GSM2476033: Input; Caenorhabditis elegans; RIP-Seq
EMB-4 RNA IPs (RIPs), were performed on extracts from gravid adult N2 worms, using 50 μl anti-EMB-4 (7K3-12, 80ng/μl). To preserve RNA integrity, 1:100 Superase-In was added to DROSO Complete buffer prior to lysate preparation, as well as to any additional DROSO complete buffer added to each IP. 4x5 mg IPs were set up for each sample (anti-EMB-4 and no antibody control). After pre-clearing, incubation with antibody and collection of immunocomplexes, beads were washed 5x5 min and all four IPs were pooled for each sample prior to the last wash. 10% of the beads were saved for IP validation by Western Blot. The remaining 90% were resuspended in 4x volume of TriReagent and RNA extraction was performed. The extracted RNA was precipitated overnight at -80°C. The Qubit HS RNA kit (Invitrogen) was used to quantify the extracted RNA and 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. The resulting PCR product DNA was quantified using the Qubit HS DNA kit (Invitrogen) and all samples were pooled in equal amounts into a 20nM solution and sequenced. 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
GSM2476033