title: GSM2401433: H3K36me3_ChIPSeq_replicate2; Mus musculus; ChIP-Seq
library_construction_protocol: For ChIP-seq of crosslink samples, sperm were crosslinked with 1% formaldehyde in 1x PBS for 10 min at RT, and the reaction was quenched with 125 mM glycine for 10 min at RT. After washing with PBS, crosslinked sperm were lysed with 5 mM PIPES, 85 mM KCl, 0.5% NP40 and 1x proteinase inhibitor on ice for 10 min. Then we performed a second fixation step by directly adding 1% formaldehyde to the lysis buffer on ice. After 10 min sperm were homogenized with a glass dounce homogenizer. Following quenching and washing, sperm cells were resuspended in RIPA buffer (1x PBS; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 1x proteinase inhibitor) and incubated on ice for 20 min. The purified sperm chromatin was sonicated to 300-1000 bp using a Diagenode Bioruptor at 25 cycles of 30 sec on and 30 sec off and the supernatant was collected. Immunoprecipitation was performed overnight at 4°C with antibodies. For ChIP-seq of Mnase samples, lysate was digested with Mnase and histone-DNA complexes were isolated with antibody. Libraries for Illumina sequencing were constructed using the following standard protocol. The fragment end were repaired using the NEBNext End Repair Module, and adenosine was added at the 3’ ends of fragment using Klenow fragment (3’ to 5’ exo minus, New England Biolabs). Precipitated DNA and input DNA were incubated with adaptors at room temperature for 1 hr with T4 DNA ligase (New England Biolabs) and amplified with Illumina primers.
identifiers.namespace: GEO
identifiers.id: GSM2401433