alias: GSM4546416
title: GSM4546416: Randomly_synthesized_oligos_rep2; Homo sapiens; OTHER
library_construction_protocol: Total RNAs from sorted cells are extracted using Trizol reagent (Invitrogen). Purified total RNAs are suspended in nuclease-free water for reverse transcription.In brief, RNA samples are mixed with 1 μl 10 mM dNTP and 2 pmol reverse primer overlap with 3'end of GFP (oligo 2) and incubated at 65 °C for 5 min, followed by incubation on ice for 5 min. The reverse transcription is carried out by incubating the reaction mixture with 1 × First-Strand Buffer, 10 mM DTT, 40 U RNaseOUT and 200 U SuperScript III at 50 °C for 60 min followed by heating at 70°C for 15minutes. First-strand cDNA is then used as the template for PCR amplification catalyzed by the Phusion High-Fidelity enzyme (NEB). The PCR is performed in a 20 μL reaction (1× HF buffer, 0.2 mM dNTP, 0.5 μM forward and reverse primers and 0.5 U Phusion polymerase) with barcoded primers (Table S1). PCR is set up based on the following condit 110 ion: 16 cycles of 98 °C, 10 s; 60 °C, 20 s; 72 °C, 10 s followed by 72 °C, 10 min. The PCR products with the expected size 163 base pairs are excised from a 8% polyacrylamide TBE gel (Invitrogen). The DNA products are recovered from DNA gel elution buffer (300 mM NaCl, 1 mM EDTA) followed by quantification using Agilent BioAnalyzer DNA 1000 assay. Equal amounts of barcoded samples were pooled together followed by deep sequencing (Illumina HiSeq).
identifiers.namespace: GEO
identifiers.id: GSM4546416