title: GSM9003473: CD-H3K36-INPUT-BR2; Saccharomyces cerevisiae; ChIP-Seq
library_name: GSM9003473
library_construction_protocol: 300ml of cells were collected for each treatment  gropu. Post-harvest, the cells were resuspenced in a small volume to help with crosslinking. Cross-linked cell pellets were lysed to isolate the DNA which was then sonicated. For INPUT samples 10% of total DNA per samples was set aside. Remaining 90% of genomic DNA was then subjected to immunoprecipitation using antibodies specific to H3K36me3 and H3K4me3 residues. The IP'ed DNA was then purified, and subjected to library construction. Cells growth (in YPD or SC) and drug administration at IC50 were performed as described for Figure S1.  ChIP was performed as described with modifications (Joshi et al, 2022).  Briefly, 300 ml of cells were collected by centrifugation, and after resuspension in 35 ml of fresh media (YPD or SC), protein-DNA crosslinking was done by the addition of formaldehyde at 1%, followed by incubation at room temperature with gentle rotation for 25 m.  The crosslinking reaction was quenched by 125 mM glycine and incubation for 5 m.  Cells were washed once each with cold 1X PBS and 50 mM HEPES, then snap-frozen prior to storage at -80°C.  To extract the DNA, the frozen cells pellets were thawed in 300 µl of lysis buffer (50 mM HEPES/KOH, pH7.5, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 140 mM NaCl, 1X Halt Protease and Phosphatase Inhibitor cocktail (Thermo Scientific)) on ice.  Each thawed cell pellet was then distributed into three screw-capped microcentrifuge tubes and volume was increased to 300 µl with lysis buffer.  To each tube 300 µL of glass beads were added and samples were treated in a homogenizer at 4°C for six cycles of 30 s each, with 3 m intermittent resting on ice.  Cell lysis was monitored by microscopy every two cycles.  At ~80% cell lysis, the cell lysates were collected by centrifugation at 1,400 g for 3 m, after puncturing the bottom of the tube with a heated needle.  Cell lysates were then sonicated for 10 m at 10 % duty factor, 200 cycles/burst, and 75 W peak power to obtain DNA fragments between 200-600 bps.  The sonicated cell lysates were centrifuged at 15,000 g for 20 m at 4°C to remove cell debris.  The volume of the supernatant was increased to 1 ml with lysis buffer, followed by collecting a 40-µl aliquot as “Input”, a 50-µl aliquot for agarose gel electrophoresis to check DNA fragment size, and the remainder 910 µl for IP.  The 50-ml aliquot was reversed crosslinked overnight in a 65°C water bath, then DNA was purified using a Qiagen PCR column purification kit.  DNA was analyzed on a 1.5% agarose gel.  The 910-ml aliquot was pre-cleared by incubation with anti-rabbit M280 magnetic beads (Invitrogen Cat # 11203D) according to manufacturer's recommendation.  The pre-cleared lysates (eluate) were then incubated with magnetic beads conjugated with 5 µg of anti-H3K4me3 (Abcam #ab8580), or anti-H3K36me3 (Abcam #ab9050), or rabbit IgG (Bethyl lab #120-101) for IP at 4°C overnight, followed by washing once each with wash buffer 1 (50 mM HEPES/KOH, pH 7.5, 1 mM EDTA, 1% Triton X100, 0.1% Sodium deoxycholate, 140 mM NaCl, 1X protease inhibitor cocktail, 1X phosphatase inhibitor cocktail), wash buffer 2 (50 mM HEPES/KOH, ph 7.5, 1 mM EDTA, 1% Triton X100, 0.1% Sodium deoxycholate, 500 mM NaCl, 1X protease inhibitor cocktail, 1X phosphatase inhibitor cocktail), wash buffer 3 (0.25 M LiCl, 0.5% NP-40, 0.5 % Sodium deoxycholate, 1mM EDTA, 10 mM Tris-HCl pH 8.0) and TE 0.1, for 3, 3, 15 and 3 m, respectively at 4°C.  The protein-bound DNA was eluted from the beads by 160 µl elution buffer (0.85X TE pH 8.0, 1% SDS, 0.25 M NaCl) and incubation in a 65°C water bath for 30 m with gentle vortexing every 5-10 mins.  Henceforth the IP and Input DNA were processed together, after adjusting the volume of Input DNA with elution buffer (~120 µl) to obtain the same volume as IP DNA.  The DNA was digested with 800 ng of DNase-free RNaseA at 37°C for 2 h, followed by 14 µl of Proteinase K (10 mg/ml) at 65°C overnight to reverse crosslinks.  The liberated DNA was purified using phenol/chloroform extraction and ethanol precipitation using 3 µl of glycogen (20 µg/µl) as a carrier.  The resulting DNA pellet was dissolved in 30 µl and 100 µl of TE 0.1 respectively, for IP and Input DNA.  DNA library construction was performed as described previously (Joshi et al, 2021) and sequencing performed on Hi-Seq 2500 (2x150 bp).  ChIP-seq data were processed similarly as for Break-seq and the relative amount of ChIP was calculated as the Log2 ratio of signal from the ChIP to that from the Input control using MACS2 and deepTools (Ramírez et al, 2016), respectively.
identifiers.namespace: GEO
identifiers.id: GSM9003473_r1