GSM8854282: RiboTag, Rbp4-Cre, GCs, P7_r1; Mus musculus; RNA-Seq
RiboTag sample preparation protocol was adapted from Sanz et al, 2019. Briefly, hippocampi were thawed in pre-chilled homogenization buffer containing 50 mM TrisCl pH 7.5, 100 mM KCl, 12 mM MgCl2, 1% NP40, 1 mM DTT, 200 U/ml RiboLock RNase Inhibitor (Thermofisher, EO0382), 1 mg/ml Heparin (Sigma, H3393), 0.1 mg/ml cycloheximide (Sigma, C4859), 1X EDTA-free protease inhibitors. Next, samples were placed in a Dounce homogenizer where they were dissociated using 2 sizes glass pestles. Lysate was centrifuged at 10000g, 4 °C for 10 min and supernatant (named hereafter as input) was kept for further processing. 40 μl Pierce™ Anti-HA Magnetic Beads (ThermoFisher, 13464229) were added to the input and incubated overnight at 4 °C on a microtube rotator with gentle mixing. Next, samples were placed on magnetic stand for at least 1 min to remove supernatant (named hereafter as unbound), and washed 4x using high salt buffer (50 mM TrisCl pH 7.5, 300 mM KCl, 12 mM MgCl2, 1% NP40, 0.5 mM DTT, 0.1 mg/ml cycloheximide). After the last wash, 350 μl RLT buffer (Qiagen RNeasy mini kit, 74104) supplemented with β-mercaptoethanol was used to elute RNA from the ribosomes. The mix was vortexed 30s and tubes were placed back in magnetic stand to collect the clear RLT buffer containing the RNA. Next, RNA was isolated using Qiagen RNeasy mini kit with DNase treatment and following manufacturer instructions. Library preparation from total RNA extraction and sequencing was performed at the VIB Nucleomics Core. Per sample, an amount of 25-100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version: #1000000040498 v00 October 2017) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally, enrichment PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. Sequence libraries of each sample were equimolarly pooled and sequenced on Illumina NovaSeq 6000 (v1.5, SE100 (100-8-8-0), 1% PhiX).