title: Illumina MiSeq paired end sequencing Rhesus macaque RBg12 IgM
design: Frozen PBMCs from five nave Indian origin RMs were obtained from Yerkes National Primate Research Center (IACUC approval YER2001036). The cells were rapidly thawed in a 37C water bath and immediately diluted into 10 mL of pre-warmed RPMI media with 10% (v/v) heat-inactivated fetal bovine serum (FBS). Cells were pelleted at 400xg for 7 minutes and resuspended in 0.5 mL FACS buffer (PBS + 1% (v/v) FBS and stained on ice for 1 hr with the panel of fluorescent antibodies listed in Table S6. A MoFlo Astrios cell sorter (Beckman Coulter), with gating for live IgM+/CD20+/CD3-/CD4-/CD8-/CD14-/CD16- cells, was used to isolate cells that were then pelleted at 600xg for 10 min, resuspended in RLT+BME buffer (Qiagen), snap frozen in a dry ice ethanol bath and stored at -80C. RNA extraction was performed using RNeasy Protect Mini kit (Qiagen) following the manufacturers instructions. The 5 rapid amplification of cDNA ends (5RACE) with template switching method was used to obtain cDNA with unique molecular identifiers (UMIs) using a protocol modified from Turchaninova et al. (PMID: 27490633). Briefly, 300 ng of RNA was used in a 5'RACE cDNA synthesis reaction. The first-strand cDNA synthesis was performed at 42C for 1 hr using the RM IgM outer reverse primer (5'-GTGATGGAGTCGGGAAGGAAG-3'), a template switch adaptor with incorporated UMIs (5'-AAGCAGUGGTAUCAACGCAGAGUNNNNUNNNNUNNNNUCTTrGrGrGrG-3'), and SMARTScribe Reverse Transcriptase (Clontech). Residual template switch adaptor was removed by incubation with 5 U of uracil DNA glycosylase (New England BioLabs) for 40 min at 37C. The resulting cDNA was purified using the MinElute PCR Purification Kit (Qiagen) following the manufacturers instructions. PCR amplification was performed using the Q5 High-Fidelity DNA Polymerase (New England BioLabs), the forward primer (5'-NNNNAAGCAGTGGTATCAACGCA-3'), and the RM IgM inner reverse primer (5'-NNNNNAGGGGGAAAAGGGTTG-3'). Illumina adaptors were added using the NEBNext UltraTM II DNA Library Prep Kit (New England BioLabs) following the manufacturers instructions. Libraries were sequenced on an Illumina MiSeq using the Illumina v3, (2x 300 bp) sequencing kit.