title: GSM8422309: HEK293T FTO knockout, biorep 2, techrep 1; Homo sapiens; OTHER
library_construction_protocol: Cellular total RNA in TRIzol LS (ThermoFisher 10296028) was extracted by Direct-zol RNA Miniprep kit (Zymo #R2070). mRNA was purified by NEBNext Oligo d(T)25 Beads (NEB #E7499) based on mRNA purification from total RNA protocol of Dynabeads Oligo (dT)25 (Ambion #61002). 1.5-5 μg oligo(dT) selected RNA was used as input. RNA was first diluted in 14 μl water. To perform glyoxal protection, 6 µl 8.8 M glyoxal and 20 μl DMSO were then added to the diluted RNA and well mixed. The 40 µl mix was first incubated at 50 ºC for 30 minutes, then 10 μl boric acid was added to the mix. The 50 µl mix was then incubated for an additional 30 minutes at 50 ºC. After protection, the 50 μl protected RNA was mixed with 50 μl deamination buffer (25 μl 1500 mM NaNO2, 4 μl 500 mM MES, pH 6.0, 10 μl 8.8 M glyoxal, and 11 μl water). The deamination reaction was performed at 16 ºC for 8 hours. After deamination, the RNA was recovered by ethanol precipitation. To remove the glyoxal adduct from the RNA, the RNA pallet was dissolved in 50 μl deprotection buffer (500 mM TEAA pH=8.6, 47.5% deionized formamide) and was incubated at 95 ºC for 10 minutes. After incubation, the reaction was brought to 250 μl with water. Converted RNA was purified by ethanol precipitation and eluted in 39 μl water for 5' end enrichment. The converted RNA was stored at -80 ºC before 5' end enrichment. To eliminate the contamination of RNA with 5'-triphosphate and 5'-monophosphate, 5 µl 10X CutSmart buffer, 5 µl Quick CIP (5 U/µl) (NEB #M0525L), and 1 µl Superase*IN RNase inhibitor (ThermoFisher #AM2696) were added to the 39 μl converted RNA to set up a dephosphorylation reaction. The dephosphorylation reaction was performed at 37 °C for 30 minutes. The reaction was cleaned up using Zymo RCC-5 column and the RNA was eluted in 42 µl water. To de-cap the m7G capped RNA, a 50 µl decapping reaction was set up by adding 5 µl 10X yDcpS buffer, 2 µl (200 U) yDcpS (NEB #M0463S), and 1 µl Superase*IN to the 42 µl dephosphorylated RNA. The decapping reaction was performed at 37 ºC for 1 hour. This unique decapping enzyme liberates m7GMP, resulting in mRNAs with a 5'-diphosphate. Reaction was cleaned and eluted as before. The reaction was cleaned up using Zymo RCC-5 column and the RNA was eluted in 33.5 µl water. The 5'-diphosphorylated mRNAs were recapped with desthiobiotin-GTP (DTB-GTP, NEB #N0761S) using vaccinia capping enzyme (5 μL VCE buffer, 0.5 μL inorganic pyrophosphatase (NEB #M0361S), 5 μL DTB-GTP (5 mM; NEB N0761S), 50 U VCE, 1 µl Superase*IN) at 37 °C for 4 hour. The reaction was cleaned up using Zymo RCC-5 column and the RNA was eluted in 30 µl water. Now the RNA is ready for streptavidin enrichment. Streptavidin beads (NEB #S1421S) were washed in a high salt wash buffer (10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA) and resuspended in the high salt buffer at 4 mg/mL. To enrich the RNA and tag the 5' and 3' end by the specific adapter, the 30 µl recapped RNA was first mixed with 30 µl streptavidin beads and incubated at room temperature for 45 minutes with agitation. Beads were washed twice in high salt buffer, twice in a lower salt buffer (10 mM Tris-HCl pH 7.5, 250 mM