title: RQq16 LLN
design: The protocol for rhesus macaque repertoire sequencing was obtained by courtesy of Dr. Daniel Douek, NIAID/VRC(Huang et al., 2016 [PMID 27851912 ], Francica et al.,2015 [PMID 25858157]). Bulk Env-specific B cells were sorted into 350uLQiagen RLT buffer. RNA was extracted using the RNeasy Micro-DNase Digest protocol (QIAGEN) on QIAcube automation platforms (Valencia, CA). RNA from samples with cell counts above 5000 was quality checked using Agilent Bioanalyzer. Reverse transcription (RT) was performed using Clontech SMARTer cDNA template switching: 5 CDS oligo(dT) (12 M) was added to RNA and incubated at 72C for 3 minutes and 4C for at least 1 minute. The RT mastermix (5xRT Buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 30 mM MgCl2), Dithiothreitol, DTT (20 mM), dNTP Mix (10 mM), RNAse Out (40U/L), SMARTer II A Oligo (12 M), Superscript II RT (200U/L)) was added to the reaction and incubated at 42C for 90 minutes and 70C for 10 minutes. First-strand cDNA was purified using AMPure XP beads (Beckman Coulter). Following RT, two PCR rounds were carried out to generate immunoglobulin amplicon libraries compatible with Illumina sequencing. All oligos were ordered from Integrated DNA Technologies. The first PCR amplification was carried out using KAPA Real-Time Library Amplification Kit (Kapa Biosciences). cDNA was combined with master mix (2X KAPA PCR Master Mix, 12 M 5PIIA and 2 M IgG/IgK/IgL Constant Primer (Francica et al., 2015)). The amplification was monitored using real-time PCR and was stopped during the exponential phase. The amplified products were again purified using AMPure XP beads. A second round of PCR amplification was carried out for addition of barcodes and Illumina adapter sequences: master mix (KAPA RT-PCR Master Mix 2x, SYBR Green 1:10K, Nuclease-free water), 10 M of P5_Seq BC_XX 5PIIA, 10 M of P7_ i7_XX IgG/IgK/IgL and were combined with amplified Immunoglobulin from the first round PCR and amplified using real-time PCR monitoring. The P5_Seq BC_XX 5PIIA primers contain a randomized stretch of four to eight random nucleotides. This was followed by purification with AMPure XP beads. A final PCR step was performed for addition of remaining Illumina adaptors by mixing master mix (KAPA PCR Master Mix 2x,10 M P5_Graft P5_seq, Nuclease-free water), 10 M of P7_ i7_XX IgG/IgK/IgL oligo and amplified products from the previous PCR step followed by purification with AMPure XP beads. The quality of library was assessed using Agilent Bioanalyzer. The amplicon libraries were pooled and sequenced on an Illumina MiSeq as a 309 Paired run.Primers: SMARTer II A:AAGCAGTGGTATCAACGCAGAGTACATrGrGrG; CDS Oligo: TTTTTTTTTTTTTTTTTTTTTTTTTVN; Constant Primer: GCGGGAAGATGAAGACAGATGGTGCAGCCACAG; P7 Primer: CAAGCAGAAGACGGCATACGAGAT CCAGCGCT GCGGGAAGATGAAGACAGATGGTGCAGCCACAG; P5 Seq 5PIIA: CACGACGCTCTTCCGATCT NNNNNNNN AATCGTCA AAGCAGTGGTATCAACGCAGAGT
identifiers.namespace: SUB5194697
identifiers.id: 8_RQq16_LILN_wk-14_IgK