title: GSM5696588: screen 2, young, day 14 reactivated [SC5_Y_RA]; Mus musculus; OTHER
library_construction_protocol: genomic DNAwas extracted with Qiagen QiaAmp DNA Blood Maxi Kit (51194), adding 5x107 cells per column and according to the manufacturer's protocol. sgRNA was amplified from the genome in 2 successive, nested PCR reactions. For the nested PCR reactions, we used either Herculase II Fusion Polymerase (Agilent, 600679) for screen 1, or Q5 DNA polymerase (Fisher Scientific, M0491L) for screens 2 and 3, and Q5 DNA polymerase for in vivo screens, according to manufacturer's protocol. In optimizing this PCR reaction, we found that Herculase II Polymerase was outperformed by Q5 polymerase outperformed Herculase II Polymerase, Q5 polymerase requiring fewer PCR cycles to get more amplicon product, which is why we switched to Q5 DNA polymerase. We used 5 μg genomic DNA in 50 μL reactions to run on thermocycler. For the first PCR, we used primers MCB1562 (aggcttggatttctataacttcgtatagcatacattatac) and MCB1563 (acatgcatggcggtaatacggttatc) (1 µM final concentration), with PCR cycles as follows: 98°C for 2 minutes, 19 cycles of [98°C/30s, 59.1°C/30s, 72°C/45s], followed by 72°C for 3 minutes. We pooled all the PCR#1 cycle products and then used 5 µL of the pool in a second PCR reaction (PCR#2) with the same conditions but using different primers; MCB1439 (caagcagaagacggcatacgagatgcacaaaaggaaactcaccct) and a barcoded primer (aatgatacggcgaccaccgagatctacacGATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXXCGACTCGGTGCCACTTTTTC, where XXXXXX is the 6-digit barcode for high throughput sequencing sample identification). The second PCR reaction was run for either 30 cycles (in vitro screen 1 and in vivo screens), or 18 cycles (in vitro screens 2 and 3).