sequencing of gRNA in a pooled library : gRNAs derived from pieces of digested pooled human genomic DNA
Human genomic DNA extracted from pooled blood was digested with micrococcal nuclease (NEB) down to a size of approximately 20 bp, followed by PAGE-gel extraction, end repair and ligation of Gibson adapters for insertion into a gRNA vector. Gibson adapters were amplified from the gRNA vector using primers using primers 5'-linker-F (5'-ttggaatcacacgacctgga and 5'-linker-R (5'-cggtgtttcgtcctttccac and 3'-linker-F (5'-gttttagagctagaaatagcaagttaaaata) and 3'-linker-R (5'-actcggtcatggtaagctcc) respectively. To prevent self-ligation of the linkers and ensure directionality of the ligation, the 5' linker (689 bp) was digested with HindIII and a 600 bp fragment purified. The 3' linker (848 bp) was digested with SacII and the resulting 300 bp fragment gel-purified. Next, the linkers were ligated to the fragments of MNase-digested genomic DNA. Ligation reactions were directly used in nick translation, supplementing with 25 micro liters Long Amp Taq 2X Master Mix (NEB) and 2.5 micro liters primer Linker-Minus450-F (10 microM, 50-GGGCAAGTTTGTGGAATTGG) 2.5 micro liters primer Linker-Plus275-R (10 microM, 50- AAGTGGATCTCTGCTGTCCC) in a 50 micro liters reaction. Three different fragments (L1, L2 and L3) were amplified from the nick-translation product, using Phusion High-Fidelity PCR Master Mix and HF Buffer (NEB) in a 25 micro liters reaction supplemented with 2.5 micro liters of 100microM primer and 1/16th of the purified product of nick translation (2.5 micro liters) as input. L3 is amplified using primers Linker-Minus150-F (5'-CCTTCACCGAGGGCCTATTT) and Linker-Plus160-R. The L3 amplification products were analyzed on a 0.8 % low-melting agarose gel. DNA of the correct size (464 bp respectively) was excised from the gel and purified and inserted into the gRNA vector using Gibson cloning, followed by electroporation of the entire reaction into freshly prepared electrocompetent TG1 E. coli cells from which the gRNA plasmid library was isolated using the HiSpeed Plasmid Maxi Kit (QIAGEN). For sequencing the gRNAs in the library, a fragment 113 bp in length and comprising the gRNA targeting sequence was amplified from the library (100 ng input) using Long Amp Taq 2X Master Mix (NEB) and 1micro liters of each primer gRNA-Upstream-50-F (10 microM, 50-AAGTATTTCGATTTCTTGGCTTTATATATCT) and gRNA-Downstream-19-R (10 microM, 50-CGGACTAGCCTTATTTTAACTTGC). Ends were phosphorylated using T4 polynucleotide kinase (NEB) and pre-annealed Mis-Seq adapters (Adapter-InPE-1.0 5'- [Phos]GATCGGAAGAGCACACGTCT, Adapter-InPE-2.0 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T) were ligated to the ends of the PCR amplicon using the Quick ligation kit (NEB) with an adapter-to-PCR-insert ratio of 10:1 using 5 micro liters ligase enzyme and incubating at 18degrees Celcius for 2 h. PCR amplification with KAPA HiFi PCR kit (Kapa Biosystems) with indexed reverse primers was used for indexing the sequencing libraries.