title: GSM3330050: small RNA from N2; Caenorhabditis elegans; ncRNA-Seq
library_construction_protocol: We used FLAG Immunoprecipitation kit (Sigma Catalog# FLAGIPT1) and followed the the manufacture's protocol to pull-down FLAG-StrepII-HRDE-1 and its bound siRNA.  Briefly, the frozen worm grind (5000-10000 adult animals, previously ground with mortar and pestle in liquid nitrogen) was first resuspended in 1ml of Lysis Buffer, 10ul of 100xHALT protease inhibitor (ThermoFisher), and 10ul of RNaseOUT (ThermoFisher).  The lysis were sonicated three times, 8 minutes each time, at 4°C using a Bioruptor (Diagenode) at high setting and 30 second on/off cycle.  Then the lysis were centrifuged at top speed for 4 minutes at 4°C.  The supernatant was mixed with ANTI-FLAG M2 affinity gel (washed and collected from 40ul resin provided by kit) and was rotated at 4°C overnight.  On the next day, the sample was centrifuged at 8,200xg for 30 seconds at 4°C and the supernatant was removed.  The resin was washed three times with 0.5ml of 1x Wash Buffer at 4°C.  FLAG-StrepII-HRDE-1 was eluted from resin with 15ug of 3xFLAG Peptide in 100ul of 1x Elution Buffer by rotating 30 minutes at 4°C. RNA was extracted from IP using TRIzol reagent. Small RNA-seq was performed using a 5'-monophosphate-independent small RNA cloning procedure described in Gent, J.I. et al. A Caenorhabditis elegans RNA-directed RNA polymerase in sperm development and endogenous RNA interference. Genetics 183, 1297–1314 (2009).