title: sRNA-seq of Caenorhabditis elegans: adult hermaphrodite
design: Total RNA was extracted from whole animals of ~100,000 synchronized young adults. Small (<200nt) RNAs were enriched with mirVana miRNA Isolation Kit (Ambion). In brief, 80 L (200-300 g) of total RNA, 400 l of mirVana lysis/binding buffer and 48 L of mirVana homogenate buffer were mixed well and incubated at room temperature for 5 minutes. Then 176 L of 100% ethanol was added and samples spun at 2500 x g for 4 minutes at room temperature to pellet large (>200nt) RNAs. The supernatant was transferred to a new tube and small (<200nt) RNAs were precipitated with pre-cooled isopropanol at -70C. Small RNAs were pelleted at 20,000 x g at 4C for 30 minutes, washed once with 70% pre-cooled ethanol, and dissolved with nuclease-free water. 10 g of small RNA was fractionated on a 15% PAGE/7M urea gel, and RNA from 17 nt to 40 nt was excised from the gel. RNA was extracted by soaking the gel in 2 gel volumes of NaCl TE buffer (0.3 M NaCl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) overnight. The supernatant was collected through a gel filtration column. RNA was precipitated with isopropanol, washed once with 70% ethanol, and resuspended with 15 L nuclease-free water. RNA samples were treated with RppH to convert 22G-RNA 5 triphosphate to monophosphate in 1x reaction buffer, 10U RppH (New England Biolabs), and 20U SUPERase-In RNase Inhibitor (Invitrogen) for 3 hours at 37C, followed by 5 minutes at 65C to inactivate RppH. RNA was then concentrated with the RNA Clean and Concentrator-5 Kit (Zymo Research). Small RNA libraries were prepared according to the manufacturers protocol of the NEBNext Multiplex Small RNA Sample Prep Set for Illumina-Library Preparation (New England Biolabs). NEBNext Multiplex Oligos for Illumina Index Primers were used for library preparation (New England Biolabs). Libraries were sequenced using an Illumina HiSeq4000 at the University of Chicago Genomic Facility.