GSM9889986: LF-09F Stool sRNA-Seq; Homo sapiens; ncRNA-Seq
Stool samples were collected in a collection tube containing a nucleic acid stabilizing solution (Norgen Biotek) for subsequent nucleic acid isolation. The returned samples were stored at -80°C. RNA was extracted from 200 µL aliquots using the stool total RNA purification kit (Norgen Biotek Corp.) according to the manufacturer's protocol. RNA quality and quantity were verified in accordance with the MIQE guidelines (http://miqe.gene-quantification.info/). RNA concentration was quantified using the Qubit microRNA assay kit (Invitrogen) on a Qubit fluorimeter (Invitrogen). Small RNA-seq libraries were prepared using the NEBNext® Multiplex Small RNA Library Prep for Illumina® (New England Biolabs, Inc.) kit used to convert small RNA transcripts into barcoded cDNA libraries. For each library, 200 ng of RNA were processed as starting material. Each library was prepared with a unique indexed primer. Multiplex adapter ligations, reverse transcription primer hybridization, reverse transcription reaction and PCR amplification were performed according to the manufacturer's protocol. After PCR amplification, the cDNA constructs were purified with the QIAQuick® PCR Purification Kit (QIAGEN), following the modifications suggested by the NEBNext® Multiplex Small RNA Library Prep for Illumina® protocol. Final libraries were loaded on the Bioanalyzer® 2100 (Agilent Technologies) using the DNA High Sensitivity Kit (Agilent Technologies) according to the manufacturer's protocol. Libraries were pooled together (34-plex) and further purified with a gel size selection. A final Bioanalyzer® 2100 run with the High Sensitivity DNA Kit (Agilent Technologies) allowed the assessment of DNA libraries quality regarding size, purity, and concentration. The obtained libraries were subjected to the Illumina sequencing pipeline on Illumina NextSeq500 sequencers (75 cycles, Illumina Inc., USA).