title: GSM8771341: FIRSTseq Induro in Magnesium Buffer; Saccharomyces cerevisiae; OTHER
library_construction_protocol: For E. coli, bacterial pellets were resuspended in 62.5 μl Buffer R [20 mM Tris-HCl pH 8.0; 80 mM NaCl; 10 mM EDTA pH 8.0], supplemented with 100 μg/mL final Lysozyme (cat. L6876, Merck) and 20 U SUPERase•In™ RNase Inhibitor (cat. A2696, ThermoFisher Scientific), by thorough vortexing. After incubating samples at room temperature for 1 min, 62.5 μl Buffer L [0.5% Tween-20; 0.4% Sodium deoxycholate; 2 M NaCl; 10 mM EDTA] were added. Samples were then inverted 10 times and incubated at r.t. for 2 min, and on ice for 2 min. 1 mL ice-cold TRIzol™ Reagent (cat. 15596018, ThermoFisher Scientific) was then added, and samples were vortexed for 15 sec. RNA was extracted as per manufacturer protocol. For MDA-MB-231, cell pellets were directly resuspended in 1 mL TRIzol™ Reagent, incubated at 70°C for 5 min, and then RNA was extracted as per manufacturer protocol.  For S.cerevisiae , cells were then quickly transferred into 50 mL pre-chilled falcon tubes, and centrifuged for 5 minutes at 3,000 g in a 4ºC pre-chilled centrifuge. Supernatant was discarded, and cells were flash frozen. Flash frozen pellets were resuspended in 700 µL TRIzol (Thermo, 15596026) with 350 µL acid washed and autoclaved glass beads (425-600 µm, Sigma G8772). The cells were disrupted using a vortex on top speed for 7 cycles of 15 seconds (the samples were chilled on ice for 30 seconds between cycles). Afterwards, the samples were incubated at room temperature for 5 minutes and 200 µL chloroform was added. After briefly vortexing the suspension, the samples were incubated for 5 minutes at room temperature. Then they were centrifuged at 14,000 g for 15 minutes at 4ºC and the upper aqueous phase was transferred to a new tube. RNA was precipitated with 2X volume Molecular Grade Absolute ethanol and 0.1X volume Sodium Acetate (3M, pH 5.2). The samples were then incubated for 1 hour at -20ºC and centrifuged at 14,000 g for 15 minutes at 4ºC. The  pellet was then washed with 70% ethanol and resuspended with nuclease-free water after air drying for 5 minutes on the benchtop. The RNA libraries for direct RNA sequencing (SQK-RNA002) were prepared with the manufacturer's protocol with some adjustments. Unprobed and DMS probed RNAs were polyadenylated using E. coli Poly(A) Polymerase, following the commercial protocol, before starting the library prep. RNA library for direct RNA sequencing (SQK-RNA002) was prepared following the ONT Direct RNA Sequencing protocol version DRS_9080_v2_revI_14Aug2019. Briefly 500 ng total of poly(A) tailed RNAs were ligated to RT adaptors (RTA) using concentrated T4 DNA Ligase (NEB, M0202T) and was reverse transcribed using TGIRT, without the heat inactivation step. The products were purified using 1.8X Agencourt RNAClean XP beads (Thermo, NC0068576) and washed with 70% freshly prepared ethanol. Then, RMX adapter, composed of sequencing adapters with motor protein, was ligated onto the RNA:DNA hybrid, and the mix was purified using 1X Agencourt RNAClean XP beads, washing with wash buffer twice. The sample was then eluted in elution buffer and mixed with RNA running buffer before loading onto a primed R9.4.1 flowcell and run on a MinION sequencer with MinKNOW acquisition software version v.3.5.5.  