title: GSM9329316: Monomac6, FB23-2 treated, biological replicate 3; Homo sapiens; OTHER
library_construction_protocol: Cellular total RNA in TRIzol LS (ThermoFisher #10296028) was extracted by Direct-zol RNA Miniprep kit (Zymo #R2070) Library strategy: CROWN-seq; To study snRNA m6Am stoichiometries, we used a modified protocol of CROWN-seq, in which 2'-O-methyladenosine (Am) is chemically converted into 2'O-methylinosine (Im) and N6,2'O-dimethyladenosine (m6Am) remains unconverted, allowing for m6Am quantification. Short RNAs (17-200 nt) were isolated from total RNA using RNA Clean & Concentrator-5 (Zymo Research, R1016), following the Purification of Small and Large RNAs into Separate Fractions protocol. 100 ng of the small RNA fraction, in 8 µl nuclease-free water, was used as input. Following the conditions from the GLORI2.0 protocol, samples were converted in deamination buffer (13μl glyoxal, 10μl DMSO, 10μl 5 M NaNO2, 5μl H3BO3, 4μl 500 mM MES (pH 6.0)) for 10 min at 50°C. Next, samples were ethanol precipitated, and the glyoxal adduct was removed with deprotection buffer (500 mM TEAA pH=8.6, 47.5% deionized formamide, 2mM EDTA) for 10 min at 95°C. RNA was collected again by ethanol precipitation, denatured for 2 min at 65°C, and spiked with 10 ng of Drosophila melanogaster embryo Poly A+ RNA (Takara Bio, #636224) for QC. To eliminate contamination of RNA species with a 5'-triphosphate or 5'-monophosphate, a dephosphorylation reaction was set up with 5 µl 10X CutSmart buffer, 5 µl Quick CIP (5 U/µl) (NEB, M0525L), and 1 µl SUPERase·In RNase inhibitor (ThermoFisher #AM2696) for 2 hours at 37 °C. The reaction was cleaned up using a Zymo RCC-5 column, and the RNA was eluted in 15.5 µl water. Next, RNA was denatured for 5 min at 65°C before removing the m7G-cap with 2 µl mRNA Decapping Enzyme (100U/µl) (NEB, M0608S), 2 µl 10X Decapping Enzyme Reaction Buffer, and 0.5 µl SUPERase·In RNase inhibitor for 1 hour at 37°C. The decapped 5'-monophosphate RNA was purified with ethanol-AMPure XP (RNA:beads:ethanol=1:2:3), washed twice in 80% ethanol, and eluted in 11.5 µl water. 5' ligation was carried out with 20 pmol of 5' adapter (rCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrNrNrNrNrNrNrNrNrUrUrA), 2 µl T4 RNA ligase 1 (ssRNA Ligase, 30U/µl) (NEB, M0437M), 4 µl 10X T4 RNA Ligase Buffer, 4 µl 10mM ATP, 0.5 µl SUPERase·In RNase inhibitor, and 16 µl 50% PEG8000 for 2 hours at 25 °C. 5' ligation product was again purified with ethanol-AMPure XP and eluted in 16 µl water. End repair was carried out with 1 µl T4 PNK (NEB, M0201L), 2 µl 10X T4 PNK Reaction Buffer, and 1 µl SUPERase·In RNase inhibitor for 30 min at 37 °C. End-repaired RNA product was purified with ethanol-AMPure XP and eluted in 12 µl water. 3' adapter incorporation and cDNA synthesis were carried out via a template switching reaction with an R2R RNA/R2R DNA heteroduplex adapter based on TGIRT-seq (since A is assumed to be converted from GLORI, the R2R DNA mix consists of A, G, or C overhangs in a 1:1:2 ratio, see Table S4). The template switching reaction was first pre-incubated at room temperature for 30 min with 2 µl 10X annealed R2 RNA/R2R DNA heteroduplex mix (100 nM final conc.), 1 µl Induro RT (200U/µl) (NEB, M0681L), 4 µl 5X Induro RT Buffer, and 1 µl SUPERase·In RNase inhibitor; next, 1 µl 10mM dNTP was added before incubating for 5 min at 25 °C, 15 min at 55 °C, 1 min at 95 °C, and holding at 4 °C. 5 µl of the cDNA product was carried forward for indexing PCR, mixed with 15 µl water, 25 µl Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531L), 2.5 µl i5 indexing primer, and 2.5 µl i7 indexing primer (NEB, E7780S). Cycling conditions were 98 °C for 30 sec, then 14 cycles of 98 °C 15 seconds, 65 °C 30 seconds, and 72 °C 15 seconds, with a final 5 min 72 °C extension. Two rounds of 0.9X AMPureXP bead purifications were performed to remove primers. 10-20 ng indexed library was obtained for each library. The libraries were mixed and sequenced by NovaSeqX