GSM5943518: VHL_r2_MZ1_500_batch1; Homo sapiens; OTHER
Genomic DNA (gDNA) was extracted from frozen cell pellets following the QIAamp DNA Mini Kit (Qiagen, 51304). VHL and CRBN were amplified via PCR from gDNA with primers CRBN_GA_fwd & rev and VHL_GA_fwd & rev for CRBN and VHL, respectively. Primer sequences are available in Supplementary Table 5 of the linked publication. The total isolated gDNA was processed in batches of 5 µg per PCR reaction with Q5 polymerase (NEB, M0491L). One PCR reaction contained 10 µl 5x reaction buffer, 10 µl 5x GC enhancer, 2.5 µl primer mix containing 10 μM forward and reverse primer each, 1 µl dNTP mix (10 μM each), 1 µl Q5 polymerase and nuclease-free water to bring the reaction volume to 50 µl. Target amplification was achieved by performing: 30 s initial denaturation at 95°C; next for 20 to 28 cycles: 15 s at 95°C, 30 s at 57°C and 2 min at 72°C; followed by a final extension for 5 min at 72°C. The cycle number for specific amplification of the 700 base-pair (VHL) and 1.4 kilo-base-pair (CRBN) targets was confirmed by agarose gel electrophoresis. PCR reactions for each treatment were pooled and purified using AMPure XP beads (Beckman Coulter, 10136224) according to standard protocol for double-sided clean up in a 0.3:1 and 1:1 ratio. The purity and integrity of the PCR products were analysed on an Agilent 2100 Bioanalyzer following manufacturer recommendations for high sensitivity DNA chips (Agilent, 5067-4626). Sequencing libraries were prepared using Nextera DNA Library Prep Kit (Illumina, FC-131-1024) following standard manufacturer instructions for amplicon libraries. After purification of the tagmented and PCR amplified DNA libraries quality control was performed by analysis on an Agilent 2100 Bioanalyzer following manufacturer recommendations for high sensitivity DNA chips (Agilent, 5067-4626). Final sequencing libraries were pooled in equimolar amounts and sequenced running 50-bp single-end reads on a HiSeq4000.