title: GSM7360904: JH_AIDKO_1; Mus musculus; OTHER
library_construction_protocol: Germinal center B cells were sorted from the spleen of NP-CGG-immunized mice at day 14. Genomic DNA was harvested using DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer's protocol, except for the elution step in which the DNA on the spin column was eluted in 50mL of elution buffer. The reverse complement primer probes (RC-probes) targeting the JH4 intron (Fwd: 5'-CATAACAAAGGTTAAAAATAAAGAC-3' and Rev: 5'-GCCTGACATCTGAGGACTC-3') were synthesized by NimaGen and used for the generation of indexed amplicons by reverse complementary PCR (RC-PCR). The addition of Illumina universal indexes and sequence adapters to the JH4-specfic RC-probes and the amplification of the intronic sequence with these RC-generated region-specific indexing primers were performed in a probe-polymerase mix. The mix was composed of 1) RC-PCR HiFi Master Mix, 2) RC-probe mix, 3) RC-PCR probe dilution buffer (all from NimaGen), and 4) 80 to 100 ng of genomic DNA. The following thermocycling program was used: 1) 98°C for 2 minutes, 2) 98°C for 10 seconds, 58°C for 10 minutes at a ramp rate of 0.1°C/second, and 72°C for 1 minute, 3) 98°C for 10 seconds, 80°C for 1 second, 58°C for 1 hour and 30 minutes at a ramp rate of 0.1°C/second, and 72°C, 4) go to step 3 once, 5) 98°C for 10 seconds, 80°C for 1 second, 58°C for 2 minutes at a ramp rate of 0.5°C/second, and 72°C for 1 minutes, 6) go to step 5 33 times, 6) 4°C hold. PCR amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter) and resuspended in 50mL of low EDTA Tris-EDTA buffer. Indexed amplicons were sequenced on a NovaSeq platform, and an average of 10 to 20 million 150 bp paired-end reads were generated for each sample (Memorial Sloan Kettering Cancer Center Integrated Genomics Operations, New York, USA).