The cDNA libraries for the direct cDNA sequencing were prepared following the direct cDNA Sequencing ONT protocol (DCB_9091_v109_revC_04Feb2019) and barcoded using the Native Barcoding Expansion kit (EXP-NBD104). Briefly, 50 ng poly(A) tailed RNA was mixed with 2.5 uL VNP oligo, 1 uL dNTPs (Thermo, R0192), and up to 11 uL nuclease-free water. After gently mixing by flicking and spinning down briefly, the mix was incubated at  65°C for 5 minutes and then placed on ice immediately. In a separate tube, 4 uL 5X RT Buffer, 1 uL RNase Inhibitor Murine (NEB, M0314S), 2 uL Strand-Switching Primer (SSP), 1 uL nuclease-free water was prepared and then two solutions were mixed. After incubating the mix at 42° C for 2 minutes, 1 uL Maxima H Minus Reverse Transcriptase (Thermo, EP0752) was added. Then the mix was incubated at 42°C for 90 minutes and inactivated by heating at 85°C for 5 minutes before moving to ice. RNAse Cocktail (Thermo Scientific, AM2286) was added to the mix in order to digest the RNA, and the mix was incubated at 37°C for 10 minutes.  The reaction was then cleaned up using 0.8X AMPure XP Beads (Agencourt, A63881). Second strand synthesis was performed by mixing the 20 uL cDNA sample with 25 uL 2X LongAmp Taq Master Mix (NEB, M0287S), 2 uL PR2 Primer (PR2), and 3 uL nuclease-free water. The reaction was incubated at 94 °C  for 1 min, 50 °C for 1 min, 65 °C 15 mins, and placed on ice. The reaction was then cleaned up using 0.8X AMPure XP Beads. End-repair and dA-tailing of the double-stranded DNA was achieved by mixing the 20 uL DNA sample with 30 uL nuclease-free water, 7 uL Ultra II End-prep reaction buffer and 3 uL Ultra II End-prep enzyme mix (NEB, E7546S), and incubating this mix at 20° C for 5 minutes and 65° C for 5 minutes. After incubating the reaction on ice for 1 min, it was cleaned up using 0.8X AMPure XP Beads. End-prepped DNA was then ligated to the Native Barcode (NB) for multiplexing with other libraries. The ligation was performed by mixing 22.5 uL end-prepped DNA, 2.5 uL Native Barcode (NB), 25 uL Blunt/TA Ligation Master Mix (NEB, M0367S) and incubating the mix at room temperature for 10 mins. The reaction was then cleaned up using 0.8X AMPure XP Beads. Once all the barcoded libraries were pooled after elution, the ligation of the sequencing Adapter Mix (AMX) was performed by mixing the 65 uL pooled barcoded DNAs (up to 200 fmol), 5 uL AMX, 20 uL 5X NEBNext Quick Ligation Reaction Buffer (NEB,B6058S) and 10 uL Quick T4 DNA Ligase (NEB,M2200S), and incubating the mix at room temperature for 10 mins. The reaction was cleaned up using 0.8X AMPure XP beads, using WSB Buffer for washing. The sample was then eluted in Elution Buffer (EB) and mixed with Sequencing Buffer (SQB) and Loading Beads (LB) prior to loading onto a primed  R9.4.1 flowcell. Library was run on MinION flowcell with MinKNOW acquisition software version v.3.5.5.  For FIRST-seq, Before starting the library preparation, 2 µL of 10 µM VNP (/5Phos/ACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTVN) and 2 µL of 10 µM Comp_DNA (GAAGATAGAGCGACAGGCAAGTA) were mixed with 1 µL 0.1 M Tris pH 7.5, 1 µL 0.5 M NaCl, and 4 ul RNase-free water. The mix was incubated at 94°C for 1 min and the temperature was ramped down to 25°C (-0.1°C/s) in order to pre-anneal the oligos. For each reverse-transcriptase, different reverse-transcription reaction was prepared with 50 ng poly(A) tailed RNA and 1 uL pre-annealed oligo. For Superscript II (Thermo, 18064014), RNA and oligos were mixed with 1uL dNTPs and RNase-free water up to 11 uL. Mixture was heated to 65°C for 5 mins and then quickly chilled on ice. Then 4 uL 5X First-strand buffer, 1 uL 0.1 M DTT, 1 uL RNAse Inhibitor Murine, and 1 uL SuperScript II were added to the mix. Reaction was then incubated at 42°C for 60 mins and inactivated by heating at 70°C for 15 mins. For Protoscript II (NEB, M0368S), same reaction was performed with the 5X ProtoScript II buffer. For Superscript III (Thermo, 18080093), RNA and oligos were mixed with 1uL dNTPs, 4 uL 5X First-strand buffer, 1 uL 0.1 M DTT, 1 uL RNAse Inhibitor Murine, 1 uL  Superscript III and RNase-free water up to 11 uL. Reaction was then incubated at 55°C for 60 mins and inactivated by heating at 70°C for 15 mins. For Superscript IV (Thermo, 18090010), RNA and oligos were mixed with 1uL dNTPs, 4 uL 5X SSIV buffer, 1 uL 0.1 M DTT, 1 uL RNAse Inhibitor