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| Status |
Public on Apr 16, 2019 |
| Title |
BY_5k |
| Sample type |
SRA |
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| Source name |
ATCC
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell type: mixture of GM12878 and A20 cells
treatment: n/a
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| Biomaterial provider |
Coriell Cell Repositories https://www.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=GM12878&Product=CC
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| Treatment protocol |
Fresh BCC biopsies were digested in 5 mL DMEM/F12 media + 250 ug/mL Liberase TL and 200 U/mL DNAse I with the gentleMACS Octo system at 37C for 3 hours at 20 rpm. After tissue pieces were fully digested, 50 uL of 500 mM EDTA was added and samples were collected by centrifugation at 300xg for 5 minutes. Single-cell suspensions were filtered through 70 um mesh and pelleted by centrifugation at 300xg at 4C for 10 minutes. Finally, cells were then resuspended in 1 mL of RPMI media and cryopreserved in FBS supplemented with 10% DMSO.Cells were gently thawed at 37C for 5 min and resuspended in media prior to FACS. Cells were stained with anti-CD45 V500 (clone HI30, cat. no. 560779, BD Biosciences), anti- CD3 FITC (clone OKT3, cat. no. 11-0037-41, Invitrogen), anti-CD8 Pacific Blue (clone 3B5, cat. no. MHCD0828, Invitrogen), anti-PD-1 APC/Cy7 (clone EH12.2H7, cat. no. 329921, BioLegend), and anti-HLA-DR eVolve 605 (clone LN3, cat. no. 83-9956-41, Affymetrix-Ebioscience). All antibodies were used at a 1:200 dilution, with the exception of anti-CD45 and anti-HLA-DR antibodies, which were used at a 1:100 dilution. Propidium iodine (cat. no. P3566, Invitrogen) was used for live/dead staining at a final concentration of 2.5 g/mL. PI-negative live cells were sorted as T cells (CD45+CD3+), non-T immune cells (CD45+CD3-), or tumor/stromal cells (CD45-CD3-). Cells were processed following 10x Demonstrated Protocol (https://assets.ctfassets.net/an68im79xiti/18T8YsKiwKcGUakQmc8U22/086d0daa06444314fb757e35828f3c08/CG000169_DemonstratedProtocol_NucleiIsolation_ATAC_Sequencing_RevA.pdf) for PBMCs and Low Cell Input Nuclei Isolation protocol was followed for samples with cell count < 100,000. Isolated nuclei were further processed for scATAC-seq.
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| Growth protocol |
Human (GM12878) and Mouse A20 (ATCC TIB-208) B Lymphocytes were acquired and cultured according to guidelines from Coriell and ATCC, respectively. Fresh PBMCs, GM12878, and A20 cells were frozen according to the instructions outlined here: https://assets.ctfassets.net/an68im79xiti/18T8YsKiwKcGUakQmc8U22/086d0daa06444314fb757e35828f3c08/CG000169_DemonstratedProtocol_NucleiIsolation_ATAC_Sequencing_RevA.pdf. Healthy volunteer PBMC and BM samples were obtained from AllCells or the Stanford Blood Center (SBC).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
All protocols to generate scATAC-seq data on the 10x Chromium platform, including sample prep, library prep, instrument and sequencing settings, are available here: https://support.10xgenomics.com/single-cell-atac. The isolation, washing, and counting of nuclei suspensions were performed according to the Demonstrated Protocol: Nuclei Isolation for Single Cell ATAC Sequencing (10x Genomics). Briefly, anywhere from 100,000 to 1,000,000 cells were added to a 2 ml microcentrifuge tube and centrifuged (300 rcf for 5 min at 4°C). The supernatant was removed without disrupting the cell pallet and 100 µl 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) was added then pipette mixed 10 times. The microcentrifuge tube was then incubated on ice, with the length of time optimized for each cell type: GM12878 and A20 cell lines were 5 min; PB and BM cells were 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 the resulting solution and pipette mixed 5 times. Nuclei were centrifuged (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 desired final nuclei concentration, an appropriate volume of chilled Diluted Nuclei Buffer (10x Genomics; 2000153) was used to resuspend nuclei. The resulting nuclei concentration was determined using a Countess II FL Automated Cell Counter. Nuclei were then immediately used to generate single cell ATAC-seq libraries as described below.
