title: GSM4733037: M52_Sperm_Atrazine_ChIP; Rattus norvegicus; ChIP-Seq
library_construction_protocol: For each sample, the epididymis was dissected free of fat and connective tissue, then, after cutting open the cauda, placed into 6 ml of phosphate buffer saline (PBS) for 20 minutes at room temperature. Further incubation at 4ºC will immobilize the sperm.  The tissue was then minced, the released sperm pelleted at 4ºC 3,000 x g for 10 min, then resuspended in NIM buffer and stored at -80ºC for further processing.   An appropriate amount of rat sperm suspension was used for ChIP analysis. Rat sperm samples were sonicated and then pretreated with 50 µl 1M DTT in each sample tube. Samples were then incubated for 2h at RT on rotation. 123 mg of NEM dissolved in 1 mL of milliQ water in a 56ºC incubator. 120 µL of NEM added to each sample and then incubated for 30 min at RT on rotation. Chromatin prepared with  400 µl "Buffer 1 Stock" (see cited ChIP protocol), 102 mg sucrose and 0.5 µl 1M DTT. Samples then centrifuged at  2,500 rpm 5 min at RT. Supernatant discarded. Sperm resuspended in 1 ml 1X PBS and centrifuged at 2,500 rpm 5 min at RT. Supernatant discarded. Sperm resuspended in 130 µl of “Complete Buffer” (“Buffer 1 stock” + detergents) and placed in 130 µl Covaris tubes. Incubate 20 minutes on ice. Covaris program set to to the desired program: Chromatin shearing 10 min, Peak power: 75.0, Duty factor: 10.0, Cycles/Burst: 200. Program run for each tube. Samples removed from Covaris tube and placed it in 1.5 ml tube and centrifuged at 12,500 rpm 10 min at RT. Supernatant then collected and protease inhibitor cocktail ((20X) by dissolving 1 tablet in 500 µl MilliQ H2O) is added (11 µl of this solution per sample). Samples run on aa 1.5 % agarose gel to check bands size before resuming the IP protocol. Each sample had 3 µl of antibody (Ab anti-histone H3, CT, pan, clone A3S Millipore cat #05-928) added and were placed at 4ºC on rotation O/N. Samples then washed with magnetic beads (ChIP grade protein magnetic beads, Cell Signal, at 4ºC). 40 µl of beads for each sample were washed: placed on DynaMag-2 for two minutes and supernatant removed without disturbing the beads. Resuspended with 1 ml of 1X IP buffer, then repeat wash process once. Supernatant removed and resuspended in 350 µl of 1x IP. 40 µl of beads added to each sample and placed on rotation at 4ºC for 3 h. Tubes were then washed on DynaMag with 1X IP 3-4 times. With the final wash, beads resuspended in 300 µl “MeDIP digestion buffer." 3 µl of Proteinase K 20 mg/ml added to each tube and placed on rotation at 56ºC, 4 h (or O/N). Samples centrifuged quickly, then supernatant placed in new 1.5 ml tubes. 300 µl of phenol/chloroform/isoamyalcohol added to each tube and Centrifuged at 12,500 rpm 10 min at RT. The aqueous phase was placed in a new 1.5 ml tube and 2 µl of Glycoblue, one tenth vol of 3 M sodium acetate (NaOAc) and 2 vol of ethanol were added to each sample. Samples then precipitated O/N at 20ºC (O/N precipitation necessary for better yields). Tubes centrifuged at 12,500 rpm 30 min at 4ºC. Supernatant removed and 500 µl of 70% EtOH added. Centrifuged at 12,500 rpm 10 min at 4ºC. Supernatant removed and samples air dried 5-10 min. Pellets resuspended in 25 µl milliQ H2O. DNA concentration measured in Qubit with brDNA kit. ChIP DNA was used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra DNA Library following the manufacturer's protocol  and indexing each sample individually with NEBNext Multiplex Oligos for Illumina.