GSM4257259: ko_vehicle_heavy_rnaseq; Mus musculus; RNA-Seq
Frozen cortical-hippocampal slices were thawed in ice-cold homogenization buffer on ice for 5min. Wide orifice tips were used to transfer slices to a pre-chilled detergent-free Dounce homogenizer. Tissues were gently homogenized by hand (3 strokes of loose pestle A, and 7 strokes of tight pestle B). Homogenates were carefully transferred to clean 1.5ml tubes with clean glass Pasteur pipets and bulbs. Homogenates were clarified by centrifugation at 2,000g 4 °C for 10min. The supernatants were collected and clarified again by centrifugation at 10,000g 4 °C for 10min. The supernatants were collected, and the amounts of nucleic acid were measured with Nanodrop (A260 units). 1% NP-40 was added to the homogenates and incubated on ice for 10min. ~1A260 unit of lysate was saved for the input RNA. ~5 A260 units of lysate were applied to 15%-45% (w/w) sucrose gradients prepared in 1X polysome buffer and with the lower block mark and long cap. After the ultracentrifugation in a SW41Ti rotor (Beckman Coulter) at 35,000 rpm (avg 151,263g) 4°C for 2 hours, gradients were fractionated at 1.5 ml/min and 20 sec collection intervals through a fractionation system (Brandel) that continually monitored A260 values. Some gradients were treated with RNase A prior to centrifugation. Based on the FMRP signals in the immunoblotting of gradient fractions, medium and heavy fractions were identified, pooled, and extracted with TRIzol LS together with saved input lysates. RNA samples were quantified with Qubit HS RNA assay kit and the integrity was examined with Fragment Analyzer. 200ng RNA was used for BIOO Rapid directional qRNA-seq library preparation following the manufacturer's instructions. 12 cycles were used for the final PCR amplification. The libraries were quantified with KAPA Library Quantification Kit and the quality was examined with Fragment Analyzer. Libraries were pooled with equal molar ratios, denatured, diluted, and sequenced with NextSeq 500/550 High Output Kit v2 (Illumina, 80bp pair-end runs for RNA-seq, #FC-404-2002) on a Nextseq500 sequencer (Illumina).