title: GSM8835735: GC38_cx_ctrl; Mus musculus; RNA-Seq
library_construction_protocol: as explained in LaClair et al.: "Tissues were homogenized in Trizol using homogenizer (Precellys). Total RNA was isolated using Direct-zol-96 RNA isolation kit (ZymoResearch) following the manufacturer's instructions. RNA quantity and quality were controlled on Agilent 2100 BioAnalyzer using Agilent RNA 6000 Nano Kit" Following extraction and QC, samples were transferred to 96-well plates and RNA-sequencing was performed with 20ng of total RNA using the prime-seq protocol as described in Janjic et al., 2022.  Briefly, reverse transcription was carried out in a 10 uL reaction consisting of 20 ng to total RNA, 30 units Maxima H- enzyme (Thermo Fisher, EP0753), 1x Maxima H- Buffer (Thermo Fisher), 1 mM each dNTPs (Thermo Fisher), 1 uM template-switching oligo (IDT), 1 uM barcoded oligo(dT) primers (IDT). The reaction was incubated at 42 C for 90 minutes. Following cDNA synthesis, the samples were pooled, cleaned, and concentrated with cleanup beads at a 1:1 ratio and eluted in 17 uL of ddH20. Residual primers were digested using Exonuclease I (Thermo Fisher, EN0581) at 37 C for 20 minutes followed by a heat inactivation step at 80 C for 10 minutes. The samples were cleaned once more using cleanup beads at a 1:1 ratio, and eluted in 20 L of ddH2O.  Second strand synthesis and pre-amplification were performed in a 50 uL reaction, consisting of 1x KAPA HiFi Ready Mix (Roche, 7958935001) and 0.6 uM SingV6 primer (IDT), with the following PCR setup: initial denaturation at 98 C for 3 minutes, denaturation at 98 C for 15 seconds, annealing at 65 C for 30 seconds, elongation at 68 C for 4 minutes, and a final elongation at 72 C for 10 minutes. Denaturation, annealing, and elongation were repeated for 5-15 cycles depending on the initial input.  The DNA was cleaned using cleanup beads at a ratio of 1:0.8 of DNA to beads and eluted with 10 uL of ddH2O. The quantity was assessed using a quant-iT PicoGreen dsDNA assay kit (Thermo Fisher, P11496) and the quality was assessed using an Agilent 2100 Bioanalyzer with a High Sensitivity DNA analysis kit (Agilent, 5067-4626).  Libraries were prepared with the NEBNext Ultra II FS Library Preparation Kit (NEB, E6177S) according to manufacturer instructions in most steps, with the exception of adapter sequence and reaction volumes. Fragmentation was performed on 2.5 uL of cDNA (generally 2 - 20 ng) using Enzyme Mix and Reaction buffer in a 6 uL reaction. A custom prime-seq adapter (1.5 uM, IDT) was ligated using the Ligation Master Mix and Ligation Enhancer in a reaction volume of 12.7 uL. The samples were then double-size selected using SPRI-select Beads (Beckman Coulter, B23317), with a high cutoff of 0.5 and a low cutoff of 0.7. The samples were then amplified using Q5 Master Mix (NEB, M0544L), 1 uL i7 Index primer (Sigma-Aldrich), and 1 uL i5 Index primer (IDT) using the following setup: 98 C for 30 seconds; 10-12 cycles of 98 C for 10 seconds, 65 C for 1 minute 15 seconds, 65 C for 5 minutes and 65C for 4 minutes. Double-size selection was performed once more as before using SPRI-select Beads. The quantity and quality of the libraries were assessed as before.  Sequencing was performed on an Illumina HiSeq 1500 flow cell with Read 1 (barcode & UMI): 28 bp, Read 2 (cDNA): 50bp.