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| Status |
Public on Jul 15, 2020 |
| Title |
R-TEn-p1-Ebox_Mut_DP_H3K4me1_2 |
| Sample type |
SRA |
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| Source name |
DP cells purified by MACS (CD4+CD8+CD69-TCRb-)
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: DP cells purified by MACS
genotype: R-TEn-p1-Eboxmut/mut
tissue: Thymus
chip antibody: anti-H3K4me1 (abcam: # ab8895)
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| Growth protocol |
For proB cell culture, B cells were enriched by B220-MACS column from total bone marrow cells. Then, B cells were cultured in PRMI 1640 with 10% fetal bovine serum, 2mg/ml sodium bicarbonate, 1 mM sodium pyruvate, 0.1mM nonessential amino acid solution, 5x10-5M 2-Mercaptoethanol, 100µg/ml streptomycine and 100U/ml penicillin in the presence of IL-7 and stem cell factor.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-seq : ATAC-seq was performed as described (Buenrostro et al., 2013) with modifications. Cells were lysed, then treated with Tn5 transposase (Nextera DNA Sample Preparation Kit). After the treatment, the tagmentated DNA was cleaned up and purified with DNA Clean & Concentrator kit (Zymo Research). The purified DNA were amplified with determined cycles of PCR based on the amplification curve, then were size-selected using Ampure XP beads. Hi-C : in situ Hi-C was performed as described (Rao et al., 2014). DP and cultured proB cells from control and deletion mutant mice were fixed with 1% formaldehyde/FACS buffer for 10 minutes at room temperature. Cells were lysed and chromatin was digested overnight using MboI. The fragment overhangs generated by the restriction enzyme were filled in with biotin-14-dATP by Klenow fragment, then proximity ligation was performed. Biotinylated DNA was sonicated to 300-500 bp on a Covaris M220. The biotinylated DNA was captured on Dynabeads MyOne Streptavidin T1 beads. After washing the beads, ends of sheared DNA were repaired and added A-tail according to the protocol. NEBNext Multiplex Oligos for Illumina (NEB) were ligated and the beads were washed. Libraries were amplified by PCR and size selected using ProNex (Promega). As to CD4SP cells, Tethered Conformation Capture (TCC) was performed as described (Kalhor et al. 2012). CD4SP cells were fixed with 1% formaldehyde/FACS buffer for 10 minutes at room temperature. Cells were lysed and chromatin was digested overnight using MboI. ChIP-seq : Cells were fixed in 1% formaldehyde/PBS . The reactions were quenched by 1.5M glycine and cells were washed with PBS and pelleted. Pelleted cells were lysed and chromatin was sonicated by Covaris M220. Diluted sheared chromatin was immunoprecipitated with each antibody which is pre-bound to Dynabead Protein G magnetic microbeads overnight at 4 °C. Samples were washed, eluted and reverse cross-linked 65°C overnight. Samples were next treated with RNase A and proteinase K and purified using DNA Clean & Concentrator kit (Zymo Research). Samples were ligated to NEBNext Multiplex Oligos for Illumina (NEB) and multiplex amplified by PCR. Amplified ChIP DNA was selected by size.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
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| Description |
ChIP-seq
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| Data processing |
Illumina bcl2fastq software used for basecalling (HiSeq 2500, NovaSeq 6000,Illumina HiSeq X Ten ).
ATAC-seq and ChIP-seq trimmed reads were mapped to the mm9 reference genome using Bowtie2 (version 1.2.7). Hi-C reads were trimmed from the 3’ end of sequences to GATC (MboI restriction enzyme site) and aligned to the mouse reference genome mm9 using bowtie2.
Tags mapped to unique DNA sequences of ATAC-seq, ChIP-seq and Hi-C were analyzed using HOMER (Lin et al., 2012, Heinz et al., 2018)
Genome_build: mm9
Supplementary_files_format_and_content: ATAC-seq and ChIP-seq : Tab-delimited BED files containing peak positions for each ATAC-seq or ChIP-seq experiment. Hi-C: Tab-delimited Text files containing PC1 values for each experiment.
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| Submission date |
Jul 15, 2020 |
| Last update date |
Jul 18, 2020 |
| Contact name |
Kazuko Miyazaki |
| E-mail(s) |
kamiyazaki@infront.kyoto-u.ac.jp
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| Organization name |
Institute for Life and Medical Sciences, Kyoto University
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| Lab |
Department of Immunology
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| Street address |
53 Kawahara-cho, Shogoin, Sakyo-ku
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| City |
Kyoto |
| ZIP/Postal code |
606-8507 |
| Country |
Japan |
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| Platform ID |
GPL21273 |
| Series (1) |
| GSE141223 |
E2A Specifies Adaptive Immunity by Instructing Large-Scale Topological Changes |
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| Relations |
| SRA |
SRX8737125 |
| BioSample |
SAMN15566678 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4672151_R-TEn-p1-Ebox_Mut_DP_H3K4me1_2_mm9_peaks.bed.gz |
616.6 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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