Amplicon-seq of Homo sapiens: HEK293T following targeted RNA-knockdown of CKB with CRISPR-Csm and crRNA2
1x10^6 HEK293T cells were transfected with 2.5 ug all-in-one CRISPR-Csm plasmid DNA using 7.5 ul FuGENE HD transfection reagent in 6-well plates, as per manufacturers instructions. Following transfection, cells were grown for 48 hr to allow plasmid expression and RNA knockdown to occur, then transfected cells (mCherry-positive) were enriched by FACS. Cells were lysed with Laird lysis buffer (10 mM Tris pH 8, 5 mM EDTA pH 8, 200 mM NaCl, 0.2% SDS, 0.2 mg/mL proteinase K) at 55C for 2 hr, and genomic DNA extracted with phenol-chloroform. The CKB locus was amplified from genomic DNA by PCR (primer sequences listed in Table S1) using PrimeSTAR GXL DNA polymerase (Takara Bio). The full-length PCR amplicon was purified from agarose gel and sheared by sonication to 200-400 bp fragments using a Qsonica Q800 Sonicator at 50% power setting, 30 sec on/30 sec off, for a total sonication time of 8 min at 4C. DNA libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, excluding reverse-transcription of cDNA generation.