GSM8547496: plasmid; Homo sapiens; OTHER
Cell pellets from samples were resuspended in 20 mL P1 buffer (Qiagen) with 100 μg/mL RNase A and 0.5% SDS followed by incubation at 37C for 30 min. After that, Proteinase K was added (100 μg/mL final) followed by incubation at 55C for 30 min. After digest, samples were homogenised by passing them three times through a 18G needle followed by three times through a 22G needle. Homogenised samples were mixed with 20 mL Phenol:Chlorophorm:Isoamyl Alcohol (Invitrogen #15593–031), transferred into 50 mL MaXtract tubes (Qiagen) and thoroughly mixed. Samples were then centrifuged at 1500 g for 5 min at room temperature (RT). The aqueous phase was transferred into ultracentrifuge tubes and thoroughly mixed with 2 mL 3 M sodium acetate plus 16 mL isopropanol at RT before centrifugation at 15,000 g for 15 min. The gDNA pellets were carefully washed with 10 mL 70% ethanol and dried at 37C. Dry pellets were resuspended in H2O and gDNA concentration was adjusted to 1 μg/uL. The degree of gDNA shearing was assessed on a 1% agarose gel and gDNA was sheared further by boiling at 95C until average size was between 10 and 20 kb. Multiple PCR reactions were prepared to allow amplification of the total harvested gDNA from a 1000x cell coverage for each sample. For the first round of two nested PCRs, the total volume was 100 μL containing 50 μg sheared gDNA, 0.3 μM forward (5′-ggcttggatttctataacttcgtatagca-3) and reverse (5′-cggggactgtgggcgatgtg-3′) primer, 200 μM each dNTP, 1x Titanium Taq buffer and 1 μL Titanium Taq (Clontech). PCR cycles were: 1x (94C - 3 min), 16x (94C - 30 s, 65C – 10 s, 72C – 20 s), 1x (68C – 2 min). All first round PCRs were pooled and a fraction was used as template for the second round PCR. The total volume of the second round PCR was 100 μL containing 2 μL pooled first round PCR, 0.5 μM forward (5'-AATGATACGGCGACCACCGAGATCCACAAAAGGAAACTCACCCTAAC-3′) and reverse (5'-CAAGCAGAAGACGGCATACGAGAT-(N)6-GTGACTGGAGTTCAGACGTG-3′) primer where (N)6 is a 6 nt index for sequencing on the Illumina HiSeq platform, 200 μM each dNTP, 1x Titanium Taq buffer and 1 μL Titanium Taq (Clontech). PCR cycles were: 1x (94C - 3 min), 16x (94C - 30 s, 55C – 10 s, 72C – 20 s), 1x (68C – 2 min). The resulting PCR product (344 bp) was extracted from a 1% agarose gel. Gel extracted bands were submitted for sequencing on an Illumina HiSeq 2500 platform using paired end 50 kits with the custom sequencing primer 5'-GAGACTATAAGTATCCCTTGGAGAACCACCTTGTTGG-3′ for reading the sgRNA sequence and the standard Truseq Illumina reverse primer to read out 20 nt unique barcode sequences and library IDs. sequencing of amplicon