GSM3290891: H3K27me3 scChIPseq HBCx-22; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Unclassified
Antigen
Unclassified
Cell type
Cell type Class
Breast
Cell type
HBCx-22
NA
NA
Attributes by original data submitter
Sample
source_name
HBCx-22
tissue
mouse PDX human tumor
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Prior scChIP-seq and scRNA-seq, PDX were digested at 37°C for 2h with a cocktail of Collagenase I (Roche, # 11088793001) and Hyaluronidase (Sigma, # H3506) as previously described (Petit et al, Lab Investigation 2013). Cells were further individualized at 37°C using a cocktail of 0.25% trypsin/Versen (ThermoFisher Scientific, #15040-033), Dispase II (Sigma, # D4693) and Dnase I (Roche, # 11284932001). Red Blood Cell lysis buffer (ThermoFisher Scientific, # 00-4333-57) was then added to degrade red blood cells. To increase the viability of the cell suspension, we removed dead cells using a Dead Cell Removal kit (Miltenyi Biotec). Cells were re-suspended in PBS/0.04% BSA (ThermoFisher Scientific, # AM2616). After immunoprecipitation targeting H3K4me3 or H3K27me3, scChIP-seq libraries were prepared as following: barcoded-nucleosomes were amplified by in vitro transcription using the T7 MegaScript kit (ThermoFisher Scientific). The resulting amplified RNA was purified using 1x RNAClean XP beads (Beckman) and reverse transcribed by random priming in cDNA. After RNA digestion, DNA was amplified by PCR using with Illumina Primers for 12 cycles and library fragments ranging from 300 to 600 bp (single-cell barcode plus nucleosomal sequence + PCR primer sequence) were size-selected on an agarose gel. Single-cell ChIP-seq libraries were sequenced on Illumina NextSeq 500 MidOutput 150 cycles