title: GSM1939608: LG+GNS; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted using the Rneasy Plus Minikit (Qiagen) as outlined by manufacturer. RNA samples with a RNA integrity Number (RIN) value of at least 9.5 or higher was used for further processing. From each 3 samples, 500ng RNA was used for PolyA mRNA library prep using NEBNext PolyA magnetic Isolation module New England BioLabs Inc., Ipswich, MA, USA) according to manufacturer's protocol. Samples were individually barcoded with unique in-house genomics service lab (GSL) primers and amplified through 10 cycles of PCR using KAPA HiFi HotStart Ready Mix (Kapa Biosystems, Inc., Woburn, MA, USA). The quality of the libraries were assessed by Qubit® 2.0 Fluorometer , and the concentration of the libraries was estimated by utilizing a DNA 1000 chip on an Agilent 2100 Bioanalyzer, respectively. Accurate quantification of the prepared mRNA libraries for downstream sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa Biosystems, Inc., Woburn, MA, USA). Each library was then diluted to a final concentration of 12.5nM and pooled equimolar prior to clustering. Cluster generation was carried out on a cBot v1.4.36.0 using the Truseq Paired-end (PE) Cluster Kit v3.0 (Illumina, Inc., San Diego, CA, USA). Paired End (PE) sequencing was performed using a 200 cycle TruSeq SBS HS v3 kit on an Illumina HiSeq2000, running HiSeq Control Software (HCS) v1.5.15.1 (Illumina, Inc., San Diego, CA, USA). Image analysis and base calling was performed using the standard Illumina Pipeline consisting of Real time Analysis (RTA) version v1.13. Raw reads were demultiplexed using a bcl2fastq conversion software v1.8.3 (Illumina, Inc., San Diego, CA, USA) with default settings.
identifiers.namespace: GEO
identifiers.id: GSM1939608