GSM8194790: C57BL/6, NOD-FMT, spleen, biological rep4; Mus musculus; RNA-Seq
Total RNA was extracted from mouse spleen, LG, and peripheral blood mononuclear cells using TRIzol reagent (Invitrogen, Waltham, MA, USA) following manufacturer's protocol. The isolated total RNAs were reverse-transcribed by mouse Heavy chain CH1 domain gene-specific primers, containing unique molecular identifier (UMI) sequences, by using the SuperScript™ IV First-Strand Synthesis System (Invitrogen). After cDNA synthesis, 1.8x volume of SPRI beads (AMPure XP, Beckman Coulter, Brea, CA, USA) were used to purify the cDNA and elute in 35 μL of DW. The purified cDNA was subjected to second-strand synthesis using V gene-specific primers and KAPA biosystems (KAPA HiFi HotStart, Roche, Basel, Switzerland). The PCR conditions were as follows: 95°C for 5 min, 98°C for 1 min, 60°C for 45 secs, and 72°C for 5 min. The synthesized second-stranded DNA was purified using 1x volume of SPRI beads. The purified VH genes were amplified by forward and reverse Illumina adaptor and ligated with Index primers by PCR. The PCR conditions were as follows: 95°C for 5 min; 25 cycles of 98°C for 30 sec, 65°C for 30 sec, and 72°C for 1 min; and 72°C for 5 min. PCR products were subjected to gel electrophoresis in 1.5% agarose gel, then the target amplicons were extracted (NucleoSpin Gel and PCR Clean‑up kit, Macherey-Nagel, Düren, Germany) and purified by 1x volume of SPRI beads as described above. The quality and concentration of the final PCR products were determined using TapeStation 4200 (G2991A, Agilent technologies, Santa Clara, CA, USA) and sequenced by Novaseq 6000 250PE platform (Illumina Inc.).