GSM9702676: DIS complex (replicate 3); Homo sapiens; RIP-Seq
The nuclear extracts for cryo-EM, RNA sequencing and biochemical assays were prepared from the human K562 cells stably overexpressing GFP-tagged DHX35 or YJU2B as described previously (Vorländer, M. K. et al. Mechanism for the initiation of spliceosome disassembly. Nature 632, 443–450 (2024)). ~25 ml of the nuclear extract of K562 cells stably overexpressing DHX35-GFP or YJU2B-GFP was applied to ~1.5 ml of GFP-Trap Agarose beads (Chromotek) equilibrated in wash buffer (20 mM HEPES-KOH pH 7.9, 100 mM KCl, 0.5 mM TCEP, 2 mM MgCl2, 0.05% v/v Igepal CA-630, 8% v/v glycerol). The beads were incubated with the nuclear extract for ~3.5 h at 4°C with gentle rotation. The beads were then washed five times in 10 ml wash buffer. The washed beads were incubated twice for ~1 h at 4°C with gentle rotation in 0.5 ml elution buffer (20 mM HEPES-KOH pH 7.9, 100 mM KCl, 0.5 mM TCEP, 2 mM MgCl2, 3% w/v sucrose, 0.1 µg/ml 3C protease). The eluted samples were then applied to a 15-30% w/v sucrose gradient (20 mM HEPES-KOH pH 7.9, 100 mM KCl, 0.5 mM TCEP, 2 mM MgCl2). The gradient samples were spun in an SW60 Ti rotor (Beckman Coulter) at 22,000 rpm for 16 h at 4°C. The gradients were fractionated manually, and each fraction was quenched with 45 mM lysine. The sedimentation coefficients were estimated using the CowSuite software, and the peak spliceosome fractions were further confirmed by SDS-PAGE. The peak spliceosome fractions were pooled and concentrated using Amicon Ultra 0.5 ml concentrators with 100 kDa molecular weight cut-off (Sigma). RNA was extracted from the fractions using Trizol LS and RNA was precipitated. The pellet was resuspended in 10 ul of water. RNA from immunoprecipitated spliceosomal complexes was extracted from the same gradient fractions used for cryo electron microscopy. For each sample, 3 ul of RNA was poly-adenylated by incubating in a reaction containing 2 ul 5xSSIV reaction buffer (18091050, Invitrogen), 0.25 ul E. coli Poly-A Polymerase (M0276S, NEB), 0.25 ul RNasin (N2511, Promega), 0.25 ul 0.1M DTT (18091050, Invitrogen) and 0.5 ul 0.5 mM ATP (18091050, Invitrogen) at 37C for 15 minutes. The samples were placed on ice, diluted with 9.25 ul water, then heated at 95C for 4 minutes to fragment the RNA. To anneal reverse transcription primers, 1 ul of 10 mM dNTPs and 1 ul of 10 uM dT(22)V-P7 primer were added, and the samples were heated to 65C for 5 minutes before cooling to 42C. To initiate reverse transcription, 2 ul of 5xSSIV reaction buffer (18091050, Invitrogen), 0.5 ul 0.1M DTT (18091050, Invitrogen) and 0.5 ul SSIV enzyme (18091050, Invitrogen) were added to the samples and the reaction was incubated at 42C for 20 minutes. The reactions were cooled to 37C and incubated with 2 ul Exonuclease I (M0293S, NEB) for 15 minutes to remove excess RT primers. To further remove excess RT primers, the samples were size selected using SPRI beads with a final PEG8000 concentration of 7.5% and a final isopropanol concentration of 22.5%. The samples were eluted from the beads, then RNA was hydrolysed by the addition of 1 ul 1M NaOH and incubation at 95C for 10 minutes. The pH of the samples was then neutralised by the addition of 1 ul 1M HCl and 10 ul 1M HEPES pH 7.9. The cDNA was then purified using 2 volumes of SPRI beads and 3 volumes of isopropanol. The cDNA was eluted in 5.3 ul water, to which 1 ul DMSO and 2 ul 10 uM L01clip2.0 adapter was added. The mixture was heated to 75C for 2 minutes then placed on ice. Adapter ligation was initiated by the addition of 2 ul 10x T4 RNA Ligase Reaction Buffer (B0216SVIAL, NEB), 0.2 ul 100 mM ATP (N0437AVIAL, NEB), 9 ul 50% PEG 8000 (B1004SVIAL, NEB) and 0.5 ul T4 RNA Ligase 1 High Concentration (M0437MVIAL, NEB) and the reaction was incubated overnight at room temperature. The reaction was size selected using 20 ul SPRI beads and 13.33 ul isopropanol (final concentration of 25%). The resulting cDNA was amplified in a 50 ul qPCR reaction using Illumina-compatible i5/i7 indexed primer pairs and NEBNext® Ultra™ II Q5® (M0544L, NEB) according to manufacturer instructions until the libraries appeared to amplify linearly. The resulting libraries were purified using 1.5 volumes of SPRI beads, then sequenced using a NovaSeqX 10B XP 2x150bp flow cell (Illumina) at the VBCF Next Generation Sequencing Facility.