GSM4257248: slice_hrt_10; Mus musculus; OTHER
Frozen cortical-hippocampal slices were thawed in ice-cold lysis buffer (20mM Tris-HCl pH7.4, 5mM MgCl2, 100mM KCl, 1mM DTT, 100μg/ml CHX, 25U/ml Turbo DNaseI, 1X EDTA-free protease inhibitor, 1% NP-40, in nuclease-free water) on ice for 5min. Tissues were dissociated by pipetting and further trituration through 25G needle for 10 times. Lysates were incubated on ice for 10min and then clarified by centrifugation at 2,000g 4 °C for 10min. The supernatants were collected and clarified again by centrifugation at 20,000g 4 °C for 10min. The supernatants were collected, and the amounts of nucleic acid were measured with Qubit HS RNA assays. Lysates containing ~40µg RNA were digested with 600ng RNase A (Ambion, #AM2270) + 75U RNase T1 (Thermo Fisher Scientific, #EN0542) /µg RNA in 0.3ml at 25°C for 30min and stopped by chilling on ice and adding 50U SUPERase In RNase inhibitor. Digested lysates were applied to 10%-50% (w/v) sucrose gradients similarly as above. 3' ligation based library construction, which is similar to miRNA-seq. Ribosome protected mRNA fragments were ligated to 3' adapters. RT was then performed. RT products were separated on a 10% TBU gel and the 200-250nt region was selected. cDNA was circularized. PCR was performed to add adapters for Illumina sequencing.   260-300bp final PCR products were size-selected on an 8% TBE gel.  The size distributions of final libraries were measured by Fragment Analyzer (Advanced Analytical, performed by Molecular Biology Core Labs at UMMS). The concentrations were quantified with KAPA Library Quantification Kit (Kapa Biosystems, #KK4835). Libraries were pooled with equal molar ratios, denatured, diluted, and sequenced with NextSeq 500/550 High Output Kit v2 (Illumina, 75bp single-end runs, #FC-404-2005) on a Nextseq500 sequencer (Illumina).