GSM4676951: WAGO-10 Input rep2; Caenorhabditis elegans; RIP-Seq
For samples in which only RNA was isolated: Typical total RNA extraction and ethanol precipitation were performed, followed by quantification using a Nanodrop 1800C spectrophotometer. For samples in which Argonaute/sRNA complexes were isolated: Protein lysates were prepared by dounce homogenization of worm samples at 4°C, lysates were cleared by 10min 13,000xg centrifugation. Input (total small RNA) samples were taken first and total RNA was extracted using a typical Tri-Reagent protocol, followed by ethanol precipitation. IPs were performed with anti-GFP (Chromotek) or anti-CSR-1a (rabbit polyclonal). In the final step, beads with Argonaute-sRNA complexes were resuspended in Tri-Reagent, followed by RNA extraction as for other samples. For sRNA libraries , RNA samples were treated with a 5' polyphosphatase prior to use in library prep with the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs) per manufactuer instructions. For mRNA libraries, samples were DNAse treated prior to use with the NEBNext Ultra II Directional RNA Prep Kit for Illumina - with Poly-A selection per manufacterer instructions.
GEO Accession
GSM4676951