title: GSM4776787: SARS-CoV-2 24 hpi rep1; Homo sapiens; RNA-Seq
library_construction_protocol: To minimize variability due to sampling and processing, each biological replicate consisted of 3 organoids and the replicates for all experimental conditions were processed in parallel for RNA-extraction, library preparation and sequencing. At the desired experimental endpoints, organoids were homogenized in TRIzol (Thermo Fisher Scientific) using a disposable pestle and handheld mortar and stored at -80 ⁰C until processing. RNA clean-up was performed using the RNA Clean & Concentrator kit (Zymo Research) after TRIzol phase separation according to the manufacturer's protocol. RNA concentration and quality were assessed using a Nanodrop 2000 (Thermo Fisher Scientific). Library preparation was performed as previously described with some minor modifications (Weng et al., 2017). About 300 ng of RNA in 3.2 µL was combined with 0.25 µL RNase inhibitor (NEB) and 1 µL CDS primer (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3') in an 8-well PCR tube strip, heated to 70 ºC for 2 min, and immediately placed on ice. 5.55 µL RT mix, containing 2 µL of 5X SMARTScribe RT buffer (Takara), 0.5 µL of 100 mM DTT (Millipore Sigma), 0.3 µL of 200 mM MgCl2 (Thermo Fisher Scientific), 1 µL of 10 mM dNTPs (Takara), 1 µL of 10 µM TSO primer (5'-AAGCAGTGGTATCAACGCAGAGTACATrGrGrG-3'), 0.25 µL of RNase inhibitor (NEB), and 0.5 µL SMARTScribe reverse transcriptase (Takara) was added to the reaction. RT was performed under the following conditions: 42 ºC for 90 minutes, 10 cycles of 50 ºC for 2 minutes and 42 ºC for 2 minutes, 70 ºC for 15 minutes, and 4 ºC indefinitely. For cDNA amplification, 2 µL of the RT reaction was combined with 2.5 µL of 10X Advantage 2 buffer (Takara), 2.5 µL of 2.5 mM dNTPs (Takara), 0.25 µL of 10 µM IS PCR primer (5'-AAGCAGTGGTATCAACGCAGAGT-3'), 17.25 µL nuclease free water (ThermoFisher), and 0.5 µL Advantage 2 DNA Polymerase (Takara). Thermocycling conditions were as follows: 94 ºC for 3 minutes, 8 cycles of 94 ºC for 15 s, 65 ºC for 30 s, and 68 ºC for 6 minutes, 72 ºC for 10 minutes, and 4 ºC indefinitely. Amplified cDNA was purified using 0.8X AMPure XP beads (Beckman Coulter), eluted in 15 µL nuclease-free water, and quantified using Qubit dsDNA HS assay kit (Thermo Fisher Scientific). cDNA was fragmented by combining 100 pg cDNA in 1 µL nuclease free water, 2X TD buffer (20 mM Tris, pH 8.0; Thermo Fisher Scientific), 10 mM MgCl2, and 16% PEG 8000 (MilliporeSigma), and 0.5 µL Tn5 (Lucigen). The mixture was heated to 55 ºC for 12 minutes, and the reaction was terminated upon the addition of 1.25 µL of 0.2% SDS (Fisher) and incubated at room temperature for 10 minutes. Fragments were amplified by adding 16.75 µL nuclease free water (Thermo Fisher Scientific), 1 µL of 10 mM Nextera i7 primer, 1 µL of 10 mM Nextera i5 primer, and 25 µL KAPA HiFi hotstart readymix (EMSCO/FISHER). Thermocycling conditions were as follows: 72 ºC for 5 minutes, 95 ºC for 1 minute, 14 cycles of 95 ºC for 30 s, 55 ºC for 30 s, and 72 ºC for 30 s, 72 ºC for 1 minute, and 4 ºC indefinitely. DNA was purified twice with 0.8X AMPure XP beads (Beckman Coulter) and eluted in 10 µL of 10 mM Tris, pH 8 (Thermo Fisher Scientific). Samples were quantified by qPCR (KAPA) and pooled at equal molar amounts. Final sequencing library fragment sizes were quantified by bioanalyzer (Agilent) with an average size of ~420 bp, and concentrations were determined by qPCR (KAPA). Samples were loaded at concentrations of 2.7 pM and sequenced on a NextSeq 550 (Illumina) using 1x72 bp reads to an average depth of 40 million reads per sample.