title: NextSeq 550 sequencing; Allelic dilution series series of C57BL6/J:CAST/EiJ RNA using Smart-seq3
design: Allelic dilution series series of C57BL6/J:CAST/EiJ RNA using Smart-seq3
library_construction_protocol: Liver tissue was isolated from 12-week old male C57BL6/J and CAST/EiJ mice, dissected into 1-2mm^2-wide samples and transferred to 1ml TRIzol isolation reagent (Thermo Scientific). The samples were thoroughly homogenized in TRIzol using a metallic tissue grinder before proceeding to RNA extraction. RNA extraction was performed according to the manufacturer's instructions. In brief, 0,2ml chloroform was added to 1ml of TRIzol and the samples were vigorously shaken. The samples were incubated at room temperature and the RNA-containing upper aqueous phase was isolated and precipitated with 0,5ml isopropyl alcohol. The samples were incubated at room temperature for 10 minutes and centrifuged at 12000 x g at 4°C for 10 minutes. The pellet was washed once with 75% ethanol and the RNA pellet was air-dried for 10 minutes. The RNA pellets were resuspended in 50ul of RNase-free water and incubated in a heat block at 55-60°C for 15 minutes before measuring the concentration using a Nanodrop 2000 spectrophotometer. A modified version of Smart-seq3 was performed. Briefly, 200pg of C57BL6/J and/or CAST/Eij liver RNA was combined at varying ratios (0, 12.5, 25, 37.5, 50, 62.5, 75, 87.5 and 100% C57) and added to 3µl lysis buffer containing 5% PEG-8000 (Sigma Aldrich), 0.5µM dNTP (Thermo Scientific), 0.1% Triton-X-100 (Sigma Aldrich), 0.5u µl-1 Takara bio recombinant RNAse inhibitor (RRI, Takara bio) and 0.5µM OligodT30VN. The samples were incubated at 72°C for 10 minutes to promote denaturation of tertiary structures. Next, reverse transcription was performed by adding 5µl of reverse transcription mix containing 50mM Tris-HCl (Sigma Aldrich), pH 8.0, 75mM NaCl (Sigma Aldrich), 1mM GTP (Thermo Scientific), 2.5mM MgCl2 (Invitrogen), 10mM DTT (Thermo Scientific), 1.6u µl-1 Takara bio RRI, 2mM template-switch oligo (TSO) and 2u µl-1 of Maxima H minus reverse transcriptase enzyme (Thermo Scientific). The reaction was carried out at 42°C for 90 minutes, followed by 10 cycles of 50°C for 2 minutes and 42°C for 2 minutes and terminated at 85°C for 5 minutes. PCR amplification was performed by adding 17ul of PCR amplification mix containing 1x KAPA HiFi HotStart MasterMix (Roche) and 0.1µM Smartseq3 forward PCR primer and 0.1µM Smartseq3 reverse PCR primer to each sample. The PCR reaction took place at 98°C for 3 minutes for initial denaturation, followed by 22 cycles of each of the following: denaturation at 98°C for 20 seconds, annealing at 65°C for 30 seconds, elongation at 72°C for 4 minutes followed by a final elongation step at 72°C for 5 minutes. For PCR cleanup, AMPure XP beads were added to each sample at a ratio of 0.8:1 (beads to sample). The samples were incubated at room temperature for 8 minutes before being placed on a magnetic rack and washed once using freshly prepared 80% ethanol solution. After ethanol removal, the beads were left to air-dry for 2 minutes, and the cDNA was eluted in 12µl of RNase-free water. Library quantification was performed using the QuantiFluor dsDNA kit (Promega) according to the manufacturer's instructions. Following quantification, tagmentation was carried out using 100pg of cDNA per sample, at a concentration of 100pg/µl and 1µl of tagmentation mastermix, consisting of 0.5µl of tagmentation buffer (40mM Tris-HCl pH 7.5, 20mM MgCl2, 20% dimethylformamide), 0.1µl Nextera XT ATM Tn5 (Illumina) and nuclease-free water. The tagmentation reaction took place at 55°C for 10 minutes. Tn5 was released from the cDNA using 0.5µl of freshly prepared 0.2% SDS solution and incubating the samples at room temperature for 5 minutes. Finally, PCR amplification of the final libraries was performed using 0.75µl of custom-designed Nextera 10bp indexes (0.2µM each) and 3.75µl of PCR mastermix containing 1x Phusion HF buffer, 0.2mM dNTP, and 0.01u µl-1 Phusion HF DNA polymerase (Thermo Scientific). The amplification reaction was performed as follows: Gap-filling at 72°C for 3 minutes, initial denaturation at 98°C for 3 minutes, 12 cycles of denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds and elongation at 72°C for 30 seconds followed by a final elongation step at 72°C for 5 minutes. For the final PCR cleanup, the libraries were pooled and cleaned using AMPure XP beads at a ratio of 0.6:1, washed twice with freshly prepared 80% ethanol and left to air-dry for 3 minutes. The pooled libraries were eluted in 45µl of nuclease-free water (Thermo Scientific).