Kapa quantitative rt pcr

ATAC-seq was performed with two independent experiments per condition^{60 (link)}. Briefly, nuclei were isolated from 50,000 sorted cells per replicate using a solution of 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl₂, and 0.1% IGEPAL CA-630. Immediately following nuclei isolation, the transposition reaction was conducted using Nextera Tn5 transposase and TD buffer (Illumina) for 45 min at 37 °C. Transposed DNA fragments were purified using a Qiagen MinElute Kit, barcoded with dual indexes (Illumina Nextera), and PCR amplified using NEBNext High Fidelity 2 × PCR master mix (New England Labs). The size distribution and molarity of the sequencing library were determined by using an Agilent Bioanalyzer and KAPA quantitative RT-PCR (KAPA Biosystems). Sequencing was performed using an Illumina HiSeq X ten platform to acquire at least ∼ 50 M fragments per sample. Paired-end reads were mapped to the hg38 reference genome using Bowtie2. Only concordantly mapped pairs were kept for further analysis. Peak calling was performed using MACS v1.4 to identify areas of sequence tag enrichment. These peaks were displayed in the IGV genome browser and further processed for annotation and for differential open chromatin detection.

To prepare ATAC-seq library, engineered α-PD-1 secreting B cells cultured at day 28 and activated primary B cells without transduction were sorted via flow cytometry. ATAC-seq was performed in biological duplicates following previous report^{69 (link)}. Briefly, nucleus were isolated from 50,000 sorted cells per replicate using a solution of 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl₂, and 0.1% IGEPAL CA-630. Immediately following nuclei isolation, the transposition reaction was conducted using Nextera Tn5 transposase and TD buffer (Illumina) for 45 min at 37 °C. Transposed DNA fragments were purified using a Qiagen MinElute Kit, barcoded with dual indexes (Illumina Nextera), and PCR amplified using NEBnext High Fidelity 2× PCR master mix (New England Labs). The size distribution and molarity of the sequencing library were determined using an Agilent Bioanalyzer and KAPA quantitative RT-PCR (KAPA Biosystems). Sequencing was performed using an Illumina HiSeq X ten platform to acquire at least 50 M fragments per sample. Paired-end reads were mapped to the GRCh38 reference genome using Bowtie2. Only concordantly mapped pairs were kept for further analysis. Peak calling was performed using MACS v1.4 to identify areas of sequence tag enrichment. These peaks were displayed in the IGV genome browser and further processed for annotation and for differential open chromatin detection.