GSM4982733: WI38fast-iCas9- p21p-mCherry pooled sgRNA cells, Serum starvation- treated cells, low mCherry sorted cells, repeat2; Homo sapiens; OTHER
PureLink genomic DNA mini kit (Invitrogene; K182000) Growth competition:  PCR reaction was conducted using iProof master mix (X2), 10µM of each primer and 20ng of genomic DNA in a 50µl total volume reaction, for 26 cycles, Tm = 64°C. The primers used to amplify the sgRNA region were:  Forward primer- GCTTACCGTAACTTGAAAGTATTTCGATTTCTTGG  Reverse primer- CTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAAC   After PCR clean-up using Wizard SV Gel and PCR Clean-Up (Promega; A9281), samples were run in 2% agarose gel to ensure that the PCR product is compose of a single amplicon in the appropriate size. Next, Illumina HiSeq 2000 libraries were prepared using the sequencing library module from (Blecher-Gonen et al., 2013). Briefly, blunt ends were repaired, Adenine bases were added to the 3' end of the fragments, barcode adapters containing a T overhang were ligated, and finally the adapted fragments were amplified. The process was repeated for each sample with a different Illumina DNA barcode for multiplexing, and then all samples were pooled in equal amounts and sequenced.   Cell cycle arrest signals:  PCR reaction was conducted using 2X KAPA HiFi HotStart ReadyMix, 10µM of each primer and 20ng of genomic DNA extracted from the samples in a 50µl total volume reaction, for 20 cycles, Tm = 58°C. We used shifted primers to increase library complexity. PCR products were purified with SPRI-beads using left-side size selection protocol in which the PCR product and beads were mixed at 1:1.5 ratio. Then, each sample was barcoded with a different Illumina barcode while the Forward primer was fixed and the reverse primer included the different barcodes. The barcoding PCR reaction that was conducted using KAPA HiFi HotStart ReadyMixPCR Kit (X2), 10µM of Illumina primers and 0.35-0.7ng of PCR product in a 10µl total volume reaction, for 15 cycles. Additional clean-up step was performed using SPRI beads with left-side size selection protocol in which the PCR product and beads were mixed at 1:1.5 ratio. Samples were pooled in equal amounts and sequenced.