GSM5857958: Ecoli_none_rep2; Escherichia coli; RNA-Seq
For HEK293T cells, TRIzol reagent (Thermo Fisher Scientific 15596026) was used to extract total RNAs at each passage by following the manufacturer's manual. For E. coli, RNA was extracted by a hot acid-phenol protocol.  Briefly, 0.5 mL of acid-buffer phenol (pH 4.5 citrate) was added to frozen samples.  Samples were incubated in a heat block with shaking at 50°C for 30 min.  The aqueous phase was extracted for another round of phenol extraction and 2 rounds of chloroform extraction before ultimately precipitating with glycoblue, 300 mM sodium acetate, and 3 volumes of ethanol.  Samples were incubated for 1 hour at -80°C, then centrifuged at maximum speed (20k RCF) for 45 min to pellet RNA.  Pellets were washed twice with 70% ethanol, then resuspended in water. Libraries were prepared as previsouly described: 33337498. Briefly, Samples were incubated overnight at 16 °C in a 50 μL ligation solution with the following final concentration: 15% PEG 8000, 1x NEB T4 RNA ligase I buffer, 50 μM ATP, 5% DMSO, 1 mM hexaamine cobalt (III) chloride, 0.8 μM barcode ligation oligo, and 1 U/μL NEB T4 RNA ligase I. After overnight ligation, 50 μL of 100 mM EDTA were added to each sample to inactivate the ligase. The samples were then combined and 8 μL of ThermoFisher Streptavidin MyOne C1 Dynabeads per sample were added to the combined samples. The biotinylated samples were allowed to bind the beads for 15 minutes. After binding, the beads were magnetized, the supernatant was removed, and the samples were washed once with a high salt Tween wash buffer (1 M NaCl, 0.1% Tween 20, and 20 mM TrisHCl, pH 7.4) and once with low salt wash buffer (100 mM NaCl, 20 mM TrisHCl, pH 7.4). Periodate treatment was included when indicated - either before first ligation, or before reverse transcription. Before reverse transcription, 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. For reverse transcitpion, 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, then incubated at 30 C overnight. 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. 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 S1), 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. 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. Following desalting, PCR products were run on 10% non-denaturing TBE gel with dsDNA size markers; lanes were cut for inserts between 20ng and 300nt, mashed by pipette tip, and then resuspended in crush-and-soak buffer