Library prep steps -RNA Flashcards
Step 1
RNA Extraction:
Goal: Isolate total RNA from cells or tissues.
The RNA extraction process removes genomic DNA and other contaminants to leave a purified RNA sample. This can be achieved using commercial RNA isolation kits.
Step 2
RNA Quality Control:
Goal: Assess the quality of the RNA sample.
RNA is typically assessed for integrity (e.g., using an Agilent Bioanalyzer or TapeStation) to ensure that it is of high quality and free from degradation.
Step 3
mRNA Enrichment (optional):
Goal: Select mRNA from total RNA.
If focusing on mRNA (rather than all RNA types), polyA selection is used to isolate mRNA molecules that have a poly-A tail (using magnetic beads or other methods). Alternatively, ribosomal RNA (rRNA) can be depleted.
Step 4
DNA Synthesis:
Goal: Convert RNA to cDNA.
RNA is reverse-transcribed into complementary DNA (cDNA) using a reverse transcriptase enzyme. For full-length transcript analysis, random primers or oligo-dT primers are typically used to initiate the synthesis.
Step 5
Fragmentation (optional):
Goal: Fragment the cDNA into smaller pieces (if required).
Similar to DNA library prep, cDNA may be fragmented using mechanical shearing or enzymatic methods to create a library of appropriate fragment sizes for sequencing.
Step 6
End Repair and Adapter Ligation:
Goal: Prepare cDNA for sequencing.
After synthesis and fragmentation, the cDNA ends are repaired, and adapters are ligated to both ends of the cDNA fragments, just like in DNA library prep.
Step 7
Amplification (PCR):
Goal: Enrich the cDNA library.
The adapter-ligated cDNA is amplified by PCR to increase the amount of library material available for sequencing.
Step 8
Size Selection (optional):
Goal: Choose fragments within a desired size range.
Size selection is performed to remove unwanted short or long fragments, ensuring that the library consists of appropriately sized cDNA fragments.
Step 9
Quality Control:
Goal: Assess the quality of the RNA library.
Similar to DNA libraries, the final RNA library is assessed for quality and quantity using methods like qPCR, gel electrophoresis, or bioanalyzer assays.
