GSM2476035: small RNA WT rep2; Caenorhabditis elegans; ncRNA-Seq
TriReagent was used to extract total RNA, followed by ethanol precipitation. Quantification and crude assessment of RNA quality was performed using Qubit HS RNA kit (Invitrogen). For small RNA library preparation, 1 μg of total RNA was used following the Truseq Small RNA Library Prep Kit (Illumina) protocol. The resulting PCR product DNA was size selected (PAGE 8% gel, 150bp), eluted overnight (10 mM Tris-Cl pH7.5, 1 mM EDTA, 0.3 M NaCl), and then precipitated with 20μg glycogen and 1 volume of isopropanol. The recovered cDNA was resuspended in 30μl ultrapure water and quantified using Qubit HS DNA kit (Invitrogen). All samples were pooled in equal amounts into a 20nM solution and sent for sequencing. For small RNA, poly(A) mRNA, and ribozero mRNA, worms were frozen in 3-5 volumes of Tri-Reagent (MRC), broken apart using a Wheaton Dounce Homogenizer, vortexed for 10 minutes at RT, then 10% BCP was added, vortexed for an additional 5 min at RT, then centrifuged at 13kXg for 5 minutes. The supernatant was transferred to a new tube, and precipitated with Isopropanol. RNA quality and concentration were assessed using Nanodrop and Qubit quantification. 1 μg of total RNA was used for small RNA library preparation using the Truseq Small RNA Library Prep Kit (Illumina), following the protocol provided by the manufacturer. For mRNA library preparation, 4 μg of total RNA were treated with DNase I (Invitrogen), ethanol precipitated and quantified with the Qubit HS RNA kit (Invitrogen). 1 μg of DNase treated RNA was used for each of rRNA depletion (Ribo-Zero Gold rRNA Removal Kit, Illumina) and Poly-A selection (Truseq Stranded mRNA Sample Preparation Kit). Library preparation was executed as described by the Truseq Stranded mRNA Sample Preparation Kit manual (Illumina). For RIPs, up to 2 μg of each sample were treated with DNase I (Invitrogen). DNase-treated RNA was then phenol-chloroform extracted, ethanol precipitated and re-quantified using Qubit HS RNA kit (Invitrogen), as well as examined for quality by BioAnalyzer (Agilent). 600 μg of DNase-treated RNA were used for subsequent sample preparation. Ribosomal RNAs were removed from each sample using Ribo-Zero Gold rRNA Removal Kit (Illumina), as per the manufacturer’s protocol. The remaining RNA was ethanol precipitated and prepared for sequencing using Truseq Stranded mRNA Sample Preparation Kit (Illumina), following the protocol provided by the manufacturer.
GEO Accession
GSM2476035