GSM5494763: ChIP-seq_SCA7_KI_H3K36me3_retina; Mus musculus; ChIP-Seq
Batch of a chromatin was prepared from 6 retinas. Following cross-linking in 1 % paraformaldehyde (PFA), glycine was added to a final concentration of 0.46 M. Samples were washed in cold PBS/PIC and homogenized in lysis buffer. Chromatin was sonicated for 7 min using a Covaris ultrasonicator and centrifuged at 13 000 rpm for 5 min. Each 100 μl of chromatin sample was diluted 10 times and stored at -80°C until use. The supernatant was used for subsequent ChIP using antibodies against RNAPII (RPB1), H3K4me1, H3K9ac, H3K27ac, H3K36me3 (RPB1-7G5 in-house; ab8895, Abcam; ab4441, Abcam; ab4729 Abcam; ab9050 Abcam, respectively). Before immunoprecipitation the chromatin was precleared with Prot A or Prot G beads for 1 hour at 4 °C, and followed by o/n incubation with 3ug of the antibody at 4°C. Next Prot A or Prot G beads were added for a 3h incubation at 4°C. Subsequently the beads were washed and DNA-protein complexes were eluted from the beads, decrosslinked, and treated with proteinase K for 2 h at 43°C. DNA fragments were purified using phenol-chloroform, precipitated, and used for sequencing. ChIP-seq libraries were prepared from 0.5-10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5' ends were ligated to the 5' end of the genomic DNA, leaving a nick at the 3' end. In the final step, the 3' ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7 to 10 cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents. The libraries were sequenced on Hiseq 4000 as single end 50 base reads following Illumina's instructions.