Illumina MiSeq paired end sequencing; BCR repertoire analysis by Next-generation sequencing of lymphocytes harvested from mice vaccinated with liposomal TLR4 and TLR7 agonists
BALB/c mice were immunized with inactivated influenza A virus plus liposomal formulation of 1V270 (TLR7 agonist) and 2B182C (TLR4 agonist) on days 0 and 21. Mice were sacrificed on day 28 and lymphocytes in the draining lymph nodes in the injected sides (inguinal lymph nodes) were collected for BCR sequencing. Total RNA was extracted from lymphocytes using RNeasy Mini Kit (Qiagen, Hilden, Germany) and the quality of RNA was confirmed by Agilent 2200 TapeStation System (Agilent, Santa Clara, CA). Total RNA was converted to complementary DNA (cDNA) with Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). Then, double strand (ds)-cDNA was synthesized and an P20EA/P10EA adaptor was ligated to the 5â€™ end of the ds-cDNA and then cut with SphI restriction enzyme. PCR was performed with KAPA HiFi DNA Polymerase (Kapa Biosystems, Woburn, MA) using P20EA and IgG constant region-specific primer mCG1 (GACAGGGMTCCAKAGTTCC). The second PCR was performed with P20EA and mCG2 (ACYGRCTCAGGGAARTAVCC) using the same PCR conditions. After Tag PCR amplification with mCG-ST1-R (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCYTTGACMAGGCAYCC) and P22EA-ST1-R, index (barcode) sequences were added by amplification with Nextera XT index kit v2 setA (illumina, San Diego, CA).