Long-amplicon seq of bovine NANOS3: sample_6
Bovine samples were produced via embryo transfer of CRISPR/Cas9 NANOS3 targeted bovine embryos. DNA was extracted using Qiagens DNeasy Blood and Tissue Kit from tail tissue (fetal samples) and blood (perinatal and live samples). A 6,274 bp region centered around the bovine NANOS3 exon 1 target location was amplified by long-range PCR. Long-range PCR products were purified using an AMPure PB Kit (Pacific Biosciences of California (PacBio), Menlo Park, CA). SMRTbell libraries were prepared with PacBio barcoded overhang adapters, which allowed for pooling of the samples (SMRTbell Express Template Prep Kit 2.0 and Barcoded overhang adapter kit 8A, PacBio). Sequencing was performed on a PacBio Sequel II system. SMRT Link v11.0.0.146107 was used to perform Circular Consensus Sequencing (CCS) analysis, generate HiFi reads, sort the HiFi reads by barcode and  convert BAM files to individual FASTQ files.