scATAC-seq libraries were prepared according to the Chromium Single Cell ATAC Reagent Kits User Guide (10x Genomics; CG000168 Rev A). Briefly, the desired number of nuclei were combined with ATAC Buffer (10x Genomics; 2000122) and ATAC Enzyme (10x Genomics; 2000123/2000138), to form a Transposition Mix which was then incubated for 60 min at 37°C. A Master Mix comprising of Barcoding Reagent (10x Genomics; 2000124), Reducing Agent B (10x Genomics; 2000087) and Barcoding Enzyme (10x Genomics; 2000125/2000139) was then added to the same tube as Transposed Nuclei. The resulting solution was loaded onto a Chromium Chip E (10x Genomics; 2000121) in a Chip Holder (10x Genomics; 330019). Vortexed Chromium Single Cell ATAC Gel Beads (10x Genomics; 2000132) then Partitioning Oil (10x Genomics; 220088) were also loaded onto the same Chromium Chip E before attaching a 10x Gasket (10x Genomics; 370017/3000072) and placing into on a ChromiumTM Single Cell Controller instrument (10x Genomics, Pleasanton, CA, USA). Resulting single-cell GEMs were collected at the completion of the run (~7 min) and linear amplification was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad; 1851197): 72°C for 5 min, 98°C for 30 s, cycled 12 x: 98°C for 10 s, 59°C for 30 s and 72°C for 1 min. Emulsions were coalesced using tje Recovery Agent (10x Genomics; 220016) then subjected to Dynabeads (2000048) and SPRIselect reagent (Beckman Coulter; B23318) bead clean-ups. Indexed sequencing libraries were constructed by combining the barcoded linear amplification product with a Sample Index PCR Mix comprising of SI-PCR Primer B (10x Genomics; 2000128), Amp Mix (10x Genomics; 2000047/2000103) and Chromium i7 Sample Index (10x Genomics; 3000262). Amplification was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module: 98°C for 45 s, cycled variable amounts depending on cell load: 98°C for 20 s, 67°C for 30 s, 72°C for 20 s with a final extension of 72°C for 1 min. The barcode sequencing libraries were subjected to a final bead clean-up SPRIselect reagent and quantified by quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms; KK4824).
Sequencing libraries were loaded on an Illumina sequencer with 2 × 50 paired-end kits using the following read length: 50 bp Read 1N, 8 bp i7 Index, 16 bp i5 Index and 50 bp Read 2N.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
The Cell Ranger ATAC Software Suite (version 1.0.0) was used for processing sequencing information and single cell barcodes
Read pairs were aligned and deduplicated to yield fragment intervals whose ends signify accessibiltiy sites along the genome where the transposase made cuts
Regions of enriched accessibility, i.e. peaks were identified along the genome
Barcodes likely associated with whole cells were identified by analyzing the number of fragments mapped near TSS regions and a peak-barcode matrix is defined
Genome_build: hg19
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| Submission date |
Apr 14, 2019 |
| Last update date |
May 29, 2019 |
| Contact name |
Preyas Shah |
| Organization name |
10x Genomics
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| Street address |
7068 Koll Center Pkwy #401
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| City |
Pleasanton |
| ZIP/Postal code |
94566 |
| Country |
USA |
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| Platform ID |
GPL16512 |
| Series (1) |
| GSE129785 |
Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion |
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| Relations |
| BioSample |
SAMN11414781 |
| SRA |
SRX5679916 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3722046_BY_5k_fragments.tsv.gz |
1.3 Gb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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