title: GSM7432247: CRISPR screen, Hoxb8 imortalised GM-CSF cell line, Adar1 E861A/E861A Day 0 rep 2 (165, S10); Mus musculus; OTHER
library_construction_protocol: Sequencing libraries were generated based on the PCR amplification of the integrated sgRNA constructs containing a barcode sequence for Illumina sequencing (protocol from Broad Institute; https://portals.broadinstitute.org/gpp/public/resources/protocols). Three time points (Day 0 (day of tamoxifen addition), 7, and 14) of the two cell lines (fl/+ and fl/E861A) were selected. To utilise the full amount of gDNA recovered from each 20 million-cell pellet, between 18 and 28 PCR reactions were performed. Each 100mL PCR reaction contained 10mg of gDNA, 10mL 10x ExTaq reaction buffer, 8mL 2.5 mM dNTP mixture, 0.75mL ExTaq polymerase, 0.5mL P5 primer mix (100mM), and, for each of the six samples, 10mL of a unique P7 index primer (5mM) in nuclease-free water. The PCR products were pooled together, and 5mL of each sample was loaded in a 3% agarose gel and run for 45 minutes at 120 V to  confirm the size of the band and check for the presence of contamination or degradation products. Pooled PCR products were then purified using AMPure XP magnetic beads to remove gDNA and PCR contaminants. In brief, 100mL of pooled PCR was mixed with 100mL of magnetic beads and incubated at room temperature for 5 minutes. Next, the tubes were placed on a magnetic plate for 5 minutes and the supernatant discarded. The beads were washed twice with 70% ethanol and the DNA eluted in 50 mL TE buffer. The purified pooled library was then quantified with Nanodrop spectrophotometer system, and 2mL of product was mixed with 2mL of nuclease-free water and 1.25mL of loading buffer and run on a 3% agarose gel for a final quality check. Finally, 50mL of the purified pooled library containing at least 3ng/mL of DNA was sent for sequencing by Illumina SE150 sequencing at Novogene (Singapore) In total, 12 libraries were generated and sequenced on the Illumina platform using 150bp single end reads by Novogene (China).