RNP-seq and IP-seq from cells expressing epitope-tagged RBM5, RBM10, or SF3A3
We isolated snRNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of lysed nuclei. These complexes contained U2 snRNP proteins and a portion of the U2 snRNA, bound with intronic branch sites prior to the first catalytic step of splicing. The complexes have similar composition, whether directly isolated from extracted material, or after selection from glycerol gradients. Here we describe sequencing libraries prepared from RNA derived from directly isolated (IP-seq), or gradient-purified complexes (RNP-seq). Libraries from deproteinized RNA were prepared after selective degradation of U2 snRNA, each in a triplicate. An additional library from RBM5-Flag RNP complex was prepared without such degradation. The majority of sequenced pre-mRNA fragments map to intronic branch sites. Overall design: High molecular weight (HMW) nuclear extracts were obtained after enzymatic solubilization with a cocktail of RNase A+T1, DNase, and Benzonase from 293Flp-in cells expressing RBM5, RBM10, or SF3A3 protein with a C-terminal Flag tag. RBM5-Flag and SF3A3-Flag extracts were sedimented through 10-30% glycerol gradients in SW41Ti rotor (Beckman) for 17 hours at 32,000 RPM. The gradients were fractionated in 0.5 mL fractions top to bottom, fractions 11-13 containing peak RNP regions were dialyzed to reduce glycerol, and the complexes were immunopurified on anti-FLAG agarose. RBM10-Flag and SF3A3-Flag extracts were also directly incubated with anti-FLAG agarose to immunoprecipitate complexes. Following elution with 3xFLAG peptide and deproteinization, the RNA was incubated with U2-antisense oligonucleotide and RNase H to remove U2 snRNA. RNA fragments of 28-55 nt-size were selected from denaturing gels and used in construction of sequencing library with in-line barcodes. The libraries are sequenced in Illumina high-throughput sequencer at single end 100 nt.