 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 09, 2024 |
| Title |
Extended young donors, BMMCs, mRNA-derived cDNA, sample 16 |
| Sample type |
SRA |
| |
|
| Source name |
Bone Marrow
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Bone Marrow
cell type: Bone Marrow Mononuclear Cells (BMMCs)
group: young donor
library type: mRNA
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Bone marrow was collected from healthy young or aged donors. Bone marrow aspirate was diluted with an equal volume of Wash Buffer (PBS, 2% FBS, 1mM EDTA). Ficoll medium was added to SepMateTM (Catalog #85460) tubes and then the diluted bone marrow sample was layered on top followed by centrifuging at 1200 g for 20 minutes at room temperature. The top layer, containing the mononuclear cells, was transferred to a new tube, which was then filled up by Wash Buffer. The mononuclear cells were centrifuged at 300 g for 8 minutes. The supernatant was discarded, and the cells were washed twice and resuspended in Wash Buffer for further enrichment or in Freezing buffer (10% DMSO in FBS). Starting with the BMMCs isolated from the previous step, we enriched CD34+ cells following EasySep™ Human Cord Blood CD34 Positive Selection Kit II (Catalog #17896). Briefly, EasySep™ Human CD34 Positive Selection Cocktail (Catalog #18096C) was added to BMMCs suspension to 100 ul/ml and incubated at room temperature for 10 minutes. EasySep™ Dextran RapidSpheres™ (Catalog #50100) was vortexed and added to each sample to 50 µl/ml. The mix was incubated for 3 minutes at room temperature. Next, Wash Buffer (7 ml) was added to the tube, The cells were then washed 4 times in "The Big Easy" EasySep™ Magnet (Catalog #18001). Finally, cells were resuspended in Wash Buffer and were centrifuged at 300g for 10 min. The CD34+ positive cell pellet was then resuspended in a freezing buffer (10% DMSO in FBS). To further enrich the hematopoietic stem cells (HSCs), an aliquot of the enriched CD34+ cells were stained by one of the following two antibody panels. 1) CD34 PerCP-Cy5.5 (Catalog #347222); CD45RA Alexa Fluor 488 (Catalog #304114); CD90 PE-Cy7 (Catalog #561558); and DAPI (Catalog #D1306) as viability dye. Or 2) CD34 BV421 (Catalog #562577); CD45RA-APC-H7 (Catalog #560674); CD90 PE-Cy7 (Catalog #561558), and 7-AAD as viability dye(Catalog #559925). The cells were further sorted using BD FACSAriaTM for CD34+CD45RA-CD90+ to enrich HSCs. BMMCs, as well as enriched CD34+ and CD34+CD45RA-CD90+ cells were cryopreserved. After thawing, cells were immediately processed for experiment as soon as possible without any culturing. Different donors are hashed and pooled together for ReDeeM experiments
RNA lib and ATAC lib prep: After tagmentation, cells were immediately processed for GEM generation & barcoding following 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Manual (version CG000338 Rev A, 10x Genomics) with Step 2. Next, the Post GEM Incubation Cleanup and Pre-Amplification PCR were performed following the same User Manual with Step 3 and Step 4. The pre-amplified library was eluted in 100ul EB buffer instead of 160ul. For RNA library prep, we took an aliquot of 35 ul pre-amplified library and followed the same User Manual (version CG000338 Rev A, 10x Genomics) from Step 6 and Step 7. We developed an in-house protocol to generate the massive ATAC library with enough material for both direct sequencing and mtDNA hybridization. For each sample, 8 reactions of the following were performed (each in 50 ul). 5 μl pre-amplified product, 25 μl NEBnext mix (Catalog M0541L), 2.4 μl 10 μM Nextera N70X primer (Supplementary Data 2), 2.4 μl 10X SI PCR primer B (Catalog #PN2000128), 0.5 μl 100X SYBR Green (Catalog #S7563), and 14.7 μl water. The thermocycler program started with 98°C 45 s., followed by the 6-12 cycles of: 98°C for 10 s., 63°C for 30 s., and 72°C for 20 s.; The actual cycle numbers are determined using the amplification curve and the qPCR was stopped when the curve started to reach plateau. The qPCR products (50ul * 8 wells) were combined and purified using SPRI beads (Catalog #B23318) with size selection 0.4X~1.55X. The expected yield is 2,000 ng to 4,000 ng.
