title: GSM3215457: s_PIMC_Rep1; Homo sapiens; RNA-Seq
library_construction_protocol: The first passage PIMC from two stable and two vulnerable atheromes were washed with PBS twice and detached from Petri dishes using 0.2 % collagenase type II in IMDM, centrifuged 10 min at 400 g, then 750 µL of Trizol reagent was added to the pellet and frozen at  80°C until RNA extraction. PIMCs in Trizol were thawed, mixed with 0.15 mL chloroform, vortexed for 15 s, and incubated at ambient temperature for 2 min. Thereafter, samples were centrifuged at 12,000g for 15 min at 4°C. Aqueous phase was transferred to a fresh tube and precipitated after adding 20 µg glycogen and 375 µL isopropanol. RNA samples were treated with RNase-free DNase I (Fermentas, Vilnius, Lithuania) and precipitated in 70 % ethanol, 0,3 М NaAc, 20 µg glycogen. The concentration of extracted RNA and RNA integrity number (RIN) and were tested using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA) and Agilent RNA 6000 Pico Kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The libraries were prepared for the sequencing using Illumina TruSeq Stranded mRNA Sample Preparation Kit and were sequenced with the HiSeq 2500.
attributes.tag: GEO Accession
attributes.value: GSM3215457