100 h5UTRs were chosen for amplicon sequencing based on coverage profiles from ENCODE E14 mESC RNA-seq data. A total of 384 primer pairs were designed across these h5UTRs using ThermoAlign and following parameters (others are left at default): primer_size_range=16-40, GC_range=25-75, Tm_range=64-72, primer_conc=300, Na=0, K=75, Tris=10, Mg=2, dNTPs=1.2, amplicon_size_min=250, amplicon_size_max=250. Primers were split into 4 pools of 96 pairs using PrimerPooler106. A final set of 380 pairs were synthesized by Eurofins Genomics. 10x106 mESCs were incubated for 10min in ATP depletion media: DMEM without glucose (ThermoFisher Scientific, A1443001), 10mM 2-Deoxy-D-glucose (MilliporeSigma, 25972), 10mM sodium azide (MilliporeSigma, 71289). The cells were washed, trypsinized and harvested using PBS, trypsin, and finally resuspended in 3.5mL mESC media all containing 10mM 2DG and 10mM NaN3. 1mL 1M bicine (Millipore Sigma, B3876), titrated to pH 8.5 at 25°C, is added to resuspended cells (250mM final bicine concentration). 500uL 16% dimethyl sulfate (MilliporeSigma, D186309) in ethanol is added (1.6% final concentration). Cells are mixed and incubated for 6min at 37°C. 2.5mL ice-cold 30% BME (MilliporeSigma, M3148) in ethanol is added to quench the reaction. Following centrifuge to remove the supernatant, the cells are lysed in Trizol (ThermoFisher Scientific, 15596026). For the untreated condition without ATP depletion treatment, all procedures are the same except for initial 10min incubation in ATP depletion media and inclusion of 2DG and NaN3 in all media. Three independent samples were collected for each condition. Total RNA is phase extracted with chloroform and aqueous phase is purified on silica columns (Zymo, R1013). 10-20ug RNA is DNase treated at 37°C for 30min using 0.2U/uL TURBO DNase (ThermoFisher Scientific, AM2238) with 1U/uL Superase-In (ThermoFisher Scientific, AM2696) in 60uL and purified again on silica column. 1ug RNA is mixed with 1uL 1uM 96x primer pool (96x1uM amplicon reverse primers for total of 1uM oligos) and denatured in 6.25uL total volume (with H2O) at 65°C for 2min, then chilled to 4°C. Reverse transcription reaction conditions are as follows: 20mM Tris-HCl pH7.5, 75mM KCl, 10mM MgCl2, 5mM DTT, 500nM TGIRT (InGex, TGIRT50), 1U/uL Superase-In; 19uL total reaction volume. The RT reaction is preincubated for 25°C for 30min, then initiated with addition of 1uL 12.5mM dNTP each. After incubation at 60°C for 1hour, 1uL 2.5M NaOH is added and the reaction is heated at 95°C for 3min. 1uL 2.5M HCl and 1uL 500mM Tris-HCl pH 7.5 is added to neutralize. 29uL SPRIselect beads (Beckman Coulter, B23318) is used for purification of cDNA; elution volume is 6uL. 4x96 pooled reactions for a total of 384 targets are performed for each sample. To each set of 96 pool cDNA, multiplex PCR is performed with 5uL cDNA, 0.2mM dNTP, 2uM 96x forward primer pool (96x2uM each primer for total of 2uM oligos), 2uM reverse primer pool, 1x SYBR Green I (ThermoFisher Scientific, S7563), Q5 Hot Start DNA polymerase (NEB, M0493S), 1x Q5 Hot Start Reaction Buffer, 1x Q5 Hot Start High GC Enhancer, in total 30uL reaction volume. Cycling conditions are: 98°C 30s initial denaturation, followed by 15-25 cycles (terminated before plateau) of 98°C 10s, 56°C 40s, 76°C 10s. PCR is run for 15-25 cycles. Each 96 pool multiplex PCR reaction is then used in a master mix of second PCR with 96 individual primer pair reactions: 0.2mM dNTP, 500nM forward primer, 500nM reverse primer, 1x SYBR Green I, Q5 Hot Start DNA polymerase, 1x Q5 Hot Start Reaction Buffer, 1x Q5 Hot Start High GC Enhancer, in total 6uL reaction volume. Cycling conditions are: 98°C 30s initial denaturation, followed by 20 cycles of 98°C 10s, 62°C 10s, 76°C 10s. 384 individual reactions are pooled and purified on silica columns (NEB, T1030S). Amplicon pool is end-prepared, Illumina adaptor sequences are ligated, adaptor-ligated DNA is size selected with SPRIselect beads for 370bp, and 3 cycle barcoding PCR is performed (NEB, E7645S). 2x150bp paired end sequencing data is generated on Illumina HiSeq 4000 at Novogene.
GEO Accession
GSM4709632