GSM4237639: HEK_R3_D0; Homo sapiens; OTHER
The tiling screens were terminated after 5 to 10 days. For cell surface proteins, cells were stained in batches of 1x107 cells for 30 min at 4°C (BioLegend: CD46 clone TRA-2-10 #352405 - 3µl per 1x10^6 cells; CD55 clone JS11 #311311 - 1.5µg per 1x10^6 cells; CD71 clone CYIG4 #334105 - 4µl per 1x10^6 cells). We collected unsorted samples for input guide RNA representation of approximately 1000x coverage for each sample and sorted at least another 1000x representation into the assigned bins based on their signal intensities. Cells were PBS-washed and frozen at -80°C until sequencing library preparation. In each case, the bin containing the lowest 20% represented the strongest target knock-down. For each sample, genomic DNA was isolated from cell pellets using the GeneJET Genomic DNA Purification Kit (ThermoFisher K0722) with 2x10^6 cells or less per column. The crRNA readout was performed using two rounds of PCR. For each sample, we performed PCR1 reactions as follows: 20 µl volume with 2 ug of gDNA in each reaction limited by the amount of extracted gDNA (total gDNA ranged from 8µg to 50 ug per sample with an estimated representation of 10^6 diploid cells per ~6.6 ug gDNA. PCR1: 4µl 5x Q5 buffer, 0.02U/µl Q5 enzyme (M0491L), 0.5uM forward and reverse primers and 100ng gDNA/µl. PCR conditions: 98°C/30s, 24x[98°C/10s, 55°C/30s, 72°C/45s], 72°C/5min). We pooled the unpurified PCR1 products and used the mixture for a single second PCR reaction per sample. This second PCR adds on Illumina sequencing adaptors, barcodes and stagger sequences to prevent monotemplate sequencing issues. (PCR2: 50µl 2x Q5 master mix (NEB #M0492S), 10µl PCR1-product, 0.5uM forward and reverse PCR2-primers in 100µl. PCR conditions: 98°C/30s, 17x[98°C/10s, 63°C/30s, 72°C/45s], 72°C/5min). Amplicons from the second PCR were pooled by screen experiment (e.g. all GFP-screen samples) in equimolar ratios (by gel-based band densitometry quantification) and then purified using a QiaQuick PCR Purification kit (Qiagen 28104). Purified products were loaded onto a 2% E-gel and gel extracted using a QiaQuick Gel Extraction kit (Qiagen 28704). The molarity of the gel-extracted PCR product was quantified using KAPA library quant (KK4824) and sequenced on an Illumina NextSeq 500 - II MidOutput 1x150 v2.5.