SAMN30338699
External Id
Peking University
INSDC center name
2023-05-17T00:00:00Z
INSDC first public
2023-05-17T07:36:15.712Z
INSDC last update
SRS14672737
INSDC secondary accession
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
SRS14672737
SRA accession
K562
cell line
4#
clone
RAD21-mAC
genotype
Homo sapiens
http://purl.obolibrary.org/obo/NCBITaxon_9606
organism
cultured cell
source_name
K562_RAD21_mAC_4_IAA_END_seq
title
END-seq were performed as described in (Wong et al., 2021), with minor modifications. Three million WT or RAD21-depleted K562 cells were mixed 30,000 spike-in cells that harbored a DSB induced by SpCas9 at the c-MYC locus. Cells were harvested, resuspended in the cell suspension buffer (PBS with 5 mM EDTA), embedded in low-melting agarose, and then transferred in to plugs (Bio-Rad Plug Molds, 1703713). After solidification, plugs were incubated with cell lysis buffer (1 mg/mL Protease K, 10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 50 mM EDTA, and 0.4% SDS) at 37ºC for 1 hour and then at 50ºC for 7 hours. The plugs were washed by wash buffer (10 mM Tris-HCl pH 8.0, 50 mM EDTA) for 3 times and then incubated with RNase A(Invitrogen) at 37ºC for 1 hour. After being extensively washed, plugs were treated with blunting, A-Tailing as described in END-seq protocol (Wong et al., 2021), and then ligated with END-seq adapter 1 (up: 5’-phos- GATCGGAAGAGCGTCGTGTAGGGAAAGAGT-3’-biotin; down: 5’-NH2- ACTCTTTCCCTACACGACGCTCTTCCGATCT-3’). After ligation, plugs were melted and treated by β-Agarase I. The recovered genomic DNA were then sheared to a length between 150-400 bp by sonication (Covaris) and the biotinylated DNA fragments were purified by streptavidin beads (MyOne C1, Invitrogen). After purification, the DNA underwent end repair, dA-Tailing, and ligated with END-seq adapter 2 (up: 5’- AGACGTGTGCTCTTCCGATCT; down: 5’-phos-GATCGGAAGAGCACACGTCT). The ligated products were tagged with Illumina adapters and sequenced on Hi-seq, 2×150 bp.
treatment