GSM9019700: Rpb1AA_Rpb3AA_G1_control_rep2; Saccharomyces cerevisiae; OTHER
Cells were broken using Freezer/Mill (Spex SamplePrep ,6875D) for 5 cycles, 2 min, 15cps. After verification of the breakage under the microscope, cells were resuspended in 450µL of XLB3 (supplemented with Protease inhibitors, A32955, Pierce) and cross-linked by addition of DSG at 6mM final (Disuccinimidyl glutarate, A35392, ThermoFisher) for 30min at 30˚C. DSG was quenched by addition of 50µL of Tris 1M for 1min. Cells where wash 3 times HS buffer (Sorbitol 1M, Hepes pH 7.5 10mM) and resuspended in CAD-buffer (Sorbitol 1M, Hepes pH 7.5 10mM, DTT 5mM, IGEPAL 0.1%, MgCl2 1mM, Protease inhibitors A32955, Pierce ) and supplemented with pre activated CAD. Digestion reaction for 1hr at 37C with 1000 rpm agitation. Cells were placed on ice for 2 min and washed twice in SB buffer (10mM Hepes pH 7.5, 0.1% IGEPAL, 1mM EDTA, 1mM DTT) and resuspended in 300µL of SB. 100µL of cells were diluted in 300µL of SB and added to 50µL of pre-activated NHS beads (Pierce, 88827) and agitated for 10min at RT. Beads binding was quenched by addition of 30µL of TrisHCl 1M for 1 min. Beads were resuspended in 500µL of SB and supplemented with 3mM ZnCl2 final for 2min. Beads were washed twice in SB and resuspended in 500µL of SB. End-Repair and A-tailing was performed with the NEBNext Ultra II DNA library Prep Kit (NEB, E7645). 30min 20˚C 1 krpm, then 30min 65˚C 1 krpm. Cells were incubated 2 min on ice and washed twice in HB ( 10mM Hepes pH7.5, 1M NaCl, 0.5% Na-deoxycholate, 0.5% Triton X-100, 0.5% IGEPAL) and twice in SB buffer. Beads were resuspended in 100 µL of Hepes pH 7.5 10mM. 20µL of beads were incubated with 2µL of linker mixture at desired ratio at 25µM and 22µL Blunt/TA ligase (NEB, M0367L) for 40min at 20˚C. Beads were washes twice in HB and twice in SB. Beads were resuspended in 20µL of Hepes pH 7.5 10mM with 2µL of seed at 10ng.µL and 22µL of Blunt/TA ligase (NEB, M0367L) for 40min at 20˚C. Beads were washes twice in HB and twice in SB and resuspended in 20µL of Hepes pH 7.5 10mM. PCP reaction were realized on 1µL and 0.25µL of beads mixture in PCP buffer (HEPES pH 7.5 40mM, 6mM MgCl2,  2.5mM dNTP, 2.5 mM rMTP, 1mM DTT, 2.5% PEG 20K, 0.5 µL RNase inhibitor (Pierce, AM2694), 1µL of Reverse Transcriptase (ProtoScript II, NEB M0368), 3µL of T7 RNA Polymerase (NEB, M0251)) for 1hr at 30˚C. PCP reaction was supplemented 20µL Tris HCl pH 7.5 100M with 0.1 µL of RNAse cocktail (Invitrogen, AM2286) for at least 2 hours at 65˚C. The decrosslinked chromatin was supplemented with 17 µL of H2O, 1.5 µL of each of the PCR primers (any of the Illumina NEBNext primers, E6440S) at 10 µM and 50µL of KAPA RM (Roche, 07958927001) and incubated in the PCR reaction for 12 cycles ( 5min 95˚C, [20 sec 98˚C, 15 sec 71˚C, 1min 72˚C] x12, 2 min 72˚C, 4˚C). Following PCP, the library was produced using Illumina P5 and P7 primer from NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB E7103)