GSM5470567: hippocampus, ko_female_input_rep2; Mus musculus; OTHER
For the library preparation, hippocampi were isolated at 4°C according to Ingolia (Ingolia et al., 2011) and Biever (Biever et al., 2020) and directly resuspended in 300 µl polysome lysis buffer containing 20 mM Tris (pH 7.5), 5 mM MgCl2, 150 mM NaCl, 1% Triton X-100, 1 mM DTT, 100 µg/ml cycloheximide, and complete protease inhibitor (Roche). Tissues were dounced ten times with a plastic pestle before being pushed through a 25G syringe ten times. The lysates were cooled on ice for 10 min before being centrifuged at high speed at 4°C for 6 min. After transferring the supernatant to a fresh tube, the centrifugation procedure was repeated. After determining the RNA concentration, 15% of the lysate (≤ 20 µg) was utilised as input (see RNA-seq library preparation section), 50% for ribosome profiling, and 20-25% for polysome profiling. Ribosome profiling libraries were prepared using 50% (150 µl) of lysate as described previously (Tuorto et al., 2018). Sequencing libraries were prepared according to protocol of the NEB NEXT Small RNA Library Prep Set for Illumina (Multiplex Compatible) E7330. 15% of polysome lysate was used for RNA-seq library preparation. RNA extraction was performed as described previously (Tuorto et al., 2018). After rRNA depletion using Ribo Zero Kit (Illumina), libraries were prepared using NEBNext Ultra II RNA Library Prep Kit (Illumina) according to manufacturer's instructions. In brief, rRNA-depleted RNAs were fragmented, yielding an insert size of ~200 nt. Following the hybridisation of random primers, first and second strand cDNA syntheses were carried out. The NEBNext adaptors were ligated after the samples were end-repaired. Uracils were eliminated from the adaptor loops and the library yield was enhanced using PCR. Briefly, rRNA depleted RNAs were fragmented and resulted in an insert size of ~200 nt. After hybridisation of random primers, first and second strand cDNA syntheses were performed. Samples were end-repaired, and the NEBNext adaptors were ligated. Uracils were removed from the adaptor loops, and the library yield was increased by PCR. Finally, barcodes/indices were included in the samples to multiplex the libraries. Sequencing was performed on a NextSeq 550 platform. Chemistry used: NextSeq 500/550 highoutput V2 Kit (75cycles), FC-404-2005, loaded on FC 1,8 pM. ncRNA-Seq