GSM5918514: Fibroblast-like synoviocytes, Sox4 - TNF ChIP; Mus musculus; ChIP-Seq
mouse FLS containing 40 million cells in each replicate were treated with or without 10ng/mL TNF for 16 h, followed by fixation with 1.5% paraformaldehyde. Chromatin was extracted and sheared into 100- to 500-bp fragments using a Diagenode Bioruptor. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). 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 Illumina HiSeq 2500 following the manufacturer's protocols.