GSM5902892: WT_A_812_sRNA_rep2_sequence; Drosophila melanogaster; miRNA-Seq
sRNA-seq libraries were constructed as described in the step-by-step protocol found at http://bartellab.wi.mit.edu/protocols.html, with some alterations. Briefly, between 1 and 5 ug of total RNA was mixed with size-selection markers (18- and 32-nt, 5′-radiolabeled RNAs) and 1 uL of quantitative standards (cel-lsy-6 and xtr-427) (ratios of total RNA:quantitative standards for each sample listed in Table S7). This mixture was size-selected on a denaturing gel, and then 2S rRNA was depleted by subtractive hybridization (Seitz et al., 2008). 3′ and 5′ adapters (each containing 4 random-sequence nucleotides at their ligation junctions) were then sequentially ligated using RNL2 truncated K227Q (NEB) and RNLI (NEB), respectively, in the presence of 10% polyethylene glycol (PEG 8000, NEB) and Superasin (Thermo Fischer Scientific), with gel purification following each ligation step. Purified RNA was reverse transcribed using SuperScript III (Thermo Fischer Scientific) and amplified using Kapa HiFi HotStart Ready Mix (Roche Diagnostics). Libraries were sequenced on an Illumina HiSeq platform with 50-nt single-end reads. RNA was extracted with TRI Reagent (Life Technologies), according to the manufacturer's protocol. Briefly, samples were homogenized in TRI reagent, and RNA was phase-separated by the addition of chloroform (J.T. Baker Analytical) at a ratio of 250 µL per 1 mL TRI Reagent. RNA was then precipitated with isopropanol, with GlycoBlue (Life Technologies) added as a co-precipitant when necessary, and pellets were washed twice with 70% ethanol prior to resuspension in water.