GSM2732784: Env VRC01 Sorted Library B; Replicate 2; Human immunodeficiency virus 1; OTHER
Total RNA was harvested from sorted cells using GeneJet RNA Purification Kit (Thermo Scientific) and cDNA prepared using Accuscript Hi-Fi (Agilent Genomics) primed with a gene-specific primer: BALgp160_libA_RT_rev (CGTTCAGCTGAACAATGATG, Library A), BALgp160_libB_RT_rev (TGTTGGACAATACCAGACAG, Library B), and EBV_reverse (GTGGTTTGTCCAAACTCATC, Library C). In the first round of PCR (18 cycles), the Env coding sequences were amplified as overlapping fragments of ~800 bp each. Primer pair (Library A Fragment 1): MiSeq_pCEP4intron_F2 (TCTTTCCCTACACGACGCTCTTCCGATCTACGACTCACTATAGGCTAGC) and MiSeq_BaLgp160_588_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTGGGTAATCACAGAAGTG) Primer pair (Library A Fragment 2): MiSeq_BaLgp160_167_F (TCTTTCCCTACACGACGCTCTTCCGATCTACACCGAGGTGCATAATGTG) and MiSeq_BaLgp160_799_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATCACCACCTCTTC) Primer pair (Library B Fragment 1): MiSeq_BaLgp160_773_F (TCTTTCCCTACACGACGCTCTTCCGATCTGCTGCTGAATGGATCCTTG) and MiSeq_BaLgp160_1368_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTAAACACCTCTGTCTTG) Primer pair (Library B Fragment 2): MiSeq_BaLgp160_971_F (TCTTTCCCTACACGACGCTCTTCCGATCTTAGACAAGCCCACTGCAAC) and MiSeq_BaLgp160_1579_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCACTGTCAGTGTCATAGC) Primer pair (Library C Fragment 1): MiSeq_BaLgp160_1492_F (TCTTTCCCTACACGACGCTCTTCCGATCTGTTGTGCAGAGAGAGAAGAG) and MiSeq_BaLgp160_2251_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAACAGGCTTCTCAAGTCCAC) Primer pair (Library C Fragment 2): MiSeq_BaLgp160_1699_F (TCTTTCCCTACACGACGCTCTTCCGATCTAAACAATTGCAGGCTCGCGT) and MiSeq_BaLgp160_2422_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACAGCTGTGGCATTAAGGAG) Primer pair (Library C Fragment 3): MiSeq_BaLgp160_1855_F (TCTTTCCCTACACGACGCTCTTCCGATCTATGACGTGGATTGAGTGGGA) and MiSeq_pCEP4_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTATCATGTCTGGATCCGG) In a second round of PCR (14 cycles), Illumina adaptors and experiment-specific barcodes were added. Primer pair for adding I5 and I7 Illumina adaptors: MiSeq_Start_Adaptamer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and MiSeq_Index_Adaptamer (CAAGCAGAAGACGGCATACGAGAT-6nt-barcode-GTGACTGGAGTTCAGACGTGTGCTCTTC) Amplicons were deep sequenced with MiSeq v3 and HiSeq 2500 instruments using a 2x250 nt paired end protocol, though data is analyzed as single reads.
GEO Accession
GSM2732784