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| Status |
Public on Apr 25, 2019 |
| Title |
mock gRNA |
| Sample type |
SRA |
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| Source name |
2B4 cell line
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| Organism |
Mus musculus |
| Characteristics |
cell line: 2B4
chip antibody: Anti-Flag M2
transfection: Mock gRNA
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| Treatment protocol |
Plat-E packaging cells were transfected with Mock_gRNA or Ccl5-promoter_gRNA in addition to dCas9 which have hCD2 or GFP as markers, respectively. After selection of Plat-E expressing both hCD2 and GFP, the culture supernatant was used to infect 2B4 cells. 2B4 cells were selected for hCD2 and GFP. Before ChIP-seq, 2B4 was stimulated by 50 ng/mL PMA and 1 ug/mL ionomycin for 24 hours.
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| Growth protocol |
2B4 cells were cultured in RPMI supplemented with 10% FCS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq using anti-Flag (Sigma, F3165), 1x10^7 2B4 cells were washed with ice-cold PBS three times and and were crosslinked by incubation with 1% formaldehyde containing 0.5 mM PMSF and protease inhibitor cocktail (Roche, 4693116001) on ice for 10 minutes. The crosslinking reaction was stopped by the addition of glycine to 0.125 M. Cells were incubated on ice for 5 minutes and then washed three times with ice-cold PBS. Cells were lysed by the incubation for 10 min on ice in 0.5 ml of 50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% TritonX-100 containing protease inhibitor cocktail. Nuclei were pelleted and lysed by resuspending in 10 mM Tris-Cl (pH 8.0), 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA containing protease inhibitor cocktail. Pelleted chromatin was resuspended in 400 μl of 10 mM Tris-Cl (pH 8.0), 300 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% TritonX-100, 0.1% sodium deoxycholate and 0.5% N-laurylsarcosine containing protease inhibitor cocktail, and was sonicated using a model XL2000 ultrasonic cell disruptor (MICROSON) to approximately 350 bp of DNA fragments. Sonicated chromatin was incubated at 4 °C overnight with anti-Flag antibody preconjugated to magnetic beads (Dynabeads). Beads were washed by RIPA buffer 6 times and DNA was eluted and reverse-crosslinked at 65 °C in 50 mM Tris-Cl (pH 8.0), 10 mM EDTA, 1% SDS overnight.
Library construction was conducted by using NEBNext ChIP-seq Library Prep Reagent Set for Illumina according to the manufacture’s protocol. Libraries were sequenced to 50 bp read length on a HiSeq2000 (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Mouse T cell line
Mock gRNA was used as a control
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| Data processing |
ChIP-seq sequences were aligned to mouse reference genome release mm10 using bowtie (v1.1.2) with default parameters. Fragment sizes were estimated and peaks were called by MACS2 (v.2.1.1) by using the following parameters; band width 300, model fold [5,50], qvalue cutoff 0.05.
Genome_build: mm10
Supplementary_files_format_and_content: MACS2 output files as bed files
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| Submission date |
Apr 24, 2019 |
| Last update date |
Apr 26, 2019 |
| Contact name |
Wooseok Seo |
| E-mail(s) |
wooseok.seo@med.nagoya-u.ac.jp
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| Phone |
0527442135
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| Organization name |
Nagoya University Graduate School of Medicine
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| Department |
Immunology
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| Street address |
65 Tsurumai-Cho, Showa-Ku
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| City |
Nogaya |
| State/province |
Aichi |
| ZIP/Postal code |
466-8550 |
| Country |
Japan |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE130235 |
Runx/CBFβ transcription factor complexes regulate Ccl5 expression through two novel enhancers to enhance tumor immunity [dataset 1] |
| GSE130600 |
Runx/CBFβ transcription factor complexes regulate Ccl5 expression through two novel enhancers to enhance tumor immunity |
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| Relations |
| BioSample |
SAMN11490126 |
| SRA |
SRX5728890 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3734262_Mock_gRNA_peak.bed.gz |
5.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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