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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 09, 2020 |
| Title |
WT CD8+ splenic T cell, target:H3, Tx: StimIL2&CD3CD28day3, Rep.2 |
| Sample type |
SRA |
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| Source name |
CD8+ splenic T cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: WT
Sex: Male
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| Treatment protocol |
For the culture of primary T cells FACS sorted cells were cultured in RPMI 1640 supplemented with 15% FBS, 100 IU/mL penicillin and 100 µg/mL streptomycin, 1% glutamine, 1% sodium pyruvate, 50 µM beta-mercaptoethanol, and were incubated at 37ºC with 5% CO2, with or without stimulation. For stimulation of CD8+ T cells, cells were treated with mIL-2 (0.2 ng/mL) and either CD3/CD28 co-stimulatory beads (20µL/mL, ThermoFisher Cat# 11456D) or plate-bound anti-CD3ε (Clone 145-2C11) and soluble anti-CD28 antibodies (Clone 37.51).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For CUT&Tag (Kaya-Okur et al., 2019) 100,000 CD8+ T cells were isolated from the spleens of wildtype age- and sex-matched animals by flow cytometry. Additionally, cells were cultured under stimulating conditions for 3 days, as described above, and 100,000 stimulated CD8+ T cells cells were isolated by flow cytometry. 3XFlag-pATn5 was prepared as described in 3XFlag-pATn5 Protein Purification and MEDS-loading (https://www.protocols.io/view/3xflag-patn5-protein-purification-and-meds-loading-8yrhxv6). CUT&Tag library preparation was carried out as described in Bench top CUT&Tag V.2 (https://www.protocols.io/view/bench-top-cut-amp-tag-z6hf9b6). The following primary antibodies were used: IgG (Cell Signaling 4E-BP2), mouse mAb anti-H1 (Santa Cruz sc-8030 AE-4), and mouse mAb anti-H3 (Santa Cruz sc-517576). The following secondary antibodies were used: guinea pig anti-rabbit (Antibodies online ABIN101961), and rabbit anti-mouse (Antibodies online ABIN101785).
Tn5-based libraries were prepared using PCR amplificatin with indexed primers. See CUT&Tag protocol (Kaya-Okur et al., 2019).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Library strategy: CUT&Tag
Sequencing and base calling was performed by Genewiz (South Plainfield, NJ). The sequencing libraries were multiplexed and clustered on a flowcell. After clustering, the flowcell were loaded on the Illumina HiSeq 4000 according to manufacturer’s instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS) on the HiSeq instrument. Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq files and de-multiplexed using Illumina bcl2fastq v. 2.17 program. One mis-match was allowed for index sequence identification.
Raw fastq files were trimmed using Trim Galore (v0.6.2, length 20, e = .1)
Trimmed Fastq files were then aligned to the mouse genome (mm10) using Bowtie2 (version 2.4.1; local, very-sensitive-local, no-unal, no-mixed, no-discordant, phred33, I=1-, X=700).
Reads were sorted and converted to BAM format and duplicates were marked using Sambamba using default parameters (version 0.6.6)
Filtering, duplication removal and read down sampling to equalize reads across samples was performed using Samtools (version 1.1; F= 3340, f = 0x0002)
HOMER (v4.10) was then used tag directories (makeTagDirectory; default parameters), bedgraphs (makeUCSCfile; default parameters), and to calculate genomic coverage for chromatin types (annotatePeaks.pl; -noann -noadj).
Stimulated samples were downsampled to have the same number of reads as unstimulated samples using samtools (Samtools v 1.1. H1 samples: samtools view -h -@ 4 -s 123.276, H3 samples: samtools view -h -@ 4 -s 123.380).
Genome_build: mm10 (GENCODE Release M20 (GRCm38.p6))
Supplementary_files_format_and_content: HOMER generated bedGraphs.
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| Submission date |
Jun 30, 2020 |
| Last update date |
Sep 09, 2020 |
| Contact name |
Michael Alton Willcockson |
| E-mail(s) |
michael.willcockson@gmail.com
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| Phone |
3477977912
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| Organization name |
Albert Einstein College of Medicine
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| Department |
Cell Biology
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| Lab |
Arthur Skoultchi PhD
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| Street address |
1300 Morris Park Ave, Chanin 402
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| City |
Bronx |
| State/province |
New York |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE141187 |
H1 linker histones regulate the balance of repressive and active chromatin domains via localized genomic compaction |
| GSE153543 |
H1 linker histones regulate the balance of repressive and active chromatin domains via localized genomic compaction [CUT&Tag] |
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| Relations |
| BioSample |
SAMN15404420 |
| SRA |
SRX8639746 |
| 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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