title: GSM9305562: miRNA-Seq, Zeb1_200M_pituitary_4_youngCohort; Mus musculus; miRNA-Seq
library_construction_protocol: Total RNA was extracted from mouse tissues using TRI Reagent (ThermoFisher) according to the manufacturer's protocol with the following modifications. Mouse tissues were rapidly dissected after CO2-based euthanasia and flash frozen in Eppendorf tubes in liquid N2. Tissue was transferred to a 15 mL conical tube, 1–2 mL of TRI Reagent was added, and the tissue was homogenized with a TissueRuptor (Qiagen) and disposable probes. Samples were transferred to Eppendorf tubes (1 mL per tube) and phase separated with 200 μL chloroform (J.T. Baker Analytical). After isopropanol precipitation and two 70% ethanol washes, total RNA was resuspended in water. Each small-RNA sequencing libraries was prepared with 3 ug of total RNA extracted from mouse pituitary. In addition, 0.5 fmoles of miR-427-5p (X. tropicalis) and 0.5 fmoles of lsy-6-3p (C. elegans) were added as spike-ins. From each sample, RNAs that co-migrated within the range of 18 and 32 nt radiolabeled internal standards were isolated on a 15% polyacrylamide urea gel. Purified RNA was ligated to a preadenylated 3' adapter (AppNNNNCTGTCTCTTATACACATCTCCGAGCddC) using T4 RNA Ligase 2 KQ mutant (NEB) in a reaction supplemented with 10% polyethylene glycol (PEG 8000, NEB). To reduce ligation biases, this 3' adapter had 4 random-sequence positions at its 5' end. After gel purification on a 8% polyacrylamide urea gel, RNA was ligated to a 5' adapter (CCUACACGACGCUCUUCCGAUCUNNNN) using T4 RNA Ligase I (NEB) in a reaction supplemented with 10% PEG. To reduce ligation biases, this adapter had 4 random-sequence positions at its 3' end. After gel purification on an 8% polyacrylamide urea gel, RNA was reverse transcribed with SuperScript III (Invitrogen), and the cDNA was amplified ~12 cycles with KAPA HiFi DNA polymerase (Kapa Biosystems). Amplified DNA was purified using 1.8X AmpureXP (Beckman Coulter) bead cleanup, followed by agarose gel on the PippinHT. A step-by-step protocol for constructing libraries for small-RNA sequencing is available at http://bartellab.wi.mit.edu/protocols.html