mtDNA lib prep: We designed 4 sets of DNA probes, with each probe set covering the whole mitochondrial genome and containing 138 biotinylated oligos that are 120 bp long. The 4 sets of probes ((v1, v2, v3, and v4) are staggered by 30 bp with each other and ensure good coverage on junction areas (Supplementary Data 2). The hybridization started with 500 ng ATAC library products for each probe set. In total, 4 independent reactions were performed in parallel and combined at the end (2,000ng input in total). The detailed incubation and wash methods largely followed the Tube Protocol in IDT xGen Hybridization Capture of DNA Libraries protocol. Briefly, 500 ng input was combined with Cot DNA and purified by SPRI beads, which was eluted by hybridization mix that contains hybridization buffer, hybridization buffer enhancer, one of the probe set, as well as xGen™ Universal Blockers NXT (Catalog #1079584). The hybridization reaction was performed as below: 95C 30 seconds followed by 65C overnight. The biotin-streptavidin Dynabeads purification was carried out on Day 2 using xGen Hybridization and Wash Kit. The Dynabeads after capturing and washing were used for final quick qPCR amplification (3-5 cycles). The reaction mix is as below: 20ul beads, 25ul KAPA HiFi HotStart ReadyMix (Catalog #KK2601), Primer 2.4ul P5 (Supplementary Data 2), Primer P7 (Supplementary Data 2), 0.5ul 100X SYBR Green (Catalog #S7563). The PCR program started with 98°C 45 s, followed by the 3-5 cycles of: 98°C for 10 s., 63°C for 30 s., and 72°C for 20 s. The final enriched mtDNA was purified by SPRI beads (1.6X)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
3' mRNA library (Cell barcodes are matchable with ATAC, and CellHash lib in sample 16): read1 (1-28nt) file contains cell barcode and UMI; read2 file contains transcript.
|
| Data processing |
ReDeeM. For each sample, all three libraries were sequenced on Illumina NovaSeq S4 300 cycle kit. The sequencing length was as below. Read1: 149nt, i7: 8nt, i5: 24nt, Read2: 149nt. We expected to sequence 20,000~30,000 raw reads per cell for RNA libraries and ATAC libraries, while 50,000~60,000 raw reads for enrich mtDNA libraries.
The data preprocessing for the joint single-cell RNA and ATAC data was performed using 10X Genomics data preprocessing software Cell-ranger-arc. The basic quality control analysis was as follows: RNA UMI: 1,000~25,000 transcripts per cell, unique ATAC fragment: 1,000 ~ 70,000, Fragment on peak minimum percentage: 10%, minimum mtDNA copies per position per cell: 10X. Finally, the possible doublets are removed by Amulet (using default parameter). Unsupervised community detection-based clustering was performed on weighted nearest neighbour (WNN) using Seurat. Cell types are firstly annotated by gene expression using the latest signature (van Galen under review) which was further validated by sorted bulk RNA-seq data (https://sankaranlab.shinyapps.io/geneExpression/). The ATAC unique fragments for each clustered cell type were further aggregated to generate pseudo-bulk ATAC tracks. The single cell motif analysis was performed using ChromVar using the JASPAR human transcription factor (TF) database. The deviation of the TF frequency was computed for each cell for the downstream analysis.
The mutation calling pipeline is in following steps using package redeemV: 1, Data Preprocessing and eUMI grouping. 2, Consensus filtering. 3, Strand bias removal. 4, Lineage informative variants identification
Assembly: hg38
Supplementary files format and content: mtDNA mutation raw genotype and depth: mtDNA.RawGenotypes.Total.StrandBalance, mtDNA.depth
Supplementary files format and content: 10x Genomics output files: filtered_feature_bc_matrix.h5, atac_fragments.tsv.gz
Supplementary files format and content: TotalSeq-A Cell hash analyzed: Hash_calls.tsv, Hash_annotation.tsv
|
| |
|
| Submission date |
Mar 07, 2024 |
| Last update date |
Mar 09, 2024 |
| Contact name |
Chen Weng |
| E-mail(s) |
cweng@wi.mit.edu
|
| Organization name |
Broad Institute and Whitehead Institute
|
| Department |
Genetics
|
| Lab |
Vijay Sankaran lab and Jonathan Weissman lab
|
| Street address |
415 Main St, Cambridge
|
| City |
Boston |
| State/province |
OH |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE219015 |
Deciphering cell states and genealogies of human hematopoiesis with single-cell multi-omics |
| GSE261078 |
Deciphering cell states and genealogies of human hematopoiesis with single-cell multi-omics [Extended donors] |
|
| Relations |
| BioSample |
SAMN40298505 |
| SRA |
SRX23867619 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8133911_Extended_Young_BMMC.filtered_feature_bc_matrix.h5 |
116.3 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
