title: GSM1466688: eat-2(ad1116) Day 1; Caenorhabditis elegans; miRNA-Seq
library_construction_protocol: RNA was isolated using Trizol (Invitrogen, USA).  Briefly, worms grown on OP50 bacteria,  were washed off the plates with M9 buffer. Then, 0.4 ml of Trizol reagent was added and the worms lysed by vigorous vortexing. RNA was purified by phenol:chloroform:isoamylalcohol extraction and ethanol precipitation. RNA integrity were confirmed by analysis on an Agilent 2100 bioanalyzer, using the RNA 6000 nano kit (Agilent) to conform RINs above 8.0. Alternatively, the quality of the ribosomal 28 S and 18 S on an agarose gel was used as a measure of integrity and the absorbance at 260/280 nm was used to determine quantity. The small RNAs libraries were constructed using  Small RNA v1.5 sample preparation kit  according to the manufacturer’s instructions (Illumina, inc. ,USA.)  Briefly  the total RNA (5µg) were ligated  3' adapter using T4 RNA ligase 2 truncated (NEB)and a 5' adapter using   T4 RNA ligase 2. The ligation products were reverse transcribed using Superscript II Reverse Transcriptase (Life Technologies) and amplified with  PCR (12 Cycle). The PCR  products were purified with electrophoresis of 6% Novex TBE PAGE Gel. The purified cDNA library for each sample was analyzed on Agilent 2100 Bioanalyzer using Agilent DNA high sensitivity kit. Deep sequencing of cDNA libraries were performed with the help of Illumina Genome analyzer IIX for 36 cycles.