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Status |
Public on May 22, 2023 |
Title |
TXG 10xv2 2 (v2 T2) |
Sample type |
SRA |
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Source name |
PBMC
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Organism |
Homo sapiens |
Characteristics |
tissue: PBMC cell type: PBMC treatment: Wild type
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cryopreserved human PBMCs from a male and female donor were puchased from AllCells and (AllCells, Lot #3030456 and 3030663). Cryopreserved PBMC were thawed according to the 10x Genomics demonstrated protocol CG00039 ("Fresh Frozen Human Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing"). Briefly, 1 mL of frozen cells was rapidly thawed in a 37 °C water bath and transferred to a 50 mL tube using a 1000 µL wide bore tip. Next, 1 mL of 37 °C pre-warmed medium supplemented with 10% FBS (Thermo Fisher Scientific) was added drop-wise while gently swirling the sample. After 1 minute of incubation at room temperature, 2, 4, 8 and 16 mL of medium with 10% FBS were added drop-wise with 1 minute of incubation at room-temperature inbetween. The cell suspension was then centrifuged at 300 g for 5 min. at room temperature. The pellet was resuspended in 10 mL of medium with 10% FBS, and cells were counted. Unless specified otherwise in technology-specific methods sections, nuclei isolation was performed according to the 10x Genomics demonstrated protocol “Nuclei Isolation for Single Cell ATAC Sequencing”. Briefly, 1 million cells from the cell mix were transferred to a 1.5 mL microcentrifuge tube and centrifuged at 500 rcf for 5 min at 4 °C. The supernatant was removed without disrupting the cell pellet and 100 µL of chilled Lysis Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 MgCl2; 0.1% Tween-20; 0.1% Nonidet P40 Substitute; 0.01% Digitonin and 1% BSA) were added and pipette mixed 10 times. Samples were then incubated on ice for 3 min. Following lysis, 1 mL of chilled Wash Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 MgCl2; 0.1% Tween-20 and 1% BSA) was added and pipette mixed. Nuclei were centrifuged at 500 rcf for 5 min at 4 °C and the supernatant removed without disrupting the nuclei pellet. Based on the starting number of cells and assuming a 50% loss during the procedure, nuclei were resuspended into the appropriate volume of chilled Diluted Nuclei Buffer (10x Genomics) in order to achieve a nuclei concentration of 925-2300 nuclei/µL, suitable for a Target Nuclei Recovery of 3000. scATAC-seq libraries were prepared according to the Chromium Single Cell ATAC Reagent Kits v2 User Guide (10x Genomics; CG000496 Rev B ). Briefly, the Transposition Reaction was prepared by mixing the desired number of nuclei with ATAC Buffer (10x Genomics) and ATAC Enzyme (10x Genomics), and was then incubated for 30 min at 37 °C. Nuclei were partitioned into Gel Bead-In-Emulsions (GEMs) by using the Chromium Controller with Chip H. Sample v2 T1 was the only sample for which Chromium X was used. After this, the DNA linear amplification was performed by incubating the GEMs at the following thermal cycling conditions: 72 °C for 5 minutes, 98 °C for 30 seconds, 12 cycles of 98 °C for 10 seconds, 59 °C for 30 seconds and 72 °C for 1 minute. GEMs were broken using the Recovery Agent (10x Genomics), and the resulting DNA was purified by sequential Dynabeads and SPRIselect reagent beads clean-ups. Libraries were indexed by PCR using the Single Index Kit N Set A, and incubating at the following thermal cycling conditions: 98 °C for 45 seconds, 8 cycles of 98 °C for 20 seconds, 67 °C for 30 seconds, 72 °C for 20 seconds with a final extension of 72 °C for 1 min. Sequencing libraries were subjected to a final bead clean-up SPRIselect reagent.]
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Library strategy |
ATAC-seq |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
Raw sequencing reads were processed to aligned reads and fragments files using the PUMATAC pipeline. Briefly, cell barcodes were corrected using a pre-existing whitelist using scToolkit. TrimGalore was used to trim reads. Alignment to GRCh38 was performed using bwa-mem2. Cell barcode mutliplets were detected using scToolkit. Aligned reads were converted to fragments.tsv.gz using scToolkit. Assembly: GRCh38 Supplementary files format and content: "FULL" fragments.tsv.gz: tab-delimited bed-like fragments file with 5 fields: chromosome, start position, end position, cell barcode and count. Derived from the full sequencing data. Supplementary files format and content: "FULL" fragments.tsv.gz.tbi: tabix index supplementary to full fragments.tsv.gz files. Supplementary files format and content: "FIXEDCELLS" fragments.tsv.gz: tab-delimited bed-like fragments file with 5 fields: chromosome, start position, end position, cell barcode and count. Derived from the sequencing data downsampled to the fixed number of cells found in the full sequencing data. Supplementary files format and content: "FIXEDCELLS" fragments.tsv.gz.tbi: tabix index supplementary to fixedcells fragments.tsv.gz files.
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Submission date |
Mar 17, 2023 |
Last update date |
Aug 02, 2023 |
Contact name |
Stein Aerts |
E-mail(s) |
stein.aerts@kuleuven.be
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Organization name |
KU Leuven
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Lab |
Lab of Computational Biology
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Street address |
O&N4 Herestraat 49 PO Box 602
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City |
Leuven |
State/province |
Vlaams-Brabant |
ZIP/Postal code |
3000 |
Country |
Belgium |
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Platform ID |
GPL24676 |
Series (1) |
GSE194028 |
Systematic benchmarking of single-cell ATAC sequencing protocols |
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Relations |
BioSample |
SAMN33818124 |
SRA |
SRX19718526 |
Supplementary file |
Size |
Download |
File type/resource |
GSM7102950_TXG_10xv2_2.FIXEDCELLS.fragments.tsv.gz |
2.0 Gb |
(ftp)(http) |
TSV |
GSM7102950_TXG_10xv2_2.FIXEDCELLS.fragments.tsv.gz.tbi.gz |
1017.4 Kb |
(ftp)(http) |
TBI |
GSM7102950_TXG_10xv2_2.FULL.fragments.tsv.gz |
2.2 Gb |
(ftp)(http) |
TSV |
GSM7102950_TXG_10xv2_2.FULL.fragments.tsv.gz.tbi.gz |
1.0 Mb |
(ftp)(http) |
TBI |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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