004898147	fq.gz	191.3 MB	Report
EGAF00004898148	fq.gz	205.0 MB	Report
EGAF00004898149	fq.gz	220.3 MB	Report
EGAF00004898150	fq.gz	238.4 MB	Report
EGAF00004898151	fq.gz	265.6 MB	Report
EGAF00004898152	fq.gz	324.7 MB	Report
EGAF00004898153	fq.gz	168.9 MB	Report
EGAF00004898154	fq.gz	180.2 MB	Report
EGAF00004898155	fq.gz	185.1 MB	Report
EGAF00004898156	fq.gz	224.6 MB	Report
EGAF00004898157	fq.gz	287.4 MB	Report
EGAF00004898158	fq.gz	296.1 MB	Report
EGAF00004898159	fq.gz	624.4 MB	Report
EGAF00004898160	fq.gz	640.3 MB	Report
EGAF00004898161	fq.gz	454.4 MB	Report
EGAF00004898162	fq.gz	447.5 MB	Report
EGAF00004898163	fq.gz	3.6 GB	Report
EGAF00004898164	fq.gz	3.9 GB	Report
EGAF00004898165	fq.gz	265.2 MB	Report
EGAF00004898166	fq.gz	318.8 MB	Report
EGAF00004898167	fq.gz	255.3 MB	Report
EGAF00004898168	fq.gz	306.8 MB	Report
EGAF00004898169	fq.gz	380.5 MB	Report
EGAF00004898170	fq.gz	346.1 MB	Report
EGAF00004898171	fq.gz	164.6 MB	Report
EGAF00004898172	fq.gz	177.7 MB	Report
EGAF00004898173	fq.gz	183.3 MB	Report
EGAF00004898174	fq.gz	216.8 MB	Report
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EGAF00004898176	fq.gz	298.3 MB	Report
EGAF00004898177	fq.gz	266.9 MB	Report
EGAF00004898178	fq.gz	263.1 MB	Report
EGAF00004898179	fq.gz	49.5 MB	Report
EGAF00004898180	fq.gz	50.7 MB	Report
EGAF00004898181	fq.gz	167.7 MB	Report
EGAF00004898182	fq.gz	200.8 MB	Report
EGAF00004898183	fq.gz	384.5 MB	Report
EGAF00004898184	fq.gz	395.1 MB	Report
EGAF00004898185	fq.gz	616.4 MB	Report
EGAF00004898186	fq.gz	639.0 MB	Report
EGAF00004898187	fq.gz	240.0 MB	Report
EGAF00004898188	fq.gz	264.5 MB	Report
EGAF00004898189	fq.gz	674.9 MB	Report
EGAF00004898190	fq.gz	783.8 MB	Report
EGAF00004898191	fq.gz	643.7 MB	Report
EGAF00004898192	fq.gz	672.4 MB	Report
EGAF00004898193	fq.gz	454.6 MB	Report
EGAF00004898194	fq.gz	465.5 MB	Report
EGAF00004898195	fq.gz	301.8 MB	Report
EGAF00004898196	fq.gz	308.3 MB	Report
EGAF00004898197	fq.gz	507.5 MB	Report
EGAF00004898198	fq.gz	512.4 MB	Report
EGAF00004898199	fq.gz	97.6 MB	Report
EGAF00004898200	fq.gz	114.7 MB	Report
EGAF00004898201	fq.gz	369.1 MB	Report
EGAF00004898202	fq.gz	375.6 MB	Report
EGAF00004898203	fq.gz	65.4 MB	Report
EGAF00004898204	fq.gz	66.5 MB	Report
EGAF00004898205	fq.gz	377.4 MB	Report
EGAF00004898206	fq.gz	424.7 MB	Report
EGAF00004898207	fq.gz	729.7 MB	Report
EGAF00004898208	fq.gz	725.0 MB	Report
EGAF00004898209	fq.gz	176.2 MB	Report
EGAF00004898210	fq.gz	204.7 MB	Report
EGAF00004898211	fq.gz	92.0 MB	Report
EGAF00004898212	fq.gz	92.8 MB	Report
EGAF00004898213	fq.gz	407.2 MB	Report
EGAF00004898214	fq.gz	473.6 MB	Report
EGAF00004898215	fq.gz	97.6 MB	Report
EGAF00004898216	fq.gz	92.9 MB	Report
EGAF00004898217	fq.gz	470.9 MB	Report
EGAF00004898218	fq.gz	561.5 MB	Report
EGAF00004898219	fq.gz	337.0 MB	Report
EGAF00004898220	fq.gz	363.9 MB	Report
EGAF00004898221	fq.gz	326.4 MB	Report
EGAF00004898222	fq.gz	331.2 MB	Report
EGAF00004898223	fq.gz	717.4 MB	Report
EGAF00004898224	fq.gz	753.1 MB	Report
EGAF00004898225	fq.gz	135.1 MB	Report
EGAF00004898226	fq.gz	165.3 MB	Report
EGAF00004898227	fq.gz	814.8 MB	Report
EGAF00004898228	fq.gz	890.8 MB	Report
EGAF00004898229	fq.gz	94.8 MB	Report
