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| Status |
Public on Apr 26, 2021 |
| Title |
Donor_4_KLF2_F8_ATAC |
| Sample type |
SRA |
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| Source name |
CD4+CD25- T cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD4+CD25- T cells
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| Treatment protocol |
CD4+CD25- T cells were isolated from 3 donors using consented Luekopaks (STEMCELL). Guide RNAs against 24 targets or 8 AAVS1 controls were assembled into Cas9 ribonucleoproteins and electroporated into the cells. Five days after electroporation cells were collected.
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| Growth protocol |
Cells were grown in RPMI (Sigma, Cat # R0883) with 10% FCS (Sigma, Cat # F0926), with 100U/mL Pen-Strep (Gibco, Cat # 15140-122), 2mM L-Glutamine (Sigma, Cat # G7513), 10mM HEPES (Sigma, Cat # H0887), 1X MEM Non-essential Amino Acids (Gibco, Cat # 11140-050), 1mM Sodium Pyruvate (Gibco, Cat # 11360-070), and 50 U/mL IL-2 (Amerisource Bergen, Cat #10101641) at a concentration of 1E6 cells/mL.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We harvested, counted, and treated each T cell culture with 200U/mL of DNase (Worthington Cat # LS002007) for 30 mins at 37 degrees. We then transferred 60,000 cells of each T cell culture into individual wells of a 96-well plate and washed cells once with PBS and once with RSB (10mM Tris HCl pH 7.5, 10mM NaCl, 3mM MgCl2). Cells were lysed in 50uL of cell lysis buffer (0.1% NP40, 0.1% Tween-20, 0.01% Digitonin in RSB) on ice for 3 min.
To the cell lysate, we then added 150ul of RSB with 0.1% Tween-20 to each well and pelleted nuclei at 500g for 10 min at 4 degree. Cells were resuspended in 50ul transposition mix (25ul 2X TD buffer, 16.5ul 1X PBS, 0.5ul 10% Tween, 0.5ul 1% Digitonin, 2.5ul Tn5 transposase, and 5ul H2O) and transposition was performed at 37 degrees for 30 min with 300rpm shaking. Transposed fragments were purified using ZR-96 DNA Clean & Concentrator-5 Kit (Zymo D4024) and libraries were generated using PCR amplification with Nextera adapters and purified using Ampure beads. ATAC-Seq libraries were sequenced on a Novaseq with paired end 100 bp reads.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
ATAC_counts.txt
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| Data processing |
Adapters were trimmed from fastq files using cutadapt version 2.10 with default settings keeping a minimum read length of 20 bp. Reads were then mapped to the human genome GRCh38 using bowtie2 version 2.4.1 with the following settings “-X 2000 --very-sensitive”. Low quality reads were filtered using samtools version 1.10 using the following command “samtools view -h -b -F 1804 -f 2 -q 30”. Reads mapping within the ENCODE blacklist region were removed using bedtools version 2.29.2 using bedtools intersect. Duplicated reads were removed using picard version 2.23.3 using the following settings “VALIDATION_STRINGENCY=LENIENT”. Reads mapping to ChrX, ChrY, and ChrM were excluded from further analysis. Reads were converted to single nucleotide ATAC insertion sites using the following command “bedtools bamtobed -i {input.bam} | awk 'BEGIN {{OFS = "\t"}} $6 == "+" {{$2 = $2 + 4; $3 = $2 + 1; print}} $6 == "-" {{$3 = $3 - 4; $2 = $3 - 1; print}}' | sort -k1,1 -k2,2n > {output.bed}”. For each sample, peaks were called using macs2 version 2.2.7.1 using the ATAC insertion site bed files as input with the following settings “--format BED --shift -75 --extsize 150 --nomodel --call-summits --nolambda --keep-dup all -B --SPMR -q 0.01”. Peaks called in individual samples were merged into a consensus peak file in the following way using single nucleotide peak summits from macs2. For each KO or control condition, peak summits that were within 75 bp of another peak summit in two out of three donors and were supported by at least 10 ATAC-Seq reads were merged into reproducible summit clusters. Across all samples, reproducible summits from each KO or controls were aggregated with other summits within 150 bp of each other. For each aggregate cluster, we calculated an average summit location based on the location of all of the summits within the cluster. Each average summit location was then extended to 350 bp peaks centered on the average summit location to generate a consensus peak list. For each sample, the number of ATAC-Seq insertion sites that overlap each consensus peak was counted using the summarizeOverlaps function in the GenomicAlignments package.
Genome_build: hg38
Supplementary_files_format_and_content: Count of ATAC insertion sites within peaks.
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| Submission date |
Apr 08, 2021 |
| Last update date |
Apr 26, 2021 |
| Contact name |
Alexander Marson |
| E-mail(s) |
alexander.marson@ucsf.edu
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| Organization name |
Gladstone-UCSF Institute of Genomic Immunology
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| Street address |
1650 Owens St
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE171736 |
Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks [ATAC-Seq] |
| GSE171737 |
Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks |
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| Relations |
| BioSample |
SAMN18676914 |
| SRA |
SRX10558078 |
| 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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