GSM3291072: Env sCD4 Sorted Library A; Replicate 1; 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, Libraries A, B and C) or SuperScript IV VILO (Invitrogen, Library A) primed with: random hexamers and oligo-dT (Library A, SuperScript), DU422gp160_libA_RT_rev2 (GGCCTAGTGCATTTAATCTC, Library A, Accuscript), DU422gp160_libB_RT_rev2 (GTTGCTGGTCCTTAAGATAC, Library B), and EBV_reverse (GTGGTTTGTCCAAACTCATC, Library C). In the first round of PCR, the gp140 coding sequences were amplified as overlapping fragments of ~800 bp each. Primer pair (Library A Fragment 1): HiSeq_DU422gp160_52_F (TCTTTCCCTACACGACGCTCTTCCGATCTTGTAGCGTCAGTGCTTGC) and HiSeq_DU422gp160_624_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACACTTCAGTATGGCGTACC) Primer pair (Library A Fragment 2): HiSeq_DU422gp160_279_F (TCTTTCCCTACACGACGCTCTTCCGATCTCATGTGGAAGAACGACATGG) and HiSeq_DU422gp160_850_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACGGACTTGTTGAGATGGAC) Primer pair (Library B Fragment 1): HiSeq_DU422gp160_808_F (TCTTTCCCTACACGACGCTCTTCCGATCTGCGAAGTGAGAACTTGACGA) and HiSeq_DU422gp160_1489_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCAACCGCTCGCTTTTC) Primer pair (Library B Fragment 2): HiSeq_DU422gp160_1035_F (TCTTTCCCTACACGACGCTCTTCCGATCTAACTGCGGGAGCATTACAAC) and HiSeq_DU422gp160_1713_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGATACCGCTCAATCGCGA) Primer pair (Library C): HiSeq_DU422gp160_1671_F (TCTTTCCCTACACGACGCTCTTCCGATCTGGGGCATAAAGCAACTG) and HiSeq_6His-Linker_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTGATGGTGATGATGTCC) In a second round of PCR, 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 on a HiSeq 2500 using a 2x250 nt paired end protocol, though data is analyzed as single reads.
GEO
GSM3291072