title: GSM3985125: JC12; Rattus norvegicus; RNA-Seq
library_construction_protocol: After defined survival times, animals were killed by terminal anesthetization and transcardially perfused with 4% paraformaldehyde, for immunohistochemistry or ice-cold PBS, for mRNA extraction and Western blotting. The lumbar spinal cords were excised, rapidly removed and the tissue corresponding to L5 spinal cord was isolated.  For mRNA extraction and Western blotting, L5 spinal cords were bi-dissected into ipsi and contralateral side to the injury, and tissue was snap frozen in liquid nitrogen and kept at -80 degrees Celcius until further use. Frozen micro dissected spinal cord samples were lysed with Teflon-glass homogenizers and  QIAshredder columns (Qiagen). Total RNA was isolated with the Qiagen RNeasy® Mini kit.  The RNA was assessed for purity and quantity using the Nanodrop 1000 spectrophotometer  and assessed for quality on the Agilent Bioanalyzer 2100. 400 ng of intact high-quality total RNA (RIN>7.9) from each sample was then used  as input to generate libraries for RNA-sequencing using the NEBNext Ultra II Directional kit  (NEB, Cat.no: E7760S) following the manufacturer's recommendations. This protocol involved an initial step of mRNA selection using a poly-A isolation module (NEB, Cat.no:  E7490) to select for mRNA with a mature polyA tail, followed by fragmentation prior to first  cDNA synthesis and barcoding second-strand cDNA synthesised with indices for Illumina  sequencing for final library amplification (10-cycles). The resulting libraries (342-421bp) were assessed on the Bioanalyzer 2100 for purity. The NEBNext Library Quant Kit for Illumina (NEB, Cat.no: E7630L) was used to calculate the quantity of each library. The quantification data was used to pool the libraries in equal molarity prior to performing a QC run on the MiSeq (MiSeq Reagent Kit v3 (150-cycle); Cat no: MS-102-3001). Further deep sequencing was performed on the pooled library over 2 lanes using a HiSeq4000 (by GENEWIZ) to generate roughly 26 million reads per sample.
identifiers.namespace: GEO
identifiers.id: GSM3985125