Rnase inhibitor

A total of 2.5 µg of fragmented and affinity captured PAN RNA was used for the 20 µL ligation reaction that included 20 pmol of RiL-19 oligonucleotide (Supplemental Table 1), 2 µL 10× T4 ligation buffer, 1.8 µL 100% DMSO, 100 mM ATP, 8 µL PEG-8000, 0.3 µL of RNase Inhibitor (12U final, NEB M0307L) and 1.8 µL of T4 RNA ligase (20U final, NEB M0204S), and incubated at room temperature for 90 min. Reverse transcription reaction was initiated by annealing 20 pmol of AR-17 oligonucleotide (Supplemental Table 1) to the sample and incubating the reaction for 5 min at 75°C and slowly cooling to 25°C. Next, 2 µL of 4SedTTP reaction buffer (500 mM Tris-HCl pH 8.0, 500 mM KCl, 50 mM MgCl₂, 100 mM DTT), 2 µL of 800 µM dATP, dCTP, dGTP, 4SedTTP (final concentration of 80 µM for each), 2 µL RNase Inhibitor (80U final, NEB M0307L) and 2 µL SuperScript III (400U final, Thermo Fisher 18080044) were added to each reaction to a final volume of 20 µL. The RT reactions were incubated at 42°C for 40 min, followed by incubation at 85°C for 15 min. The 100 nt long unmodified and m⁶A modified in vitro transcripts were treated in parallel with the experimental samples (Supplemental Table 1). Negative controls included fragmented, affinity captured PAN RNA (at 0–72 h pi) that was ligated to AR-17 oligonucleotide and directed to control RT reactions.

To reduce the signal derived from RNA fragmentation during the CMC reaction, ±CMC-treated RNA in 6.5 µL H₂O was mixed with 0.5 µL RNase inhibitor (NEB, M0307L), 1 µL of 10× T4 PNK reaction buffer A, 1 µL of 1 mM ATP, 1 µL of T4 Polynucleotide kinase (PNK), and then incubated at 37°C for 30 min. To ligate the RNA-5 oligo (/5AmMC6/rArCrCrCrA; Integrated DNA Technologies), 1 µL of 10× T4 RNA Ligase Reaction Buffer, 1 µL of 100 µM RNA-5 oligo, 1 µL of 1 mM ATP, 1 µL of RNase inhibitor, 3 µL of DMSO, 2 µL of H₂O, and 1 µL of T4 RNA ligase I (NEB, M0437M) were added to this mixture and incubated at 16°C for 16 h. The reaction was terminated upon the addition of 1.2 µL of 200 mM EDTA.

The RNA insert (10 μM) was hybridized to the DNA splint (10 μM) by heating the mixture to 95°C and slow cooling to 25°C at 0.1°C/s rate in 10 mM tris-HCl (pH 7.8) buffer with 100 mM NaCl. Cleavage of viral RNA was performed as described above. After cleanup, cleaved viral genome was mixed with the RNA insert: DNA splint duplex (0.4 pmol) and 5 U of T4 PNK (NEB) in 10 μl of reaction buffer [50 mM tris-HCl (pH7.5), 1 mM DTT, and 1 mM MgCl₂] supplemented with 1 mM ATP and RNase inhibitor (1 U/μl; NEB). After 1 hour at 37°C, 5 μl of a mix containing 5 U of T4 Rnl2 (NEB), 1 mM ATP, 3 μl of 50% PEG 8000, and RNase inhibitor (1 U/μl; NEB) in reaction buffer [50 mM tris-HCl (pH7.5), 1 mM DTT, and 1 mM MgCl₂] was added and incubated for 1 hour at 25°C to perform ligation.

IVT 10× MS2-tagged RNA (15 nM; full-length HOTAIR or Anti-Luc) was rotated (end over end) at room temperature for 15 min with 80 nM recombinant hnRNP B1 or A2 in EMB 300 Buffer [10 mM Hepes (pH 7.9), 300 mM NaCl, 3 mM MgCl₂, 0.5% NP-40, 10% glycerol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM dithiothreitol (DTT)], RNase inhibitor (New England Biolabs), and 20 μg of competitor yeast transfer RNA (Roche) in a total volume of 300 μl per sample. At the same time, 300 nM MS2-MBP was prebound to 20 μl of amylose resin (New England Biolabs) in EMB 300 Buffer, RNase inhibitor (New England Biolabs), and 1% BSA and rotated at room temperature for 15 min. The MS2-MBP amylose resin was then added to each IVT-hnRNP sample and incubated for an additional 15 min rotating at room temperature. Resin was washed 4× in 800 μl of EMB 300 Buffer, and then protein association was analyzed by Western blot using antibody for hnRNP A2/B1 (Abcam, #ab6102). In addition, 10% of each sample was used for RNA analysis, where RNA was isolated by phenol/chloroform extraction, purified by ethanol precipitation, and quantified by RT-qPCR.

For cDNA synthesis, RNA from single cells was reverse transcribed at 37°C for 55 min with 150 ng random hexamer primer (Integrated DNA Technologies), 0.5 µl dNTP mix (10 mM each, Fermentas), 1 µl of 0.1 M dithiothreitol (Invitrogen), 0.5% (vol/vol) Nonidet P-40, 5.6 U RNase inhibitors (New England Biolabs, Inc.), and 50 U Superscript III reverse transcription (Invitrogen) in a total volume of 14 µl. Heavy and light chain genes from single cells were amplified by PCR from cDNA using two rounds of reactions with previously published nested primers (Smith et al., 2009 (link); 40 µl reactions included 10 pmol of each primer and 0.4 µl of JumpStart Taq DNA polymerase [Sigma-Aldrich]). Aliquots of second PCR products were purified by incubation with 10 U alkaline phosphatase, calf intestinal (CIP; New England Biolabs, Inc.), and 20 U exonuclease I (New England Biolabs, Inc.) in 1× NEB 3 buffer for 15 min at 37°C and sequenced using the reverse primers at the University of Chicago sequencing core. VH, DH, JH, VL, and JL usage was identified using IgBLAST.