title: GSM2061462: T-REx cells with HBS1LV3 KD replicate B; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was isolated from HEK293 Flp-In T-REx cell lines using TRI Reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Total RNA was treated with Turbo-DNase (Life Technologies). Then, rRNA was removed from samples using a Ribo-Zero Kit (Epicentre), according to the manufacturer’s protocols with minor modifications. To control for experimental performance, external RNA (ERCC RNA Spike-In Mix; Life Technologies) was added to the samples. RNA libraries were prepared using a TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s manual, with the following changes: i) the RNA sequencing sample was obtained from total RNA, ii) SuperScript III Reverse Transcriptase was used instead of SuperScript II Reverse Transcriptase (Life Technologies) for first strand cDNA synthesis, iii) an alternative master mix that contained dUTP was added to the sample to synthesize the second strand, and dUTPs were incorporated into the emerging second strand to allow for the subsequent digestion of this strand and to obtain strand-specific libraries, iv) before PCR amplification of double stranded cDNA, the second strand was digested with 0.04 U/μl USER enzyme (Uracil-Specific Excision Reagent; BioLabs) for 30 min at 37°C; and v) a PCR amplification alternative mix was used.
identifiers.namespace: GEO
identifiers.id: GSM2061462