GSM8973554: prg-1 (adult) - 25°C, replicate 3; Caenorhabditis elegans; RNA-Seq
For immunoprecipitation, ~100,000 synchronized 1-day-old adult animals were collected in IP Buffer (50 mM Tris-Cl pH 7.4, 100 mM KCl, 2.5 mM MgCl2, 0.1% Igapal CA-630, 0.5 mM PMSF, cOmplete Protease Inhibitor Cocktail, and RNaseOUT Ribonuclease Inhibitor), frozen in liquid nitrogen, and homogenized using a mortar and pestle. After further dilution into IP buffer (1:10 packed worms:buffer), insoluble particulate was removed by centrifugation and a sample was taken as “input.”  The remaining lysate was used for the immunoprecipitation. Immunoprecipitation was performed at 4°C for 1 hour, then washed at least 3 times in immunoprecipitation buffer. Trizol reagent was added to the remainder of each sample, followed by chloroform extraction, and isopropanol precipitation. Small RNAs (18 to 30-nt) were size selected on denaturing 15% polyacrylamide gels from total RNA samples. Small RNAs were treated with 5' RNA polyphosphatase and ligated to 3' pre-adenylated adapter with Truncated T4 RNA ligase. Small RNAs were then hybridized to the TruSeq reverse transcription primer, ligated to the 5' adapter with T4 RNA ligase), and reverse transcribed with Superscript III. Small RNA libraries were amplified using Q5 High-Fidelity DNA polymerase and size selected on a 10% polyacrylamide gel. Library concentration was determined using the Qubit 1X dsDNA HS Assay kit and quality was assessed using the Agilent BioAnalyzer.