NaCl, 1 mM EDTA), and twice in PNK wash buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.2% Tween). To remove 3' phosphates resulting from fragmentation during conversion, beads were resuspended in 50 µl PNK reaction without ATP (5 µl 10X PNK buffer, 1 µl T4 PNK (#M0201S), 1 µl Superase*IN, 43 µl water), and incubate at 37 °C for 30 minutes with agitation. The beads were then washed once in PNK wash buffer, once in 2 M NaCl wash, and twice in PNK wash. Next, RNA was ligated to a 74 nt-long 3' adapter (/5'rApp/AGATCGGAAGAGCACACGTCTGAACTCCAGTCACAAAAAAAAAAAAAAACCCCCCCCCCAAAAAAAAAAAAAAA/3AmMO/) in the following 40 μl 3' ligation mix: 4 μL T4 RNA ligase buffer, 2 μl T4 RNA ligase 2 truncated KQ (NEB #M0373L), 1 µl Superase*IN, 2 μl extended-L7 adapter (20 μM stock, see Key Resource table), 16 μl of 50% PEG-8000) and incubated at 25 °C for 2 hours. After incubation, the reaction buffer was removed by washing once with high salt buffer and twice with PNK wash buffer. To remove the exceeded adapter, the beads were incubated in 50 μl adapter digestion reaction (40 μl waater, 5 µl 10X RNA ligase buffer, 1 µl RecJf (NEB #M0264S), 1 5' Deadenylase (NEB #M0331S), 1 µl Superase*IN) at 30 °C for 15 minutes then at 37 °C for 15 minutes. The beads were washed once with PNK wash, once with high salt buffer and twice with low salt buffer. To elute the DTB labeled RNA, beads were then suspended 30 µl low salt wash buffer containing 1 mM free D-biotin (ThermoFisher #B20656) and incubate at room temperature for 1 hour. The DTB labeled RNA were purified with ethanol-AMPure XP (RNA:beads:ethanol=1:2:3) and elute in 30 µl water. To increase stringency, the streptavidin bead enrichment was repeated omitting enzymatic steps and instead washing three times with high salt and then three times with lower salt wash, and the eluate cleaned again by ethanol-AMPure XP. The DTB-GTP cap was removed using 0.5 U/µl RppH in 1X ThermoPol buffer (NEB #M0356S) and incubating at 37 °C for 1 hour. The resulting 5'-monophosphate RNA fragments were purified by ethanol-AMPure XP and eluted in 10 μl water. 1 μl (10 pmol) reverse transcription primer (AGACGTGTGCTCTTCCGATCT) was pre-annealed to the templates by heating up to 90 °C for 30 seconds, then 65 °C for 5 minutes, and chilled at 4 °C for over 30 seconds. 30 pmol of a 5' adapter was ligated for 3 hours at 25 °C with 2 U/μl T4 RNA ligase 1 (NEB #M0437M). This RNA adapter contains an 11 nt UMI followed by a fixed sequence (AUAU) at its 3' end. The UMI allows robust duplicate removal, and the fixed sequence provides an anchor point to correctly identify the first nucleotide of the mRNA. 40 μl ligation product was used. Reverse transcription was carried out at 50 °C for 45 minutes in a 50 μl reaction containing 0.5 mM dNTPs, 5 mM DTT, 20 U RNaseOUT, 50 mM Tris-HCl pH 8.3, 75 mM KCl, and 300 U SuperScript III. To perform indexing PCR, 40 μl Phusion master mix (NEB # M0532L) was added to the reverse transcription product, along with 5 μl i5 indexing primer and 5 μl i7 indexing primer (NEB #E7600S). Cycling conditions were typically 98 °C 2 minutes, then 16 cycles of 98 °C 15 seconds, 65 °C 30 seconds, 72 °C 30 seconds, with a final 5 minute 72 °C extension. Two rounds of 0.9X AMPureXP beads purifications were performed to remove primers.