title: GSM2977817: Cu_Whole_Cell_Extract_replicate2; Cryptococcus neoformans var. grubii H99; ChIP-Seq
library_construction_protocol: After 3 hours of treatment, formaldehyde was added and cells incubated for 20 minutes for crosslinking and crosslinking quenched with glycine for 5 minutes. Cells were pelleted and washed once in 10 mL of PBS containing glycine. The washed pellets were resuspended in 1 mL ChIP lysis buffer and lysed by bead beating three times 1 minute with a Mini Bead Beater 16 (BioSpec). The lysate was aspirated added to a 15 ml conical tube with 1 ml of ChIP lysis buffer and sonicated 40 seconds, 20 seconds on, 20 seconds off. Lysates were cleared by two successive centrifugations for 10 minutes at 20,000 x g at 4˚C. The resulting supernatant is now the input for the ChIP experiment. 30 µl were transferred to a new tube as the Whole Cell Extract (WCE). To prepare immunoprecipitation samples 250 µl lysate was added to 250 µl ChIP lysis buffer and 120 µl 3.6 M NaCl. To these samples, protein A agarose beads (Protein A Plus Agarose, Pierce) were added and incubated for 1 hour at 4˚C with rotation to clear protein that non-specifically binds beads. Lysates with agarose beads were then centrifuged at 10,000 x g for 1 minute at 4˚C, the supernatant transferred to a new tube and FLAG antibody beads (EZview Red Anti-FLAG M2 Affinity Gel, Sigma) blocked in PBS with 5% milk for 1 hour and washed in ChIP lysis buffer were added to the lysates and incubated for 16 hours at 4˚C while rotating. After beads washed several times, The immunoprecipitated Cuf1-2xFLAG was eluted by adding 150 µl of ChIP elution buffer and incubated for 10 minutes at 65˚C, centrifuged at 10,000 x g for 1 minute and 125 µl of the supernatant was transferred to a new tube. 95 µl of ChIP elution buffer was added to 30 ul WCE. The IP and WCE tubes were incubated at 65˚C overnight to reverse formaldehyde crosslinks, treated with 5 µl of proteinase K (BioLine) at 37˚C for 90 minutes and samples purified using a Qiagen PCR purification kit as per manufacturer's instructions and eluted in 50 µl of water. Libraries were prepared using the Kapa BioSystem HyperPrep Library Kit. libraries were cleaned using SPRI beads, quantified using Qubit 2.0 and size checked using the Agilent Bioanalyzer. Four libraries of replicate 1 were pooled together and sequenced by Illumina HiSeq 2000 sequencer at 100 bp single end. the other eight files from replicate 2 and 3 were sequenced by Illumina HiSeq 4000 sequencer at 51 bp single end.