title: GSM7875078: mouse11, F, striatum; Mus musculus; RNA-Seq
library_name: GSM7875078
library_construction_protocol: For each sample, we transferred ~30 mg of each brain region (or the entire brain region, if less than 30 mg) into an MP Biomedical Lysis D Matrix, 2ml tube (#6913500) containing 500μl of TRIzol reagent (Invitrogen #15596018) and lysed cells from each tissue on the FastPrep-24 5G bead beating grinder and lysis system (MP Biomedical #116005500). After lysis, we added 100μl of chloroform to the tube and centrifuged at 12,000×g for 15 minutes, and then transferred the clear top layer of the supernatant into a fresh tube. We next added an equivolume amount of isopropanol to the clear liquid and centrifuged at 12,000×g for 10 minutes. We decanted the supernatant, washed the pellet twice with 75% ethanol, and resuspended the air-dried pellet in RNAse-free water. We incubated the final RNA product with TURBO DNase (Invitrogen #AM1907) for 30 minutes and assessed for RNA quality using a Qubit fluorometer and Agilent Fragment Analyzer. All RNA extractions had an RNA quality number (RQN) score >7. We processed RNA samples for nanopore sequencing using the PCR-cDNA Barcoding Kit (SQK-PCB111.24) in two batches (20 samples each, batches A and B) according to manufacturer instructions and prepared libraries in equimolar amounts based on fragment length and concentration to make 15 fmol of cDNA library per flow cell, or 105 fmol of pooled cDNA library in a total volume of 77 μl for six flow cells (11μl per flow cell + 1 extra).