title: GSM9059309: Ipsilateral primary cortex from mouse 2; Mus musculus; ncRNA-Seq
library_name: GSM9059309
library_construction_protocol: RNA was extracted from fresh tissue. Tissue for RNA extraction was isolated from V1 contralateral and ipsilateral to the sutured eye after 3-day MD (at P28). A sterile scalpel blade and forceps were used to surgically micro-dissect the appropriate tissue section. The dissected tissue was immediately placed into a 2-mL lysing tube containing 1.4 mm spherical ceramic beads (MPBio, Lysing Matrix D, Cat. No. 116913050) and pre-filled with 1 mL of ice-cold TRIzol (Ambion, Cat. No. 15596-026) and then immediately homogenized with a bead-based homogenizer (FastPrep-24 5G, MPBio) using the preset settings for mouse brain tissue (8.0 m/sec, 30 sec). Once foaming has subsided at room temperature, the homogenate was transferred into 2 mL Phase Lock Tube (Heavy; 5Prime Bio, Cat. No. 2302830) pre-filled with 200 µL chloroform and briefly vortexed. Samples were centrifuged at 12,000 x g for 10 min at 2-4°C. The colorless upper aqueous layer was then transferred into a new 2.0 mL LoBind tube that was pre-filled with 1.5 volumes of 100% EtOH (~600 µL), then mixed thoroughly by pipetting up and down several times, and then briefly centrifuged. The sample was furthered purified using Zymo RNA Clean & Concentrator-5 (Zymo Research, R1016) according the manufacturer's recommended protocol and always performed with the optional in-column DNase I treatment. RNA was eluted twice with ≥6 μL 1x TE buffer (pH 7.5). The approximate concentration and relative purity of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Indexed cDNA libraries were generated using the SMARTer Stranded Total RNA-Seq Kit v2 according to the manufacturer's protocol.