GSM5008919: CAST-seq centromeric CCR5#2 CRISPR/Cas9 treated CD34+ cells, duplicate Day4 non-treated control; Homo sapiens; OTHER
Genomic DNA of at least 5 x 105 cells was isolated at the indicated time points using QIAamp® DNA Blood Mini Kit (Qiagen, Cat.# 51306) DNA fragment by sonication (M220 Focused Ultrasonicator) or enzymatic digestion (NEBNext® Ultra™ II FS DNA Library Prep Cat.# E7805S) to obtain average fragment lengths of 350 bp. The DNA was end repaired and a protruding 3'-A nucleotide was added according to the manufacturer's instructions (NEBNext® Ultra™ II FS DNA Library Prep Cat.# E7805S) in order to enable the ligation of a linker sequence with a protruding 3'-T. After DNA purification (Qiagen, PCR purification kit, Cat.# 28104), two rounds of PCR utilizing Q5 polymerase (NEB, Cat.# M0493S) were performed using the following conditions: 20 cycles at 95°C for 15 s, 63°C (first reaction) or 68°C (second reaction) for 20 s, 72°C for 20 s. The first reaction was performed with primers complementary to the linker sequence and to a sequence in close proximity to the on-target site (bait primer). One or two decoy primers were introduced to reduce full-length amplification of the fragments which contain the on-target sequence without a chromosomal aberration event. The second PCR utilized nested primers to reduce the amount of mis-amplified fragments Third PCR introduced the barcoded Illumina adapter for sequencing (NEB, NEBNext Multiplex Oligos for Illumina, Cat.# E7335). Denatured amplicons at 6-10 pM were loaded into the Illumina MiSeq Reagent Kit V2-500 cycle (Illumina, Cat.# MS-102-2003) according to the manufacturer's instructions.