GSM5081971: WT-LK-CTCF-ChIP-seq; Mus musculus; ChIP-Seq
RNA samples extracted with Trizon and treated with Dnase I.  ChIP Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with CTCF, SA1, SA2, RAD21, H3K4me3 and H3K27me3 antibody (antibody: Anti-CTCF, Cell Signaling Technology, Cat# 2899; Anti-SA1, Abcam, Cat# ab4457; Anti-SA2, Abcam, Cat# ab4464;  Anti-RAD21, Abcam, Cat# ab992;  Anti-H3K4me3, Millipore, Cat No. 04-745;   Anti-H3K27me3, Millipore, Cat No. 07-449).  ATAC-seq samples derived according to Nextera Tn5 Transposase kits. DNA:RNA immunoprecipitation sequencing (DRIP-seq) was performed as described previously with s9.6 antibody (Anti-DNA-RNA Hybrid [S9.6] Antibody, Kerafast, Cat# ENH001). ChIRP libraries were prepared using Illumina's TruSeq ChIP Sample Preparation Kit according to the manufacturer's instructions. The quality of the library was checked with Agilent Bioanalyzer. NG-Capture-C assay was performed as previously described. Hi-C assay was performed to generate a genome-wide interaction as described previously with Arima-HiC Kit (Cat: A410030) (https://arimagenomics.com/) with minor modifications. Single cells were generated using Chromium Controller (10x Genomics), and scRNA-seq and scATAC-seq libraries were constructed using chromium single cell 3' reagent kits v2 (10x Genomics, California USA) according to the manufacturer's recommendations. RNA-seq libraries were prepared according to TruSeq Stranded mRNA Library Prep (#20020594). ChIRP libraries were prepared using Illumina's TruSeq ChIP Sample Preparation Kit according to the manufacturer's instructions. The quality of the library was checked with Agilent Bioanalyzer.  ChIP-seq libraries were prepared according to Illumina's instructions accompanying the TruSeq ChIP Library Preparation Kit (#IP-202-1012). ATAC-seq libraries were prepared according to Nextera DNA Library Prep Kit (#FC-121-1030). NG-Capture-C-seq libraries were prepared according to  using the Herculase II PCR kit (Agilent). . Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols. single cell RNA-seq, ATAC-seq, ChIP-seq, ChIRP-seq, DRIP-seq, DRIPc-seq, NG-Capture-C-seq and HiC-seq
GEO Accession
GSM5081971