title: GSM1859886: Heart_C57BL/6J_spliceJunctions_CaptureSeq; Mus musculus; RNA-Seq
library_construction_protocol: Mouse tissue samples were obtained from adult C57BL/6J mice (AEC Approval Number IMB/030/08/ARC). Mouse brain samples were dissected in ice cold PBS, snap frozen in liquid nitrogen and RNA extracted with TRIzol® (Life Technologies). Non-brain organs were removed and placed in ice cold PBS, where any contaminating tissue was removed and the required organ cut into pieces <30mg and placed in RNAlater® (QIAGEN). RNA was extracted using the TissueRuptor® and RNeasy® Mini purification kit (QIAGEN). Purity of all mouse samples was validated by Nanodrop (Thermo scientific). RNA was DNase treated with Turbo™ DNase (Life Technologies), purified and confirmed DNA-free by PCR for gDNA as previously described (Mercer et al. 2014 ,PMID:24705597). Intact RNA was confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies). Ribo-Zero™ (Epicentre) rRNA depletion was performed on 10 μg of each sample. For non-brain samples 5μg of purified RNA from 2 organ pieces from the same mouse was ribodepleted and then pooled to ensure good coverage across the tissue. Efficient ribodepletion was confirmed by Bioanalyzer (Agilent 2100) Pico chip. Capture sequencing was performed as previously described (Mercer et al. 2015, PMID:25561518) combining the NimbleGen SeqCap EZ Library SR User’s Guide V3.0 and the NimbleGen Arrays User’s Guide: Sequence Capture Array Delivery v3.2 with the following specifications. RNA sequencing libraries were created using the TruSeq® Stranded mRNA Sample Preparation Kit (Illumina). All samples contained ERCC spike-in mix no1. or no2 at a 1/100 dilution. Test libraries to estimate yield were amplified for 15 cycles as per “Enrich DNA fragments” protocol. Ten cycles of precapture LMPCR was performed for all samples except brain and liver (11 cycles). Capture hybridization were performed as a 4-plex with 250ng of brain or non-brain libraries pooled together. Post-LMPCR was performed for 17 (non-brain samples) or 16 (brain samples) cycles respectively.
attributes.tag: GEO Accession
attributes.value: GSM1859886