title: GSM2944672: Expression Sorted  NT Library; Replicate 2; 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: SERT_NT__rev (__________, NT Library) and EBV_reverse (GTGGTTTGTCCAAACTCATC, CT Library). In the first round of PCR (20 cycles), the SERT coding sequences were amplified as overlapping fragments of ~700 bp each in the case of the NT Library or ~600bp for the CT library. Primer pair (NT Library Fragment 1): MiSeq_pCEP4intron_F2 (5`TCTTTCCCTACACGACGCTCTTCCGATCTACGACTCACTATAGGCTAGC3`) and MiSeq_SERT_1080_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGAAAAGAAGATTTGCGCGG3`). Primer pair (NT Library Fragment 2): MiSeq_SERT_191_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGATACACGGCACAGCATTC3`) and MiSeq_SERT_902_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATGTCTTTACTCCCTTCCAG3`). Primer pair (NT Library Fragment 3): MiSeq_SERT_426_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGTATCCCGCTCTTCTACATG3`) and MiSeq_SERT_698_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGTCCTCCTCGCTAATCAG3`). Primer pair (CTLibrary Fragment 1): MiSeq_SERT_1041_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGGCAGAAGCTTCTTGAAACC3`) and MiSeq_SERT_1592_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCAATGCCACGGTCAGAAC3`). Primer pair (CT Library Fragment 2): MiSeq_SERT_1250_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGCTGAGATGAGGAACGAAGA3`) and MiSeq_SERT_1808_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCCAGGATAATTGACCAG3`). Primer pair (CT Library Fragment 3): MiSeq_SERT_1376_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGATGCTGATAACCCTTGG3`) and MiSeq_pCEP4_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTATCATGTCTGGATCCGG3`). 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 with MiSeq v3 and HiSeq 2500 instruments using a 2x250 nt paired end protocol, though data is analyzed as single reads.