Similar protocols

Pipeline publication

[…] ocedures were carried in accordance with national and international laws and policies., RNAs from the THP-1 cell line with stable expression of sPRDM16-WT or sPRDM16–K568R or a mock-transfected cell line were purified using TrizolTM method and subsequently cleaned using RNAeasy Kit (Qiagen). The polyadenylated RNAs purified from the cells were used for the construction of a sequencing library using the ScriptSeq Complete Gold Kit (Epicentre, Illumina). Cluster generation and sequencing were carried out using standard procedures in Hi-Seq 2500 Illumina platform. We used a single-end sequencing protocol to generate a 50 nt read at each end. RNA-seq reads were aligned to the human genome using TopHat (Johns Hopkins University, Baltimore, MD, USA). Cufflinks was employed to normalize Data and perform relevant comparisons among the different samples. Gene ontology analysis was performed using DAVID GO analysis software to search for enriched pathways., Total RNA was extracted by Trizol kit (Invitrogen) and treated with DNase (Promega). Complementary DNA was reverse transcribed using M-MLV reverse transcriptase and random hexamers according to the manufacturer’s protocol (Takara). All experiments were performed with Power SYBR® Green PCR Master Mix (Applied Biosystems) using the LightCycler® 480 Real-Time PCR System (Roche). PCR was carried out in triplicate and standard deviations representing experimental errors were calculated. Differences in cDNA input were normalized to GAPDH. All data were analyzed by the LightCycler® 480 software (Roche). The following PCR primers were used […]

Pipeline specifications

Software tools TopHat, Cufflinks, DAVID