title: GSM5946960: HEK293T_polysomepolyARNA_heatstress_longRT_minusDM_rep3; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were collected and total RNA was extracted using TRIzol (ThermoFisher, 15596026) by following the manufacturer's protocol when cells reached 80-90% confluency. Poly(A)-selection was done with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490S) according to manufacturer's instructions: 1 µg of input polyA+ RNA samples and 100 ng polysome polyA+ RNA samples were used to build sequencing libraries. The RNA fragmentation and end repair steps are the same as the CMC sequencing libraries construction. Briefly, polyA+ RNA in 18 µl were added to PCR tubes and 2 µl Magnesium RNA fragmentation buffer (NEB, E6150S) was added to each tube. The tubes were incubated at 94°C in a thermocycler for 5 mins to fragment the RNA to ~200 nt. The tubes were transferred to ice and 2 µl RNA fragmentation stop solution was then added to each tube to stop the fragmentation. Samples were spun down and diluted to 50 µl using sterile H2O. The fragmented RNA was purified using Zymo RNA clean and concentrator columns and eluted in 16 µl sterile H2O. 2 µl T4 PNK buffer and 2 µl T4 PNK were added to the tubes and the tubes were incubated at 37 °C for 30 mins to repair the ends of the fragmented RNA. MSR-seq method was then used to build sequencing libraries with the fragmented RNA. Slight modifications were made to the MSR-Seq protocol. After the first ligation step, ligation reaction was quenched by adding 50 mM EDTA. Samples were combined and ligation products over 200nt long was purified using Zymo RNA clean and concentrator columns twice. Target cDNA products were purified using AMPure Total RNA was prepared for library construction by first deacylating in a solution of 100 mM Tris-HCl, pH 9.0 at 37 °C for 30 minutes, then neutralized by addition of sodium acetate, pH 4.8 to a final concentration of 180 mM. Deacylated RNA was then ethanol precipitated and resuspended in water, or desalted using a Zymo Research Oligo Clean-and-Concentrator spin column. First bar-code ligation: Depending on the nature of the experiment described in the main text, input RNA or total nucleic acid samples were either deacylated or had undergone one-pot deacylation and β-elimination as described above. Up to 1 µg of total RNA input was used in a ligation reaction of 50 µL with the following components: 1 U/µL T4 RNA ligase I (NEB), 1x NEB T4 RNA ligase I buffer, 15% PEG 8000, 50 µM ATP, 1 mM hexaamine cobalt chloride, and 5% DMSO. After adding the ligation mix to the sample, the capture hairpin oligo (CHO, Table S5) was added to a final concentration of 1 µM and the samples were incubated at 16 °C overnight (12+ hours). Binding to Dynabeads: The ligation mixture was diluted by adding an equal volume of water to reduce the viscosity of the solution. Next, streptavidin-coated MyOne C1 Dynabeads (ThermoFisher) were added to each sample in a 1.2:1 excess over CHO (for example, a 50 µL reaction had 50 pmol capture hairpin oligo; beads were supplied at 10 mg/ml and had binding capacity of 500 pmol biotinylated oligo per mg, so use 12 µL beads). The bead-sample mixture was incubated at room temperature for 15 minutes. After binding, supernatants were removed, and the beads washed once with high salt wash buffer (1 M NaCl, 20 mM Tris-HCl, pH 7.4) and once with low salt wash buffer (100 mM NaCl, 20 mM Tris-HCl, pH 7.4). After washing, multiple individually barcoded samples can be combined for downstream steps. At this stage, enzymatic or chemical treatments can be incorporated to the library preparation protocol such as AlkB demethylase reaction or CMC treatments (see below).Dephosphorylation: A 50 µL dephosphorylation mix containing the following was added to the multiplexed sample on bead: 0.04 U/µL calf intestine phosphatase (Roche), 10 mM MgCl2, 0.5 mM ZnCl2, 20 mM HEPES, pH 7.3. The sample was incubated at 37 °C for 30 minutes. The sample was then washed once with high salt wash buffer and once with low salt wash buffer, then resuspended in 20 µL water. Reverse transcription: 5 µL of SuperScript IV VILO 5x master mix (ThermoFisher) was added to the dephosphorylated sample (final volume of 25 µL) and then incubated at 55 °C for 10 minutes. The sample was then washed once with high salt wash buffer and once with low salt wash buffer. Identical results were obtained upon extending the reaction time to 60 minutes. For overnight reverse transcription, after the initial 10 min incubation at 55 °C, the samples are further incubated at 35 °C overnight. RNase H digestion: Beads were then resuspended into 50 µL of RNase H master mix containing 0.4 U/uL RNase H (NEB) and 1x NEB RNase H buffer and incubated at 37 °C for 15 minutes. The sample was then washed once with high salt wash buffer and once with low salt wash buffer. The sample was then resuspended in 40 µL water. Periodate oxidation: 10 µL of 250 mM freshly prepared sodium periodate, 0.5 M sodium acetate, pH 5 were added to the RNase H digested sample and incubated at room temperature for 30 minutes. Afterwards, ribose was added to a final concentration of 167 mM to quench excess periodate at room temperature for 5 minutes. The sample was then washed once with high salt wash buffer and once with low salt wash buffer. Second ligation: Beads were resuspended into 50 µL of a ligation master mix with the following components: 2U/µL T4 RNA ligase I (NEB), 1x NEB T4 RNA ligase I buffer, 2 µM second ligation oligo (Table S5), 25% PEG 8000, 50 µM ATP, 7.5% DMSO, and 1 mM hexaamine cobalt chloride. After incubation at room temperature overnight (12+ hours), the reaction was diluted with 50 µL water to reduce viscosity, washed once with high salt wash buffer and once with low salt wash buffer, and then resuspended in water. The amount of water was 6 µL per sample in the first ligation reaction, before pooling the barcoded samples. For example, if the second ligation mixture contains a pool of 6 samples, the amount of water used for resuspension would be 36 µL. Samples can be stored at 4 °C or frozen at -20 °C; both can be used for the next PCR step. PCR: A 50 µL PCR reaction was run using 5-10% of the bead slurry products from the second ligation reaction using Q5 DNA polymerase (NEB) and following the manufacturer's instructions: 0.02 U/µL Q5 DNA polymerase, 1x Q5 reaction buffer, 0.2 mM dNTPs, 0.5 µM Illumina index primer (Table S5), and 0.5 µM Illumina multiplex primer (Table S5). Typical PCR cycles were 9-15 cycles at 10 seconds at 98 °C, 15 seconds at 55 °C, and 72 °C for 20 seconds. PCR reactions were then processed through the DNA Clean and Concentrate kit (Zymo Research). TBE-PAGE gel extraction: Following desalting, PCR products were run on 10% non-denaturing TBE gel with dsDNA size markers; lanes were cut according to the desired product size, mashed by pipette tip, and then resuspended in crush-and-soak buffer (500 mM sodium acetate, pH 5.0). The gel fragments were extracted overnight and then ethanol precipitated. MSR-seq, mRNA-seq, DM-tRNA-seq