title: GSM4586560: Round Spermatids-H3K4me3; Rattus norvegicus; ChIP-Seq
library_construction_protocol: Round spermatid cell lysates were prepared from nuclei by sonication and specific Histone modification antibodies were used for histone-DNA chromatin immunoprecipitation (ChIP) assays using the SimpleChIP Enzymatic Chromatin IP kit (9003, CST, USA) according to the protocol provided by the manufacturer. Immunoprecipitated DNA fragments were subjected to library preparation using TrueSeq DNA Kit (FC-121-2001, Illumina, USA). NEB Next index set 3 was used for barcoding and adapter ligation (E7710S, NEB, USA).  Briefly, 5 – 10 ng of ChIP DNA was subjected to end repair which converts the overhangs resulted from fragmentation into blunt ends using an end repair mix. This end repair mix has 3' to 5' exonuclease activity which removes the 3' overhangs and the polymerase activity fills in the 5' overhangs. After this step, a single 'A' nucleotide is added to the 3' ends of the blunt fragments which prevent the end repaired fragments from ligating to each other which is called as A tailing. Following this step, adapter ligation was performed where adapters having indexes were ligated to the ends of the DNA fragments, enabling them for hybridization onto a flow cell. Purification of adapter ligated fragments followed by enrichment of library was done to amplify DNA fragments having adapter sequences. DNA sequencing was performed in Illumina NextSeq 500 platform (Illumina Inc, USA) with a read length of 75 bp and paired end chemistry.