title: NextSeq 500 paired end sequencing; iMUT-seq analysis of over 20 different DSB repair siRNA mediated knockdowns or small molecule inhibitors
design: iMUT-seq analysis of over 20 different DSB repair siRNA mediated knockdowns or small molecule inhibitors
library_construction_protocol: 60,000 AID-DIvA cells were seeded into 6-well plates, incubated for 24 hours and then transfected as described earlier. Transfected cells were incubated for 24 hours, their media was then replaced, and they were then incubated for a further 48 hours. Cells were then treated with or without 300nM 4-hydroxytamoxifen (OHT) for 4 hours to induce DSBs, then washed twice with pre-warmed PBS and then once with pre-warmed media containing 500g/mL IAA, and then replaced with media containing 500g/mL IAA and incubated for 14 hours to degrade the AsiSI fusion protein and allow complete DSB repair.   ATMi (KU-55933, Merck SML1109), ATRi (VE-821, Merck SML1415) and DNA-PKi (NU7441, Tocris 3712) were all used at 10M and administered in a 1 hour pre-treatment and maintained throughout the period of the experiment.   Cells were placed on ice, washed once in PBS and lysed in cytoplasmic lysis buffer (50mM HEPES pH7.9, 10mM KCl2 , 1.5mM MgCl2, 0.34M sucrose, 0.5% triton, 10% glycerol, 1mM DTT) for 10 minutes. Cells were then washed once in cytoplasmic lysis buffer and then the nuclei were lysed in genomic extraction buffer (50mM Tris pH 8.0, 5mM EDTA, 1% SDS, 0.5mg/mL Proteinase K). The nuclear lysates were transferred tor 2mL DNA LoBind microcentrifuge tubes (Fisher Scientific, 0030108426) and incubated in a thermomixer at 60C for 40 minutes with 500rpm agitation. 0.1 volumes of 3M sodium acetate pH 5.2 was then added followed by 2.5 volumes of 100% ethanol, the tubes were inverted several times to mix an then incubated on ice for 1 hour to precipitate the genomic DNA. The DNA was then pelleted at 19000g for 20 minutes at 4C, washed in 75% ethanol and re-pelleted for 10 minutes twice. The ethanol was then aspirate and the DNA pellet allowed to dry at RT before resuspended in water and quantifying the DNA concentration via nanodrop.  For each condition, 3 50L PCR reactions were run each containing 0.7L Phusion polymerase (NEB, M0530L), 1X Phusion HF buffer, 2M betaine, 1.5% DMSO, 400M dNTPs, 40nM multiplexed genomic primers, 1g genomic DNA. The reaction was then carried out as follows: 98C 5mins, 12 cycles of 98C 60s, 62C 120s, 72C 120s, then 72C 5mins. All 3 reactions were combined and 100L of the total reaction volume was taken forward for size selection. 60L of SPRISelect beads (Beckman, B23318) was added to the 100L PCR reaction mixture, mixed by pipetting and then incubated for 5mins at RT. The beads were collected on a magnetic rack and the supernatant was transferred to a fresh tube. 70L of beads were then added to the supernatant and mixed by pipetting and incubated for 5mins at RT, binding the amplicons. Beads were then collected on the magnet, the supernatant was discarded and the beads were washed twice in 85% ethanol for 30s. The beads were then dried until most ethanol had evaporated, but the beads were still wet, and the beads were then resuspended in 50L 10mM Tris pH 8.0 and incubated for 10 minutes at 37C with 1000rpm agitation. The 50uL of eluted amplicons were then transferred to a fresh tube and the process of bead selected was repeated but with half the volumes (30L and 35L of beads) and then eluted in 25L of 10mM Tris pH 8.0. 10L of these amplicons were then used for final library preparation using the NEB Ultra II DNA library prep kit according to manufacturer instructions using a 1/25 adapter dilution and 5 PCR cycles.