![]() (C) Left: A schematic of a gel image where RT products (lane 3) are resolved on UREA-PAGE and visualized via SYBR–gold staining along with a low range DNA ladder (lane 1) and unextended RT primer (lane 2). ![]() The RT reaction includes biotinylated dATP and dCTP to specifically label the extended RT products for enrichment in the next step. (B) Ligation product from A is then used for reverse transcription reaction (in the same tube as ligation) using the special RT primer that includes a region complementary to mirCat33 adapter, binding sites for read two primer binding sequence linked via polyethylene glycol linker (Sp18) to read one primer binding sequence, the 8-nucleotide random sequence that serves as unique molecular identifier (UMI) and two Gs at the 5′-end that conform to the preferred base for the CircLigase used in a subsequent step. (A) The first step consists of ligation of a pre-adenylated mirCat33 adapter to the dephosphorylated 3′ end of RNA footprints with T4 RNA Ligase 2, truncated K227Q. A known amount (0.1pmole) of single-stranded RNA oligo (lane 1) is radio-labeled and run on the gel to estimate RIPiT RNA concentration.Īn overview of the main steps in cDNA library prep for high-throughput sequencing. Size distribution of the RNA footprints can be estimated via comparison with radio-labeled base-hydrolyzed poly-U 45 RNA ladder (lane 2) and DNA ladders (lane 4 and 5). ![]() Shown image was spliced at the vertical dotted lines. (B) An example of RNA footprints from FLAG-CASC3:EIF4A3 containing complexes following 5′-end-labeling with ATP and resolution on a 26% Urea-PAGE gel. As seen in the figure, different complexes have different composition, as reflected by different levels of UPF2 protein in the three complexes. Cell lines expressing the endogenously tagged protein used in RIPiT are indicated above each lane. Western blots showing levels of the proteins (on the right) in various fractions labeled on the top (EL, elution FT, flowthrough). (A) An example of RIPiT validation via western blots. Visualization of proteins and RNAs enriched via RIPiT. With a 20–30% insertion efficiency and relatively small amplicon, there should be a clear shift of amplicon containing inserted tag (two replicates shown in lanes 2 and 3, labeled tagged locus) as compared to wild-type endogenous locus (single band in lane 1). ![]() (B) A bulk-level (i.e., in a pool of cells) analysis of CRISPR-mediated insertion efficiency by amplifying the insertion site with PCR 48–72h after guide RNA-Cas9 RNP complex electroporation. Primers (labeled Fwd and Rev) flanking the insertion site are used in the PCR reaction with genomic DNA as a template to identify the insertion event. Single-stranded oligo donor (ssODN) for homology-directed repair is on the top where FLAG tag sequence and a flexible linker (amino acid sequence GGGS) is flanked by 35–50nt DNA sequence with perfect homology with the DNA sequence surrounding the cut site (triangle) induced by guide RNA-Cas9 RNP complex. (A) Scheme of CRISPR-Cas9-mediated insertion of affinity tag (FLAG) into endogenous locus of a gene. All rights reserved.ĬRISPR-Cas9-mediated insertion of affinity tag and bulk assessment of tag insertion. High-throughput sequencing RIPiT RNA footprint RNA-binding protein Ribonucleoprotein.Ĭopyright © 2021 Elsevier Inc. This updated custom library preparation protocol is compatible with commercial PCR multiplexing systems for Illumina sequencing platform for simultaneous and cost-effective analysis of large number of samples. We present a modified protocol for library preparation for high-throughput sequencing so that it exclusively uses equipment and reagents available in a standard molecular biology lab. In this protocol, we have used CRISPR-Cas9 to introduce affinity tag to endogenous protein of interest to capture a more representative state of an RNP complex. Here, we provide an updated and improved protocol for RIPiT-seq. To fill this gap, we have previously developed RNA Immunoprecipitation in Tandem followed by high-throughput sequencing (RIPiT-seq) to characterize RNA targets of compositionally distinct RNP complexes by sequentially immunoprecipitating two proteins from the same RNP and sequencing the co-purifying RNA footprints. While powerful techniques are available to identify binding sites of individual RBPs at high resolution, it remains challenging to unravel binding sites of multicomponent ribonucleoproteins (RNPs) where multiple RBPs or proteins function cooperatively to bind to target RNAs. The ability to identify RNA targets bound by RBPs is critical for understanding RBP function. RNA-binding proteins (RBPs) regulate all aspects of RNA metabolism.
0 Comments
Leave a Reply. |