alias: GSM3376168
title: GSM3376168: P23-1_S10; Mus musculus; RNA-Seq
library_construction_protocol: RNA from tissue samples is extracted using Precellys soft-tissue beads and TriZol. First, TriZol is pipetted on the frozen tissue sample. The tissue is transferred in TriZol to Precellys soft-tissue bead tube and homogenized in Precellys 24 Lysis & Homogenization (Bertil Technologies) machine. Chloroform is added to the homogenized sample and after the upper aqueous phase and lower organic phase have separated, the aqueous phase containing RNA is collected. The RNA is then precipitated using isopropanol and washed using 75 % ethanol. The formed RNA pellet is resuspended in RNase free water after which the RNA is purified using RNeasy® Mini Kit (Qiagen). SureSelect Strand-Specific RNA Library Prep Kit is used for RNA-sequencing library preparation for next generation sequencing. Before the cDNA synthesis the total RNA is treated with Ribo-Zero treatment that removes the ribosomal RNA from the total RNA (1µg). The ribosomal depleted RNA is purified with RNeasy mini Elute columns (Qiagen) and fragmented to generate inserts around 300bp. The first-strand cDNA is synthesized and purified after which the second strand cDNA is synthesized and end repaired in one reaction. The created double-stranded cDNA is purified with AMPure XP beads. The double-stranded cDNA's 3'end is adenylated and adaptors are ligated. After AMPure XP beads purification adaptor-ligated cDNA library is amplified and purified with AMPure XP beads
identifiers.namespace: GEO
identifiers.id: GSM3376168