Amplitaq360

Single B cell cDNA was generated with random hexamers using SSIII. The antibody heavy- and light-chain variable regions were PCR amplified using AmpliTaq360 master mix (Applied Biosystems). PCR products were purified (Qiagen, Valencia, CA) and sequenced by Genewiz. Gene rearrangements, clonal relatedness, unmutated common ancestors, and intermediate ancestor inferences were made using Cloanalyst (13 (link)). The DH677 clonal lineage tree was generated using FigTree.

Genomic PCR was performed using AmpliTaq 360 (Applied Biosystems), KAPA HiFi, or KAPA Taq Extra (KAPA Biosystems) on a Veriti 96-well Thermal Cycler (Applied Biosystems) or Biometra Trio (Analytik Jena) according to the manufacturer’s instructions. Specific PCR conditions are available upon request.
Primers used to verify gene targeting events are described in Figs. 2 and 4 and Supplementary Figs. 10 and 11, and Supplementary Table 7 and 8. Sequencing of the junction regions was used to ensure the fidelity of the flanking µH and CRISPR-Cas9 protospacers. PCR products from cassette-excised iPSC clones were sequenced directly, while PCR products from populations were cloned using the Zero Blunt TOPO PCR Cloning Kit for Sequencing (Invitrogen), and then amplified and sequenced from the resulting bacterial colonies with Ex Taq DNA Polymerase (Takara) and T3/T7 primers. Primer design for exons 1–9 of HPRT1 (Accession NG_012329.1) and exons 1-5 of APRT (Accession NG_008013.1) was performed using the NCBI Primer-BLAST with optional settings for human repeat filter, SNP handling, and primer pair specificity checking to H.sapiens (taxid:9606) reference genome, and are listed in Supplementary Table 9.