Illumina NovaSeq 6000 paired end sequencing; Antibody repertoire RNA-seq of BALB/c and C57BL/6 baseline mice (blood and spleen)
Antibody repertoire RNA-seq of BALB/c and C57BL/6 baseline mice (blood and spleen)
Groups of specific pathogen free (SPF) female BALB/c and C57BL/6 mice aged 4 to 6 weeks were purchased from Guangdong Experimental Animal Center. The mice were euthanized and their peripheral blood was collected to isolate peripheral blood monouclear cells (PBMC) with Ficoll; their spleens were collected for splenocyte isolation with RBC lysis buffer. Total RNA was isolated from PBMCs and splenocytes (about 106 cells from each sample) using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instruction. RNA concentrations were determined using a NanoDrop 2000 spectrophotometer. About 50ng of total RNA was reverse transcribed by 5' rapid amplification of cDNA ends (RACE) using the SMARTer PCR cDNA synthesis kit (Clontech, USA) as previously described (PMID: 24391640). The 10µl-reaction was performed as below. Firstly, RNA template was mixed with mice B cell receptor (BCR) heavy chain primers (IgG: CCCTTGACMAGGCATCCYAG, IgM: CTGGTAGTTCCAGGTGAAGG, IgA: CCAGGTCACATTCATCGTGC) and dNTP. To denature RNA and anneal the priming oligonucleotides, RNA was incubated at 72˚C for 3 min and then on ice immediately for 2 minutes. The SMARTer IIA oligonucleotide and SMARTScribe reverse transcriptase were added for 5'-template switching and the cDNA synthesis reaction, incubated at 42˚C for 60 min, terminate the reaction by heating at 70˚C for 15min. Two-round PCR was performed for BCR library preparation. The first round of PCR amplification was performed using 2µl of cDNA, the universal primer Smart20, and the BCR IgH chains specific primers (IgG: CCAGGGGCCAGTGGATAGAC, IgM: CCACCAGATTCTTATCAGAC, IgA: ATCAGGCAGCCGATTATCAC). The reaction was performed for 22 cycles using the following regimen: 95˚C for 20s, 62˚C for 20s, and 72˚C for 40s. The first-round PCR products was purified by magnetic beads (Beckman, USA). One fifth of the purified product was used in the second round PCR with IgH chains specific primers (IgG: TAGACAGATGGGGSTGTYGTT, IgM: AAGACATTTGGGAAGGACTG, IgA: GTCAGTGGGTAGATGGTGGG). The reaction was performed for 22 cycles with the following regimen: 95˚C for 20s, 65˚C for 20s, and 72˚C for 20s. PCR products were identified on 2% agarose gels, and bands centered at 500–700 bp were excised and purified using Qiaquick Gel Extraction kit (Qiagen, Germany).