title: GSM1182953: 8 weeks Unconventional prefoldin RPB5 interactor overexpression, replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: Freshly taken liver tissues were snap frozen in liquid nitrogen, lysed in 1.5 ml of TRIZOL solution using precellys homogenizer at 25,000 rpm for 30 s. The mixture was mixed vigorously by vortexing. 0.4 volume of chloroform was added, homogenised by hand for 15 s and incubated at room temperature for 5 min. Then, a centrifugation step for 15 min at 12,000 speed was performed and the upper aqueous phase was transferred into a fresh Eppendorf tube containing 60% volume of Trizol. The upper phase was collected and a second chloroform extraction step was performed. Subsequently, RNA was precipitated by adding 0.8 volumes of 100% isopropanol to the aqueous phase, mixed and incubated at room temperature for 10 min. The RNA-isopropanol mixture was centrifugated for 13.000 rpm for 20 min and total RNA was precipitated. The supernatant was decanted and the pellet was washed with freshly prepared 80% ethanol. 1.5 µg of total RNA samples was used as provided by the user. RNA Integrity Numbers were 8.3 on average (range 7.5 to 9.2) when assayed by Lab-chip technology on an Agilent 2100 Bioanalyzer. PolyA+ RNA fraction was extracted and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq RNA Sample Preparation Guide" (Part # 15008136 Rev. A). Adapter-ligated library was completed by limited-cycle PCR with Illumina PE primers (10 cycles). The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer's protocols.