GSM1567923: Intestine_10wkA; Homo sapiens; RNA-Seq
Fetal tissue samples <100 mg in size were placed into DNase- and RNase-free 1.5 ml microfuge tubes containing 1 ml of RNAlater RNA Stabilization Reagent (Qiagen) within 1 hour of the pregnancy termination procedure. After storage at room temperature for a period of 24-72 h, excess RNAlater was removed from the microfuge tubes and the samples were placed in the -80oC freezer for storage until RNA isolation was performed. The tissues were lysed in mirVana (Life Technologies) lysis buffer, using a Mini-Beadbeater-16 (Biospec), with agitation for 1 min in the presence of 1 mm zirconia beads. Samples were then centrifuged at maximum speed in a tabletop microcentrifuge for 1 min and the lysed solution was transferred to a fresh microfuge tube. The remainder of the extraction was per the manufacturer's protocol for the mirVana kit (Life Technologies). Following RNA isolation (mirVana miRNA Isolation Kit, Life Technologies, Inc.), the RNA was quantified (Qubit RNA Assay Kit, Life Technologies, Inc.), and quality controlled (RNA6000 Nano Kit and BioAnalyzer 2100, Agilent). 200 ng to 1000 ng was used as input for the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold sample prep kit (Illumina, Inc.) and sequencing libraries were created according to the manufacturer’s protocol. Briefly, first both cytoplasmic and mitochondrial rRNA was removed by selectively hybridizing biotinylated probes to target sequences and using magnetic beads to capture the bound products. Following rRNA removal, the RNA was fragmented and copied into first strand cDNA using random primers and reverse transcriptase. Next, second strand cDNA synthesis was completed using DNA Polymerase I and RNase H. The cDNA was then ligated to Illumina supplied adapters and enriched with PCR to create the final cDNA libraries. The libraries were then pooled and sequenced on a HiSeq 2000 (Illumina, Inc.) instrument as per manufacturer’s instructions. Sequencing was performed up to 2 X 101 cycles.