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| Status |
Public on May 11, 2021 |
| Title |
scATACAseq_MSKCLL156_6hours_DMSO |
| Sample type |
SRA |
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| Source name |
Peripheral Blood Mononuclear Cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MSKCLL-156
timepoint: 6 hours
treatment+dose: DMSO
assay: scATAC-seq
hashtag oligos: N/A
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| Treatment protocol |
For scATAC-seq,The peripheral blood mononuclear cells (PBMC) fraction was isolated from a CLL specimen by Ficoll Paque Plus (Amersham Biosciences, Piscataway, USA). Buffy layers containing the PBMCs were removed and the red blood cells were lysed using red blood cell lysis buffer (10mM KHCO3, 150mM NH4Cl and 0.1mM EDTA, pH 8.0) for 5 minutes at 37oC followed by a wash with sterile phosphate buffer saline (PBS). Cells were then cultured in RPMI-1640 media supplemented with 10% heat inactivated bovine calf serum, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin for 6 hours in the presence of A-485 (1µM) or DMSO vehicle. Following 6 hours incubation, samples for snATAC-seq were first subjected to nuclei isolation with lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% Digitonin, 1% BSA) and wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) in accordance with the 10x Genomics Nuclei Isolation for Single Cell ATAC Sequencing protocol (CG000169, Rev D). Nuclei number and quality was assessed using Trypan blue and the Countess II Automated Cell Counter (ThermoFisher). Following QC, nuclei were then resuspended at 5,000 nuclei/µL in 1X diluted nuclei buffer (10x Genomics) and used as input for the 10x Genomics Chromium Next GEM Single Cell ATAC Library Kit (PNs-1000176, 1000162, 1000212).
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| Growth protocol |
For scRNA-seq, The peripheral blood mononuclear cells (PBMC) fraction was isolated from a CLL specimen by Ficoll Paque Plus (Amersham Biosciences, Piscataway, USA). Buffy layers containing the PBMCs were removed and the red blood cells were lysed using red blood cell lysis buffer (10mM KHCO3, 150mM NH4Cl and 0.1mM EDTA, pH 8.0) for 5 minutes at 37oC followed by a wash with sterile phosphate buffer saline (PBS). Cells were then cultured in RPMI-1640 media supplemented with 10% heat inactivated bovine calf serum, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin for 6 hours in the presence of A-485 (1µM) or DMSO vehicle. Following 6 hours incubation, samples for scRNA-seq were first subjected to Fc receptor blocking (using the Human TruStain FcX reagent; BioLegend) and barcoded using TotalSeq-C cell hashing antibodies (TotalSeq-C0251 for DMSO treatment and TotalSeq-C0252 for A-485 treatment; BioLegend), following the manufacturers recommendations. Samples were then pooled and resuspended at 1000 cells/µL in PBS+0.04% BSA and passed to the Integrated Genomics Operation (IGO; Memorial Sloan Kettering Cancer Center) for library preparation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For scATAC-seq, Nuclei were incubated with transposition mix for 60 minutes at 37°C and then loaded onto Chromium Chip. GEM generation and library preparation of up to 10,000 nuclei proceeded using the Chromium Single Cell ATAC Library & Gel Bead Kit according to the manufacturer’s protocol with 11 cycles of PCR.
For scRNA-seq, the single cell suspension was loaded onto Chromium Chip A (10X Genomics PN 230027) and GEM generation, cDNA synthesis, cDNA amplification, and library preparation of 6,481 cells proceeded using the Chromium Single Cell 5’ Reagent Kit (10X Genomics PN 1000006) according to the manufacturer’s protocol. cDNA amplification included 11 cycles and 41ng of the material was used to prepare sequencing libraries with 14 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a 28/91 paired end paired end run using the NovaSeq 6000 S1 Reagent Kit (100 cycles) (Illumina).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
scATAC_meta_data.RDS
scATAC_pmat.RDS
scATAC_bmat.RDS
A485_DMSO_merged_narrow_peaks.bed
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| Data processing |
For scRNA-seq, Pre-processing of scRNA-seq data was performed using Cell Ranger v4.0.0. Files were processed using the cell ranger count function mapping to the hg38 genome (arguments: --chemistry=fiveprime). Clustering was performed using Seurat V3 and cells were filtered based on nFeature_RNA greater than 200 and less than 2500, percent of mitochondrial reads less than 10 and nCount_RNA greater than 1000. The two samples were integrated using the standard worklow.
For scATAC-seq, Pre-processing of snATAC-seq were performed using the cell ranger v4.0.0 and reads were mapped to the hg38 genome. Cell ranger output was converted to snap files as outlined in Snaptools. Cells were filtered based on read count greater than 3000 and less than 30000 and fraction of reads in promoters greater than 20%. Samples were integrated using Harmony (Korsunsky et al., 2019) in the SnapATAC framework using binary counts in 5kb bins across the genome
For scRNA-seq,Differential gene expression between conditions was performed using the FindMarkers function (|logFC| > 0.75 and adjP < 0.05). Heatmap visualization based on normalized genes counts across cells in each condition for each cluster inferCNV of the Trinity CAT project was used to analyze large scale copy number variations (Patel et al., 2014; Tirosh et al., 2016). T-cells and myeloid cells were used as normal reference and the following parameters were used for the run function: cutoff=0.1, denoise=TRUE, HMM=TRUE.
For scATAC-seq, Visualization based on UMAP from SnapATAC pipeline and cells were colored based on label transferred from scRNA-seq using Seurat V3. Integration of scRNA-seq and snATAC-seq data was based gene activity score for snATAC-seq data and prediction score greater than or equal to 0.5. Peak calling for snATAC-seq performed using the runMACS function in SnapATAC with the following parameters: --nomodel --slocal 1000 --call-summits --qval 0.01 -B –SPMR.
Genome_build: Hg38
Supplementary_files_format_and_content: scATAC_meta_data.RDS (R Data file- scATAC-seq data)
scATAC_pmat.RDS (R Data file- peak by cell matrix for scATAC-seq data)
A485_DMSO_merged_narrow_peaks.bed (BED file of merged peaks for scATAC-seq data)
scATAC_bmat.RDS (R data file- binary 5kb bin matrix for scATAC-seq data)
scRNA_Seurat_obj.RDS (Seurat object for scRNA-seq data)
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| Submission date |
May 04, 2021 |
| Last update date |
May 11, 2021 |
| Contact name |
Simon J Hogg |
| Organization name |
AbbVie
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| Department |
Oncology Discovery Research
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| Street address |
1000 Gateway Boulevard
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| City |
South San Francisco |
| State/province |
California |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE172051 |
Targeting P300/CBP reveals discrete regulation of transcription and chromatin accessibility by histone acetylation |
| GSE173811 |
Targeting P300/CBP reveals discrete regulation of transcription and chromatin accessibility by histone acetylation [singlecellseq] |
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| Relations |
| BioSample |
SAMN19008794 |
| SRA |
SRX10769424 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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