GSM2544678: 26_335_CTGG_0N_E; Caenorhabditis elegans; OTHER
Synchronized Embryos or L4 larval worms were washed several times to avoid contamination with bacteria, snap-freezed at liquid nitrogen, and grinded to powder in a liquid nitrogen -prechilled mortar and pestle. High-molecular weight and Low-molecular weight RNA fractions were isolated using miRVana miRNA isolation kit Applied Biosystems (Ambion). The protocol includes ligation of a total pool of small RNAs first to 3’ adapter pre-adenylated 3’ adapter without ATP, followed by ligation to a 5’-adapter containing 4-nt RNA barcode to allow multiplexing and 8 random nucleotide UMI, reverse transcription and amplification using primers complementary to the 5’ and 3’ adapters, baring Illumina barcodes. 3 size-selection steps are performed, separation of small RNA (20-30b) from longer RNA species and separation, after each one of the two ligations, of small RNA ligated to adapter(s) from free adapter/adapter dimer. All the separations performed on SPRI beads using a proprietary combination of two crowding agents, PEG and Isopropanol. The resulting Libraries were sequenced using 50 bp SR sequencing mode on the HiSeq 2500 platform (Illumina).