GSM2837156: SubAcute_1_CA1; Equus caballus; miRNA-Seq
Isolation of RNA from tissue was performed using RNeasy Mini Kit (Qiagen, Gaithersburg, MD, USA), per manufacturer's instructions.  After extraction, RNA was analyzed by NanoDrop® (Thermo Fisher Scientific) and Bioanalyzer® (Agilent, Santa Clara, CA, USA) to evaluate concentration, purity and integrity.  All samples had a 230/260 ratio > 1.8, a 260/280 ratio > 2.0 and an RNA integrity number > 8.0.  Serum RNA was isolated using miRNeasy Micro Kit (Qiagen) per the manufacturer's instructions for cell culture, with chloroform increased to 300 mL and the optional RWT wash included.  Bioanalyzer® analysis was performed to ensure there was no cellular RNA contamination. Library preparation was performed using a modified small RNA sequencing library construction protocol 72. In brief, the in-house protocol used adapters with four degenerate bases at each ligation site.  Modified adapter sequences are based on Illumina adapter sequences and are listed in Supplementary Table 6.  The adapters were purchased from Integrated DNA Technologies (Coralville, IA, USA).  The 3' adapter ligation reaction includes 5 ml of RNA, 1 ml of 3' adapter at 10 nM, 1 ml of RNase inhibitor (Thermo Fisher Scientific), 1 ml of modified T4 RNA Ligase 2 (NEB, Ipswich, MA, USA) and 1 ml of 10 X ligase buffer.  The ligation was performed for 1 hour at 25oC, and was facilitated by the addition of 15% PEG 8000.  Before adding the 5' adapter, 1 µL of single-strand binding protein (1µg/µl) (Promega, Madison, WI, USA), 1 uL of 5'-deadenylase (NEB) and 1uL of RecJ (NEB) were added, mixed and incubated at 37oC for 1 hour to remove adapter dimers.  For 5' adapter ligation, 1 µl of 5' adapter at 25 µM, 1 µL of T4 RNA Ligase (NEB, Ipswich MA), and 1 µL of 10 mM ATP were added then incubated for 1 hour at 25 oC. Complementary DNA (cDNA) was generated from ligated product with SuperScript III reverse transcriptase (Thermo Fisher Scientific) using primer complimentary to the 3' adapter sequence.  The resulting cDNA was amplified with Illumina universal primer and indexed-primer.  Library size-selection was performed using a PippinHT instrument (Sage Science, Boston, MA, USA) with either a 3% agarose cassette or with 6% polyacrylamide gel. The library with proper insert size was sequenced with the NextSeq 500 (Illumina, San Diego, CA).
GEO Accession
GSM2837156