Dynabeads myone streptavidin t1 magnetic beads

5-ethynylcytosine was synthesized as previously described [14 (link)]. Biological triplicate samples were prepared by carrying out 5EC feeding and RNA processing independently. Larvae were reared at 25 °C and fed 1 mM 5EC from 72 – 84 h after hatching prior to RNA extraction (pulse samples) or transferred to media with 10 mM uridine for 3, 6, or 12 h prior to RNA extraction (chase samples). Crudely dissected central nervous system RNA was extracted using Trizol. For each genotype and timepoint, duplicate 20 mg RNA samples were biotinylated using Click-iT Nascent RNA Capture reagents (ThermoFisher), purified on Dynabeads MyOne Streptavidin T1 magnetic beads (ThermoFisher) and used for “on bead” RNA-seq library synthesis, as previously described [15 (link)].

Equal volumes of the individual monkey serum sample and 600 mM acetic acid were combined and incubated at room temperature for 1 h. The acidified serum samples were then diluted with HBS-EP buffer + 0.2% Tween-20. Biotinylated REGN-P or REGN-N (produced in-house, 2 μg/sample) was added to each serum sample (final volume of 500 μL) and incubated for 1 h at room temperature to form a complex with ADA-PC or ADAs. Ten (10) microliter of Dynabeads MyOne™ Streptavidin T1 magnetic beads (Thermo Fisher Scientific, Waltham, MA) were incubated with 3% BSA in HBS-EP + 0.2% Tween-20 buffer for 30 min before being added to the serum samples and subsequently incubated at room temperature for 15 min. The samples were then washed twice with 3% BSA in HBS-EP + 0.2% Tween-20 buffer followed by 2 washes with HBS-EP + 0.2% Tween-20 buffer and elution with 0.1% formic acid (FA) and 50% acetonitrile.

HEK293 cells were transfected with Rbm38-Myc plasmids using Lipofectamine 2000 reagent (Thermo Fisher Scientific). At 48 h post-transfection, cells were harvested, washed with cold PBS buffer, suspended in Buffer D (20 mM HEPES (pH 7.9), 50 mM KCl, 0.2 mM EDTA, and 20% glycerol), sonicated for 20 s, and centrifuged (13,000× g, 10 min, 4 °C) to remove debris. Biotin-labeled SMEK2 intron 4 was transcribed with the SMEK2 intron 4 plasmid and the MEGAscript T7 transcription kit (Thermo Fisher Scientific), in accordance with the manufacturer’s instructions. The biotinylated substrate mRNA (10 pmol) was immobilized with 5 µL of Dynabeads MyOne StreptavidinT1 magnetic beads (Thermo Fisher Scientific), in accordance with the manufacturer’s instructions. The immobilized pre-mRNA beads were incubated at 30 °C for 20 min in 100 µL of HEK293 whole extract, RNase inhibitor (Takara Bio, Otsu, Japan), and nuclease-free water. Then, NET2 buffer (50 mM Tris-HCl (pH7.5), 150 mM NaCl, and 0.05% Nonidet P-40) was added to a final volume of 800 µL and incubated at 4 °C for 1 h. The incubated beads were washed five times with cold NET2 buffer and then boiled in 1× SDS-PAGE sample buffer for analysis by Western blotting.

HeLa cells were treated with 100 μM cisplatin for 2 h. After 0, 6 or 24 h of incubation in complete medium, the damaged cells were further cultured in serum-free DMEM containing 100 μM 5-ethynyluridine for 1 h. Similarly, Sf9 cells were incubated in Sf-900 II SFM (Thermo Fisher Scientific) containing 100 μM 5-ethynyluridine for 1 h. After the cells were counted and total RNA was extracted by Sepasol-RNA I Super G, a spike of Sf9 RNA was added to each RNA sample prepared from the same number of HeLa cells. Nascent RNA was biotinylated by using a Click-iT Nascent RNA Capture Kit (Thermo Fisher Scientific), purified with ethanol precipitation, immobilized to Dynabeads MyOne Streptavidin T1 magnetic beads (Thermo Fisher Scientific), and reverse-transcribed by using Superscript III Reverse Transcriptase (Thermo Fisher Scientific) with seven gene- specific primers on the beads. qPCR reactions were carried out by using KAPA SYBR FAST qPCR Kit Master Mix (2×), an ABI Prism (KAPA Biosystem), the appropriate primer set (Supplementary Table S1), and a StepOnePlus Real Time PCR System. The ΔΔCt method was employed to normalize the results to Spodoptera frugiperda GAPDH as a reference gene.

VariantBaits™ Target Enrichment Library Prep Kit (VBS95-1023-96D01; LC-Bio Technology, Co., Ltd. Hangzhou, China); VariantBaits™ Target Enrichment Purification Beads (NO. VBS95-0518-96D01; LC-Bio Technology, Co., Ltd. Hangzhou, China); VariantBaits™ Target Enrichment Probe (NO. VBS95-7336-96D01; LC-Bio Technology, Co., Ltd. Hangzhou, China); VariantBaits™ Target Enrichment Amplification Kit (NO. VBS95-1229-96D01; LC-Bio Technology, Co., Ltd. Hangzhou, China); VariantBaits™ Target Enrichment Wash Kit (NO. VBS95-0128C01-96D01; LC-Bio Technology, Co., Ltd. Hangzhou, China); VariantBaits™ Target Enrichment Hybridization Kit (NO. VBS95-0128C02-96D01; LC-Bio Technology, Co., Ltd. Hangzhou, China); VariantBaits™ Target Enrichment Adapter & Block Oligo A (NO. VBS95-0128C03A-96D01; LC-Bio Technology, Co., Ltd. Hangzhou, China); VariantBaits™ Target Enrichment Adapter & Block Oligo B (NO. VBS95-0128C03B-96D01; LC-Bio Technology, Co., Ltd. Hangzhou, China); DynabeadsMyOne Streptavidin T1 Magnetic Beads (NO. 65602; Invitrogen, US); High Sensitivity DNA Kit (NO. 5067-4626; Agilent, US); Qubit 1x dsDNA HS Assay Kit (NO. Q33230; Invitrogen, US); Platinum™ SYBR™ Green qPCR SuperMix-UDG (NO. 11733038; Invitrogen, US); Nuclease-Free Water (NO. AM9930; Ambion, US); Pipette head, 10 μL,200 μL,1000 μL, (Axygen,US); Eppendorf Tube,200 µL,600 µL,1.5 mL,2.0 mL (Axygen,US).