Schematic of the acrip-seq procedure

ACRIP-Seq (Antibody-Coupled RNA Immunoprecipitation followed by Sequencing) is a technique used to analyze RNA-protein interactions.

Here is a step-by-step Schematic of the acrip-seq procedure.

Steps in ACRIP-Seq procedure

1. Cell Lysis and RNA-Protein Crosslinking
   • Cells are lysed under conditions that preserve RNA-protein complexes.
   • Crosslinking (optional, using UV light or chemical agents) stabilizes RNA-protein interactions.
2. Immunoprecipitation
   • An antibody specific to the target protein is used to capture RNA-protein complexes.
   • The antibody-protein-RNA complex is immobilized on beads (e.g., magnetic or agarose beads coated with Protein A/G).
3. Washing
   • Unbound RNA, proteins, and other contaminants are washed away to purify the RNA-protein complexes.
4. RNA Recovery
   • RNA is released from the immunoprecipitated complexes, usually by protein digestion or heat treatment.
   • Crosslinks are reversed if applied.
5. RNA Purification
   • Extracted RNA is purified using standard methods (e.g., phenol-chloroform extraction, column-based purification).
6. Library Preparation
   • RNA is converted to complementary DNA (cDNA) via reverse transcription.
   • cDNA is amplified, adapter-ligated, and indexed for sequencing.
7. High-Throughput Sequencing
   • Prepared libraries are subjected to next-generation sequencing (NGS).
8. Data Analysis
   • Sequencing reads are aligned to the genome/transcriptome to identify RNA molecules interacting with the protein of interest.
   • Enrichment analysis and bioinformatics tools are used to infer RNA-protein interaction sites and functional relevance.

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Illustration Elements for the Schematic

1. Step 1 (Cell Lysis): Depict cells being lysed with symbols for RNA and proteins. Optionally show crosslinking (UV/chemical).
2. Step 2 (Immunoprecipitation): Draw beads with antibodies attached to the target protein and RNA.
3. Step 3 (Washing): Illustrate impurities being washed away while RNA-protein complexes remain bound to the beads.
4. Step 4 (RNA Recovery): Show RNA strands being separated from proteins.
5. Step 5 (RNA Purification): Depict a clean RNA strand post-purification.
6. Step 6 (Library Preparation): Include an image of RNA being reverse transcribed and prepared for sequencing.
7. Step 7 (Sequencing): Display a sequencer with cDNA strands being read.
8. Step 8 (Data Analysis): Add a graphical representation of bioinformatics analysis, such as aligned reads mapped to a genome.