EGFR drug resistance screen with ABE8e in MCF10A cells_Osimertinib_replicate2
Genomic DNA isolation was performed using the Blood & Cell Culture DNA Midi Kit (Qiagen #13343) according to the manufacturers protocol. Illumina library preparation was performed in two steps. First, 4 g of genomic DNA were used in each 50L PCR1 reaction with primers amplifying the sgRNA library and containing universal Illumina adapters. Forward primers were ordered from IDT technologies to include three different stagger lengths and were pooled in equal amounts. The final PCR mixes had the following composition: Kapa Taq ReadyMix (Merck #TAQKB) 25 L, 6 L Primer mix (2.5 M each), 4 g genomic DNA and H2O to 50 L. PCR cycling conditions: 3 min at 95C, [20 s at 98C, 15 s at 60C, 30 s at 72C] for 18 cycles followed by a final extension 30 s at 72C. PCR1 products from the same sample were then pooled together. For each sample, a single PCR2 reaction was then performed to add P5 and P7 Illumina adapters as well as demultiplexing barcodes. The reaction mix was as follows: Kapa Taq ReadyMix (Merck #TAQKB) 10 L, 2.5 L Primer mix (4 M each), 1 L PCR1 product and 6.5 L H2O. PCR cycling conditions: 3 min at 95C, [20 s at 98C, 15 s at 61C, 30 s at 72C] for 8 cycles followed by a final extension 30 s at 72C. Barcoded PCR2 products were then pooled, desalted and 700 ng of DNA was loaded in each lane of a 2% Agarose EGel (InVitrogen, #A42135). DNA bands were extracted using the Qiagen PCR purification kit (Qiagen, #28104).