title: GSM7865489: 2E_03, Brain_stem, 5xFAD , 2E-treated; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated from PFA-fixed OCT-embedded frozen brain sections using an in house developed method that digest proteins to free crosslinked RNA(Farris et al., 2017; Nichterwitz et al., 2016; Thomsen et al., 2016). In brief, frozen brain sections (14 μm) were taken out of -80°C and incubated at RT for 5 min, avoiding drying out. 50 µL RNA isolation buffer per sample was prepared in advance and kept on ice, containing 38.75 µL of PKD buffer (157014133, Qiagen) with 10 µL proteinase K solution (19131, Qiagen) and 1.25 µL ERCC (1:60k) spike-in synthetic RNAs (Life Technologies). The target brain sections were covered with 50 µL isolation buffer and incubated at room temperature for 30 s. Target brain sections were scratched and the brain lysates were collected by pipetting with 200 µL tips, and snap-frozen in liquid nitrogen and stored at -80°C. Samples were thawed at room temperature for 3 min, then vortexed and spun down, followed by the incubation of samples at 56°C for 4 hours in a thermal cycler with the lid set at 66°C, checking hourly if the sample was dissolving. After the incubation, vortexing and spinning down the samples, they were transferred into pre-cooled 1.5 mL tubes on ice. Prepared oligo dT25 magnetic beads (61005, Invitrogen) with three washes of 1x hybridization buffer (HB), which contains 2x SSPE (Life Technologies, Cat#15591-043), 0.05% Tween-20 (1610781, Bio-Rad), 0.05% RNase Inhibitor (2313A, Takara). Resuspend the beads with half of the original volume in 2x HB for later usage. 10 µL washed dT25 beads (0.1 mg) was added into each sample to reverse crosslinked samples and heated the samples at 56°C for one minute. Placed the samples on ice after 10 min incubation at room temperature to allow mRNA hybridization. Then washed the beads two times in 100 µL of ice-cold 1x HB, followed by a subsequent wash using ice cold 1x PBS with 0.1% RNase Inhibitor inside. Removed PBS and resuspended with 15 µL RNase-free water. Incubated the sample-beads mixture at 80°C for two minutes to elute mRNA, then immediately pelleted on a room temperature magnet. Rapidly removed the supernatant containing mRNA and transfer to a new tube and store at -80°C. 0.1-0.5 µg mRNA from each sample was taken and continued with Smart-seq2 protocol Libraries were prepared with SmartSeq2 protocol as described previously in (Safaiyan et al., 2021). mRNA-Seq (SmartSeq2)