title: GSM5915157: Chondrocytes miR-26a KO; Mus musculus; RNA-Seq
library_construction_protocol: Cultured chondrocytes were lysed using TRIZOL reagents. 1) Add 1 mL of TRIZOL to each tube and lyse the cells by pipetting. 2) Leave the tube at room temperature for 5 minutes. 3) Transfer the sample to a 1.5 mL tube and leave it at room temperature for 5 minutes. 4) Add 200 μL chloroform, stir by vortexing, and leave it at room temperature for 3 minutes. 5) Centrifuge at 12,000 g for 10 minutes at 4°C. 6) Transfer the aqueous layer to a new tube; (6) Collect the aqueous layer in a new tube and add 1 µL of 20 mg/mL Glycogen; (7) Add 500 µL 2-propanol, stir, and leave at room temperature for 10 minutes; (8) Centrifuge at 12,000g, 4°C, for 10 minutes (9) Discard the supernatant and add 1 mL of 75% ethanol; (10) Centrifuge at 7,500g, 4°C, for 5 minutes; (11) Discard the supernatant and leave the pellet to dry at room temperature for 5 minutes (12) Suspend in 10 μL of nuclease free water.RNA quality check using Agilent 2100 Bioanalyzer G2939A (Agilent Technologies) and Agilent RNA6000 Nano kit 5067-1511 (Agilent Technologies) QuantSeq 3'mRNA-Seq Library Prep Kit for Illumina (FWD) <015.384> (LEXOGEN) was used. (1) Total of 500 ng RNA was used for each sample; (2) Synthesize cDNA using oligo (dT) primers; (3) Remove template mRNA; (4) Double-stranded cDNA synthesis using random primers; (5) 12-cycle PCR using indexed adapters; (6) Purification using the beads;