GSM8516006: g34_bcl11a; Homo sapiens; OTHER
CD34+ HSPCs edited with Cas9-3xNLS targeting HBG promoter, cultured in X-VIVO media with cytokines, and harvested cells at days 5 and 14 after electroporation (pre-GMP engineering runs, n=3). Genomic DNA extracted from control and edited cells using Agencourt DNAdvance according to manufacturer's protocol. A custom Tn5-transposome with the full-length Illumina forward (i5) adapter, a sample barcode, and unique molecule identifier was used for the genomic DNA tagmentation reaction. After genomic DNA tagmentation, two rounds of PCRs were performed. The first PCR was performed using the target-specific HBG promoter anchor primer, and the second round of PCR added with the reverse (i7) Illumina adapter and an additional sample barcode. Two primers targeting HBG1 and HBG2 gene promoters were used in parallel for the first PCR. For positive control, gene-specific primers designed for both BCL11A and HBG promoter complete libraries were then size selected for 400–850 bp using PippinHT, quantified by qPCR, and sequenced on Illumina MiSeq with paired-end 301-8-18-301 cycles.