|Public on Dec 31, 2019
|Foreskin fibroblasts, Hi-C reversal of senescence rep1
|cell line: human diploid BJ fibroblasts
cell state: reversal of senescence
|BJ fibroblasts stably expressing dcas9/KRAB-rtTA were treated with bleomycin (40 μg/ml; Biorbyt). On the 12th day after treatment, senescent cells were infected with TP53 sgRNA lentivirus and treated with Dox (100ng/ml) to induce dcas9/KRAB expression. Then cells were selected by puromycin (2 μg/ml) for 3 days and cultured for next 12 days
|Human diploid BJ fibroblasts purchased from American Type Culture Collection were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin.
|1-2 million of cells were fixed with 1% formaldehyde at room temperature for 10 min. Formaldehyde was quenched with glycine for 10min at room temperature.
cells were lysed and digested with MboI overnight with rotation. MboI was then inactivated at 62℃ for 20 minutes. After filling in with biotin-14-dCTP, the fragments were ligated at room temperature for 5.5 hours with rotation. Then reversal of crosslink and DNA purification was done. The biotin-labeled DNA was sheared to 300-500bp with Covaris M220 and then pulled down with Dynabeads MyOne Streptavidin C1 (Life Technology). Sequencing library preparation was performed on beads, including end-repair, dATP tailing and adaptor-ligation. 9 cycles of PCR amplification were performed with Extaq (Takara). Finally, size selection was done with AMPure XP beads and fragments ranging from 300bp to 500bp were selected.
|HiSeq X Ten
|Hi-C reads were processed using HiC-Pro 2.7.0, raw and normalized contact maps at various resoultions were obtained both in biological replicates and pooled condtions.
Fit-Hi-C were used to call significant contacts with raw contacts maps of 20kb resolution and biases files from HiC-Pro with noOfPasses of 1 and distUpThres of 2000000.
RNA-seq reads were mapped to hg19 genome assembly with Tophat 2.1.1,and transcript abundance (FPKM) and counts were calculated based on GENCODE annotation using cufflinks
Supplementary_files_format_and_content: *.raw_contact_map.txt and *.iced_contact_map.txt were the representation of sparse contact matrix, contact scores were stored as 'bin number 1/bin number 2/score'; Bed files showed the corresponding between bin numbers in contact map and genomic regions; *.FPKM.txt and *.Counts.txt showed the transcription abundance of genes and repeated elements in each sample
|Jun 25, 2019
|Last update date
|Dec 18, 2020
|The loss of heterochromatin is associated with multiscale three-dimensional genome reorganization and aberrant transcription during cellular senescence