title: GSM4572512: NAI, even set (SARS-CoV-2), replicate #1; Severe acute respiratory syndrome coronavirus 2; OTHER
library_construction_protocol: Probed RNA was purified on an RNA Clean & Concentrator-5 column (Zymo Research, cat. R1013). For multiplex SHAPE-MaP, 70 oligonucleotide pairs, tiling the entire length of the SARS-CoV-2 genome (29,903 nt), were automatically designed using Primer3 and the following parameters: amplicon size between 480 and 520 bp, maximum poly(N) length between 2 and 3, minimum/optimal/maximum oligonucleotide size of 20/25/30, minimum/optimal/maximum Tm of 56/60/62 degrees, minimum/optimal/maximum GC content of 30/50/65 %. Also, pairs were designed in such a way that the target amplicon would include the target region of the reverse primer from the previous set and of the forward primer of the following set. Primers were then searched against the GENCODE v33 human transcriptome, keeping only those with less than 60% predicted base-pairing, or more than 60% predicted base-pairing and more than 2 mismatched bases at the 3' end. For reverse transcription, all reverse primers were equimolarly pooled to yield a 100 μM RT primer mix. An additional anchored oligo-dT primer (TTTTTTTTTTTTTTTTTTTTVN) was added to the mix. For each sample, two 20 μl reverse transcription reactions were carried out. 3 μg of total RNA were mixed with 0.5 μl RT primer mix and 1 μl dNTPs (10 mM each), incubated at 70°C for 5 min and then transferred immediately to ice for 1 min. Reactions were then supplemented with 4 μl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 2 μl DTT 0.1 M, 1 μl MnCl2 120 mM, 20 U SUPERase•In™ RNase Inhibitor and 200 U SuperScript II RT (ThermoFisher Scientific, cat. 18064014). Reactions were incubated at 42°C for 2 h and then RT was heat-inactivated at 75°C for 15 min. RNA was then degraded by addition of 5 U RNase H (New England Biolabs, cat. M0297S) and cDNA from each 2 reactions was purified on a single RNA Clean & Concentrator-5 column and eluted in 36 μl NF H2O. For targeted amplification, primer pairs were split into 2 non-overlapping sets, odd and even. For each of these sets, pairs were pooled in smaller sets of 3-4 pairs, as follows: 1-19-37-55, 2-20-38-56, 3-21-39-57, 4-22-40-58, 5-23-41-59, 6-24-42-60, 7-25-43-61, 8-26-44-62, 9-27-45-63, 10-28-46-64, 11-29-47-65, 12-30-48-66, 13-31-49-67, 14-32-50-68, 15-33-51-69, 16-34-52-70, 17-35-53, 18-36-54. The complete primer list can be found in Table S1. PCR reactions were carried out in 50 μl, using 2 μl of eluted cDNA, 0.3 mM final each dNTP, 0.1 μM final each primer in the set and 2.5 U TaKaRa Taq™ DNA Polymerase (TaKaRa, cat. R001A). Cycling was performed using a touch-down approach. Briefly, for the first 10 cycles the annealing temperature was lowered by 0.5°C per cycle, starting at 58°C. Then, 20 additional cycles were performed at the lowest temperature. PCR products were purified using 1 volume of NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), checked on a 2% agarose gel. Each primer set gave a single specific band of the expected size. PCR products were equimolarly pooled to yield the final odd and even sets. 500 ng of each set were fragmented in a final volume of 20 μl, using 2 μl of NEBNext dsDNA Fragmentase (New England Biolabs, cat. M0348), supplemented with 1 μl 200 mM MgCl2, by incubating at 37°C for 25 min. This yielded fragments in the range of ~50-200 bp. Reactions were purified using 1 volume of NucleoMag NGS Clean-up and Size Select beads. 5 ng of fragmented DNA were then used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), as per manufacturer instructions.