GSM9743685: Whole-tissue lysate RNA from mice cortex and hippocampus at P7, replicate 1 [1w-Cre-Input]; Mus musculus; RNA-Seq
Upon tissue collection, whole brains were rapidly removed on ice, and the cerebral cortex and hippocampus were carefully dissected. Tissues were homogenized in salt lysis buffer supplemented with RNase inhibitor, DTT, cycloheximide, and protease inhibitors. After clarification by centrifugation, a portion of the lysate was saved as the Input fraction, while the remaining lysate was incubated with anti-GFP magnetic beads to immunoprecipitate eGFP-Rpl10a–associated ribosomes. Following several washes with high-salt buffer, ribosome-associated RNA from the IP fraction was isolated using the RNeasy Micro Kit for subsequent RNA sequencing. Library construction was performed using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina® (#NR605). Fragmented mRNA was reverse-transcribed into first-strand cDNA using random primers and M-MuLV reverse transcriptase, followed by second-strand synthesis. After end repair, A-tailing, and adaptor ligation, ~250-300 bp fragments were size-selected with VAHTS DNA Clean Beads and PCR-amplified. Library quality was assessed using a Qubit 3.0 Fluorometer and an Agilent 2100 Bioanalyzer, and libraries with an effective concentration >2 nM were sequenced on an NovaSeq X Plus platform.