GSM5901001: Hnf1b, ChIP-seq; Mus musculus; ChIP-Seq
RNA was isolated using the NucleoSpin RNA plus kit following the manufacturer's protocol (Macherey Nagel, Dueren, Germany). ChIP: Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Total RNA was used for the preparation of the RNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (KAPA Biosystems, Wilmington, MA, USA). In short, oligo hybridization and rRNA depletion, rRNA depletion cleanup, DNase digestion, DNase digestion cleanup, and RNA elution were performed according to protocol. Fragmentation and priming were performed at 94 °C for 6 min. First strand synthesis, second strand synthesis and A-tailing were performed according to protocol. For the adaptor ligation, a 1.5 mM stock was used (NextFlex DNA barcodes, Bioo Scientific, Austin, Tx, USA). First and second post-ligation cleanup was performed according to protocol. A total of 11 PCR cycles were performed for library amplification. The library amplification cleanup was done using a 0.8 x followed by a 1.0 x bead-based cleanup. Library size was determined using the High Sensitivity DNA bioanalyzer kit, and the library concentration was measured using the dsDNA High Sensitivity Assay (DeNovix, Wilmington, DE, USA). Paired-end sequencing reads of 50 bp were generated using an Illumina NextSeq 500. ChIP-sequencing: 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 Genome Analyzer following the manufacturer's protocols.