Targeted PCR Amplification and NGS

Genomic DNA was extracted from cells using an ammonium acetate-based method as described previously [27 (link)]. To control for representation, PCR with gRNAs sequence primer listed in Additional file 6: Table S1b was performed on genomic DNA equivalent to 500 cells per guide, corresponding to a total of 231 μg from 35 million cells (assuming 6.6 pg in a single diploid cell). This input DNA was split into 77 reactions with 3 μg per reaction. For the verification of amplified SAM gRNA library, 10 ng of input plasmid DNA was used. PCR was conducted using Phusion Flash High-Fidelity PCR master mix (Life Technologies) for 28 cycles as 98 °C for 90 s, 98 °C for 1 s, 60 °C for 5 s, 72 °C for 15 s, followed by final extension of 72 °C for 1 min. To enable multiplexing during NGS, an 8 bp unique barcode was added at the beginning of the forward primer as TCGCCTTG, ATAGCGTC, GAAGAAGT, ATTCTAGG or CGTTACCA. All PCR products were pooled, ethanol precipitated and resuspended in 100 ul. This was subjected to electrophoresis on a 2% agarose gel and the band of interest was extracted using Monarch DNA Gel extraction kit (NEB GmbH, Frankfurt, Germany) according to manufacturer’s instructions. NGS libraries were prepared from amplicons by GATC Biotech (Konstanz, Germany) and 125 bp paired end sequencing was carried out on an Illumina HiSeq 4000 with 15 million reads per condition.