title: GSM8457326: EtOH_2; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted using the Quick-RNA FFPE MiniPrep kit. RNA-seq was performed as previously described (Cubillos et al., 2024). RNA of differentiating NSCs was isolated using the Quick-RNA MiniPrep kit (Zymo Research, #R1008). Transcriptome libraries were prepared using an adapted version of the SmartSeq2 protocol (Picelli et al, 2013). Isolated total RNA from an equivalent of one 24-well was denatured for 3 minutes at 72°C in 4 μL hypotonic buffer (0.2% Triton-X 100) in the presence of 2.4 mM dNTP, 240 nM dT-primer (Table EV1) and 4 U RNase Inhibitor (NEB, M0314L). Reverse transcription was performed at 42°C for 90 min after filling up to 10 µL with RT buffer mix for a final concentration of 1X Superscript II buffer (Invitrogen), 1 M Betaine, 5 mM DTT, 6 mM MgCl2, 1 µM TSO-primer (Table EV1), 9 U RNase inhibitor and 90 U Superscript II. The reverse transcriptase was inactivated at 70°C for 15 min. For subsequent PCR amplification of the cDNA, the optimal PCR cycle number was determined with an aliquot of 1 μL unpurified cDNA in a 10 μL qPCR containing 1X Kapa HiFi Hotstart Readymix (Roche), 1X SybrGreen and 0.2 μM UP primer (Table EV1). The residual 9 μL cDNA were subsequently amplified using Kapa HiFi HotStart Readymix (Roche) at a 1X concentration together with 250 nM UP-primer (Table EV1) under the following cycling conditions: initial denaturation at 98°C for 3 min, 22 cycles [98°C 20 sec, 67°C 15 sec, 72°C 6 min] and final elongation at 72°C for 5 min. Amplified cDNA was purified using 1X volume of Sera-Mag SpeedBeads (GE Healthcare) resuspended in a buffer consisting of 10 mM Tris, 20 mM EDTA, 18.5% (w/v) PEG 8000 and 2 M sodium chloride solution. The cDNA quality and concentration were determined using a Fragment Analyzer (Agilent).  For library preparation, 2 µL amplified cDNA was tagmented in 1X Tagmentation Buffer using 0.8 µL bead-linked transposome (Illumina DNA Prep, (M) Tagmentation, Illumina) at 55°C for 15 min in a total volume of 4 µL. The reaction was stopped by adding 1 µL of 0.1% SDS (37°C, 15 min). Magnetic beads were bound to a magnet, the supernatant was removed, beads were resuspended in 14 µL indexing PCR Mix containing 1X KAPA Hifi HotStart Ready Mix (Roche) and 700 nM unique dual indexing primers (i5 and i7), and subjected to a PCR (72°C 3 min, 98°C 30 sec, 12 cycles [98°C 10 sec, 63°C 20 sec, 72°C 1 min], 72°C 5 min). Libraries were purified with 0.9X volume Sera-Mag SpeedBeads, followed by a double size selection with 0.6X and 0.9X volume of beads, and a final 0.9X purification to obtain a fragment size distribution of 200–700 bp. Sequencing was performed after quantification using a Fragment Analyzer on an Illumina Novaseq 6000 in 100 bp paired-end XP mode with an average sequencing depth of 40 million fragments per library.