EGAF00004898230	fq.gz	94.0 MB	Report
EGAF00004898231	fq.gz	139.0 MB	Report
EGAF00004898232	fq.gz	135.6 MB	Report
EGAF00004898233	fq.gz	339.2 MB	Report
EGAF00004898234	fq.gz	348.5 MB	Report
EGAF00004898235	fq.gz	312.4 MB	Report
EGAF00004898236	fq.gz	287.0 MB	Report
EGAF00004898237	fq.gz	558.2 MB	Report
EGAF00004898238	fq.gz	559.7 MB	Report
EGAF00004898239	fq.gz	381.0 MB	Report
EGAF00004898240	fq.gz	390.9 MB	Report
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EGAF00004912108	fq.gz	1.3 GB	Report
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EGAF00004913022	fq.gz	960.9 MB	Report
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EGAF00004915480	fq.gz	81.6 MB	Report
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EGAF00004915488	fq.gz	163.5 MB	Report
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EGAF00004915510	fq.gz	6.4 GB	Report
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EGAF00004916612	fq.gz	2.9 GB	Report
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EGAF00004922476	fq.gz	1.1 GB	Report
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EGAF00004922692	fq.gz	3.7 GB	Report
EGAF00004922693	fq.gz	4.1 GB	Report
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EGAF00004922959	fq.gz	322.4 MB	Report
EGAF00004922966	fq.gz	2.2 GB	Report
EGAF00004922967	fq.gz	2.5 GB	Report
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EGAF00004922990	fq.gz	5.8 GB	Report
EGAF00004922991	fq.gz	6.5 GB	Report
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EGAF00004923007	fq.gz	914.3 MB	Report
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EGAF00004923077	fq.gz	3.4 GB	Report
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EGAF00004923340	fq.gz	522.5 MB	Report
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EGAF00004923346	fq.gz	726.9 kB	Report
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EGAF00004923350	fq.gz	418.5 MB	Report
EGAF00004923351	fq.gz	4.8 GB	Report
EGAF00004923352	fq.gz	5.9 GB	Report
EGAF00004923353	fq.gz	99.3 MB	Report
EGAF00004923354	fq.gz	94.5 MB	Report
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EGAF00004923357	fq.gz	444.7 MB	Report
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EGAF00004923365	fq.gz	215.3 MB	Report
EGAF00004923366	fq.gz	251.6 MB	Report
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EGAF00004923370	fq.gz	94.1 kB	Report
EGAF00004923371	fq.gz	435.8 MB	Report
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EGAF00004923376	fq.gz	295.9 MB	Report
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EGAF00004923379	fq.gz	172.1 MB	Report
EGAF00004923380	fq.gz	170.9 MB	Report
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EGAF00004923384	fq.gz	186.6 MB	Report
EGAF00004923385	fq.gz	294.6 MB	Report
EGAF00004923386	fq.gz	303.3 MB	Report
EGAF00004923387	fq.gz	685.7 MB	Report
EGAF00004923388	fq.gz	766.7 MB	Report
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EGAF00004923390	fq.gz	87.3 kB	Report
EGAF00004923391	fq.gz	132.3 MB	Report
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EGAF00004923890	fq.gz	582.5 MB	Report
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5268 Files (2.3 TB)