Murine, 1 uL  Superscript IV and RNase-free water up to 11 uL. Reaction was then incubated at 60°C for 30 mins and inactivated by heating at 80°C for 10 mins. For Maxima H Minus RT, RNA and oligos were mixed with 1 uL dNTPs, 4 uL 5X RT buffer, 1 uL RNAse Inhibitor Murine, 1 uL  Maxima enzyme and RNase-free water up to 14 uL. Reaction was then incubated at 60°C for 30 mins and inactivated by heating at 85°C for 5 mins. For TGIRT (InGex), RNA and oligos were mixed with 4 uL 5X TGIRT Reaction Buffer (2.25 M NaCl, 25 mM MgCl2, 100 mM Tris-HCl, pH 7.5), 1 uL 0.1 M DTT, 1 uL RNAse Inhibitor Murine, 1 uL  TGIRT and RNase-free water up to 9.75 uL. Mix was pre-incubated at room temperature for 30 minutes, then 1.25 uL dNTP was added. Reaction was then incubated at 60°C for 60 mins and inactivated by heating at 75°C for 15 mins. For Induro RT (NEB, M0681S),  RNA and oligos were mixed with 1uL dNTPs, 4 uL 5X Induro buffer, 1 uL RNAse Inhibitor Murine, 1 uL  Induro and RNase-free water up to 11 uL. Reaction was then incubated at 60°C for 30 mins and inactivated by heating at 95°C for 10 mins. For Marathon RT (Kerafast), RNA and oligos were mixed with 1uL dNTPs, 10 uL 2X Marathon buffer (400 mM KCl, 4 mM MgCl2, 100 mM Tris-HCl, pH 8.3, 10 mM DTT, 40% Glycerol), 1 uL RNAse Inhibitor Murine, 1 uL  Marathon and RNase-free water up to 6 uL. Reaction was then incubated at 42°C for 30 mins and inactivated by heating at 95°C for 1 mins. For the manganese buffer conditions of Protoscript II, Superscript II, Superscript III, Superscript IV, Maxima, and TGIRT, buffers disclosed by the manufacturer were prepared by replacing Magnesium with the same amount of Manganese Chloride (Thermo, AAJ63150AD). For Induro, since the manufacturer does not disclose the RT buffer composition, the TGIRT buffer (with Manganese) was used. For Marathon RT, as suggested in literature 24, 2X Marathon RT buffer was prepared, replacing Magnesium with 1 mM Manganese. Once the reverse transcription reaction was performed, RNAse Cocktail was added to the mix in order to digest the RNA, and the mix was incubated at 37°C for 10 minutes. The reaction was then cleaned up using 0.8X AMPure XP Beads. In order to be able to ligate the sequencing adapters to the first cDNA strand, 1 µL 25 µM Comp_DNA was re-annealed to the 15 µL cDNA in a tube with 2.25 µL 0.1 M Tris pH 7.5, 2.25 µL 0.5 M NaCl and 2 µL nuclease-free water. The mix was incubated at 90°C for 1 minute and the temperature was ramped down to 25 °C (-0.1°C/s) in order to anneal the complementary to the first strand cDNA. The ligation of the barcode was performed by mixing 22.5 uL end-prepped DNA, 2.5 uL Native Barcode (NB), 25 uL Blunt/TA Ligation Master Mix (NEB, M0367S) and incubating the mix at room temperature for 10 mins. The reaction was then cleaned up using 0.8X AMPure XP Beads. Once all the barcoded libraries were pooled after elution, the ligation of the sequencing Adapter Mix (AMX) was performed by mixing the 65 uL pooled barcoded DNAs (up to 200 fmol), 5 uL AMX, 20 uL 5X NEBNext Quick Ligation Reaction Buffer and 10 uL Quick T4 DNA Ligase, and incubating the mix at room temperature for 10 mins. The reaction was cleaned up using 0.8X AMPure XP beads, using WSB Buffer for washing. The sample was then eluted in Elution Buffer (EB) and mixed with Sequencing Buffer (SQB) and Loading Beads (LB) prior to loading onto a primed  R9.4.1 flowcell. Library was run on MinION flowcell with MinKNOW acquisition software version v.3.5.5. A detailed step-by-step DMS-FIRST-seq protocol is provided as an additional file, both for R9 and R10 flowcells (File S1). DMS-FIRST-seq libraries of in vitro DMS modified RNAs were prepared using TGIRT in manganese buffer. In vivo DMS modified RNAs, Induro in manganese buffer was used (TGIRT not commercially available).
identifiers.namespace: GEO
identifiers.id: GSM8771341_r1