title: GSM2476038: poly(A) mRNA WT rep1; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: 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 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 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). Samples were quantified by the Qubit HS DNA kit (Invitrogen), pooled 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.
attributes.tag: GEO Accession
attributes.value: GSM2476038