title: GSM3573735: CXCR4-VC Surface Expression Sort Replicate 2 [X4VC_expression2]; Homo sapiens; OTHER
library_construction_protocol: 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 (EBV_reverse). In the first round of PCR (18 cycles), the CCR5 and CXCR4 coding sequences were amplified as three overlapping fragments of ~800 bp each. Primer pair: CCR5 fragment A: MiSeq_CCR5_myctag_F (TCTTTCCCTACACGACGCTCTTCCGATCTCGAGCAAAAGCTGATCTCAG) and MiSeq_CCR5_652_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAGGATTCCCGAGTAGCAG) Primer pair: CCR5 fragment B: MiSeq_CCR5_176_F (TCTTTCCCTACACGACGCTCTTCCGATCTAAGGCTGAAGAGCATGACTG) and MiSeq_CCR5_875_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGATGCAGCAGTGCGTCAT) Primer pair: CCR5 fragment C: MiSeq_CCR5_388_F (TCTTTCCCTACACGACGCTCTTCCGATCTCGTCCATGCTGTGTTTGCT) and MiSeq_Venus_468_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATGCCGTTCTTCTGCTTG) Primer pair: CXCR4 fragment A: MiSeq_CXCR4_myctag_F (TCTTTCCCTACACGACGCTCTTCCGATCTGCCGAGCAGAAATTGATCAG) and MiSeq_CXCR4_682_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGTGTGACAGCTTGGAGATG) Primer pair: CXCR4 fragment B: MiSeq_CXCR4_169_F (TCTTTCCCTACACGACGCTCTTCCGATCTGATTGGTCATCCTGGTCATG) and MiSeq_CXCR4_882_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACAGTGGAAGAAAGCTAGGG) Primer pair: CXCR4 fragment C: MiSeq_CXCR4_375_F (TCTTTCCCTACACGACGCTCTTCCGATCTATCCTGGCCTTCATCAGTCT) and MiSeq_Venus_468_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATGCCGTTCTTCTGCTTG) In a second round of PCR (15 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 on a HiSeq 2500 using a 2x250 nt paired end protocol, though data is analyzed as single reads.