title: Illumina HiSeq 2500 sequencing; microRNA profiling of female and male mouse pituitary gland across pubertal transition
design: microRNA profiling of female and male mouse pituitary gland across pubertal transition
library_name: Pd22M_2_s
library_construction_protocol: Male and female mice were euthanized using automated Euthanex Carbon Dioxide (CO2) lids followed by decapitation between 9:30 am and 11:30 am. Pituitary glands were dissected on postnatal days 12, 22, 27, 32. Collected tissues were directly placed in RNAlater (containing 10% w/v sodium citrate tribasic dihydrate and 60% w/v ammonium sulphate following dissection, and stored at −20 °C until RNA extraction. Total RNA was extracted from mouse pituitary gland with the NucleoSpin® miRNA kit (Macherey Nagel) in combination with TRIzol lysis (Invitrogen) following manufacturers' protocols, allowing for collection of small RNA (< 200 nt) and large RNA (> 200 nt) simultaneously into separate tubes from total RNA. Prior to RNA extraction, tissue samples were placed into bead mill tubes containing six 1.4 mm ceramic beads (MoBio Laboratories), and homogenized for 30 s at 6.5 m/s at 4 °C using Omni Bead Ruptor 24 bead mill. RNA quantity was determined using Nanodrop and the quality was assessed by Agilent 2100 Bioanalyzer. Small RNA-seq libraries were constructed from RNA extracted into the small RNA fraction using NEBNext Small RNA Library Prep Kit (New England BioLabs) according to manufacturer's protocol. Libraries were size selected using Ampure (Beckman Coulter) beads.