Illumina HiSeq 2500 sequencing; Whole-Genome GeCKO CRISPR screen in Malignant Rhadoid Tumour cells +/- SMARCB1 expression.
Whole-Genome GeCKO CRISPR screen in Malignant Rhadoid Tumour cells +/- SMARCB1 expression.
Cells were trypsinised from cell culture flask and resuspended in ice-cold PBS ready for extraction.  The cell line STA-WT1 was provided to us by Prof. Peter Ambros of St Anna Kinderkrebsfrschung, CCRI, Vienna Austria. It was generated at the CCRI and is not available through catalogued collections it is a SMARCB1 mutated Malignant Rhabdoid Tumour derived from the Kidney as first described in Nature. 1998 Jul 9;394(6689):203-6. The SMARCB1 re-expression arms were split once (1 in 2) 3 days after the re-expression virus was added. Following this media was replaced and cells split as required (i.e when they achieved 70-80% confluence) and maintained at a minimum of 3.5 x 107 cells. 3.5 x 107cells were harvested at day 7 and 14. Two biological repeats were performed. MRT cells were infected with the GeCKO viral library (MOI 0.3 gives ~1 x sgRNA/cell) at 900x coverage per sgRNA and selected with puromycin. Given that two-thirds of cells die following selection, 300x coverage i.e. >3.5x107 cells were harvested 48 hours after infection with GeCKO virus (Day 0 Baseline), the following day flasks were split into 2 arms representing 300x coverage and either SMARCB1 re-expression or control virus was added (MOI 10). 24 hours after the addition of the second virus cells were selected with neomycin.  At each time point 3.5 x 107 cells genomic DNA (gDNA) was extracted using Qiagen Maxi Kit. gDNA was then pooled and quantified by NanoDrop. Integrated sgRNA from genomic DNA were amplified using FastStart High Fidelity PCR system (Roche) using the following thermocycler conditions: 98°C for 10 minutes, 23 cycles of 98°C for 30 seconds, 59°C for 30 seconds, 72°C for 1 minute and finally 72°C for 3 minutes and 4°C hold. Primers were modified from the original Zhang protocol to amplify integrated sgRNA sequences. A universal forward primer containing the Illumina P5, Illumina sequencing primer binding site and NNNNN (directly preceding the sgRNA) was designed and used with 12 reverse primers containing the Illumina P7, Illumina sequencing site and a unique barcode sequence. 10-15μg of DNA per PCR reaction was amplified using 100μl volume PCR reactions according to manufacturer's instructions. PCR product purification was performed using AMPure XP SPRI beads (Beckman Coulter). Pooled product was mixed 0.65:1 with room temperature AMPure XP beads, incubated at room temperature for 5 mins then left to clear on a magnetic stand. gDNA was bound to the beads so they were discarded, 150μl of supernatant was transferred to a fresh tube and beads added at a 1:1 ratio, mixed and incubated for 5 mins then left to clear on a magnetic stand. The required product was bound to the beads which were then washed 2x with 500μl 80% ethanol then left to air dry for 5 mins. Beads are vortexed with 50μl H2O, incubated for 5 mins then placed on a magnetic stand and left to clear. 45-48μl of product was recovered, concentration assessed by Nanodrop (ThermoFisher Scientific), product was diluted to 30-40ng/μl and quality checked by running 1μl on a DNA 1000 chip in an Agilent 2100 Bioanalyzer. Products were pooled/diluted where required to a final molarity of 20-30 nmol/L.