RNA-Seq Sample Preparation & Library Construction

Full RNA-seq Workflow Overview

• Entire analytical pipeline
  • Begin with a clearly stated, directional, testable hypothesis involving $\ge 2$ variables.
  • Execute biological experiment according to that hypothesis.
  • Collect samples → isolate & purify RNA → quantify RNA.
  • Wet-lab finishes with library preparation and high-throughput sequencing.
  • Bio-informatics (covered in later course sections):
  • Raw read quality control.
  • Alignment to a reference genome.
  • Quantification of gene expression per sample.
  • Differential-expression testing between conditions.
  • Biological interpretation & visualization.

Experimental Design & Planning

• Core considerations (apply to all assays):
  • Sample type, size, collection method, preservation strategy.
  • Assay choice dictates statistical power requirements.
  • RNA-seq often achieves significance with $n\approx4$ replicates per group.
  • Bone µCT might need $n=10\text{–}12$.
  • Think through variables & controls (organism, tissue, genotype, environmental perturbation, etc.)
  • Plan for result interpretation before you start.

Spaceflight-specific nuances

• Timing post-perturbation (e.g. microgravity, radiation):
  • Splash-down to lab can take $\approx 72\text{ h}$ → gene expression drifts back toward $1\,g$ baseline.
  • Best practice: dissect/collect on-orbit if true microgravity signature is desired.
• Timing post-euthanasia:
  • RNases rapidly degrade RNA once physiological safeguards stop.
  • Delays between euthanasia and tissue freezing → dramatic RIN drop.

Sample Preservation Choices

• Snap-freeze in liquid N$_2$ vs. RNA-stabilizing reagents (e.g. RNAlater).
• Storage duration (e.g. months on ISS) must be reconciled with RNA stability.

RNA Integrity & RNase Avoidance

• RNase ubiquity: endogenous & on surfaces.
• Best practices:
  • Wipe benches/tools with RNase-Away.
  • Consistent glove use; change often.
  • Keep samples on ice (≈$0^\circ$C); final isolated RNA at $-80^\circ$C.
• High-quality input RNA ⇒ high-quality sequencing output.

RNA Extraction Methodologies

• Classical organic separation (TRIzol/phenol-chloroform):
  • Homogenize tissue → lyse → centrifuge → three-phase separation.
  • Bottom organic (proteins, pink hue).
  • Interphase (DNA/chromatin, white disk).
  • Top aqueous (RNA, clear).
  • Carefully aspirate RNA; treat with DNase to remove carry-over.
• Column-based kits:
  • Silica membrane binds RNA; DNase treat on-column; elute with RNase-free buffer.
  • Tissue-specific optimization mandatory.

Homogenization Tools

• Mechanical homogenizers (bones, hard tissues).
• Direct lysis in chaotropic solvents (cell culture).

RNA Quality Control (post-extraction)

• Agarose gel visualization:
  • Distinct $28\,S$ and $18\,S$ rRNA bands (plus faint $5\,S$).
• Bioanalyzer / TapeStation electropherograms:
  • Generate RNA Integrity Number (RIN) $\in[1,10]$.
  • Criteria:
  • $\text{RIN}\ge 7$ required for most mRNA-seq.
  • Partially degraded: shallow $28\,S$, extra low-MW peaks.
  • Severely degraded: no $28\,S/18\,S$ peaks, RIN $\le 3$.

Cellular RNA Composition

• $\sim 90\%$ ribosomal RNA.
• Minor fractions: tRNA, snoRNA, miRNA, siRNA, lncRNA.
• mRNA (poly-adenylated, coding) = $1\text{–}5\%$ → library prep must enrich it.

Library Preparation – Step-by-Step

1. Enrich or deplete input RNA
   • Poly-A selection (preferred):
     • Beads with oligo-dT probes capture poly-A tails under magnetic field.
     • Result: $\approx78\%$ exonic/coding content.
   • Ribo-depletion:
     • Probes hybridize rRNA; bound fraction discarded.
     • Exonic content $\approx48\%$ but retains non-poly-A species.
2. Random fragmentation of RNA (heat/chemical):
   • Generate unbiased size distribution.
   • Fragment RNA, not cDNA, to avoid restriction-site bias.
3. First- & second-strand cDNA synthesis
   • Use random hexamer primers (6-mers) + reverse transcriptase → ds-cDNA matching original fragments.
4. Size selection (magnetic beads):
   • Remove fragments <!200\text{ bp} to improve cluster generation & read quality.
   • Poor-quality/degraded RNA loses many fragments here.
5. A-tailing & adapter ligation
   • Add a single-stranded $A$ overhang → ligate Illumina P5/P7 adapters.
   • Watch for adapter dimers (≈$100\text{–}125\text{ bp}$). Must be minimized.
6. Library amplification (PCR, ≈$15$ cycles):
   • Introduces flow-cell binding sequences, sample indices.
   • Over-amplification → coverage bias; use minimal cycles.
7. Final QC of library
   • Fragment size distribution: target $200\text{–}500\text{ bp}$, mean $\approx250\text{ bp}$.
   • Concentration quantification: qPCR, Qubit, etc.
   • Adapter dimer proportion: keep <$5\%$ ideally.

Adapter Geometry Recap

• Final molecule = $\text{[P5]} + \text{[Index 1]} + \text{Insert} + \text{[Index 2]} + \text{[P7]}$.
• If using paired-end $100$ ( $2\times100$) sequencing and adapter lengths $\approx 65$ bp each:
  • Desired library size $L$:
    $L = 2\times100\,\text{(read)} + 65+65\,\text{(adapters)} - \text{overlap} \approx 300\,\text{bp}.$ (Allow overlap $\approx 30\text{–}40$ bp.)

Avoiding Bias Across Workflow

• Random fragmentation + random primers → uniform transcript representation.
• Remove small/degraded fragments → consistent insert size.
• Limit PCR cycles → avoid amplification bias & chimera formation.
• Continual QC checkpoints guard against data loss and misinterpretation.

Key Reagents, Enzymes & Tools

• Reverse Transcriptase, DNA ligase, RNase-free DNase.
• PCR polymerase, primers.
• Magnetic beads for both purification and size selection.
• Indexing adapters (single or dual indices).
• Restriction enzymes (limited role; avoided for fragmentation).

Practical / Ethical / Philosophical Notes

• Ethical responsibility to design statistically sound experiments, minimizing animal use while ensuring rigorous conclusions.
• In spaceflight studies, logistics of animal handling intersect with the ethics of both humane care and scientific validity.
• Proper sample stewardship (RNase control, storage) protects irreplaceable biological data—especially when retrieval opportunities (e.g. ISS down-mass) are scarce.

Connections to Previous Lectures

• Builds on earlier discussion of gel electrophoresis principles for nucleic-acid QC.
• Sets stage for upcoming module on sequencing chemistry & flow-cell clustering.

Recap Checklist

• [ ] Hypothesis framed & variables defined.
• [ ] Adequate sample size (e.g. $n\ge4$ for RNA-seq).
• [ ] Collection timing & preservation optimized.
• [ ] RNase-free extraction completed; RIN $\ge7$ confirmed.
• [ ] Chosen enrichment method (poly-A or ribo-depletion) executed.
• [ ] Fragmentation, cDNA synthesis, size selection performed without bias.
• [ ] Adapters ligated; dimers removed.
• [ ] Library amplified within $\le15$ cycles.
• [ ] Final QC: fragment $\approx250$ bp, minimal adapter dimers, quantified for flow-cell loading.
• Ready for next lecture: sequencing principles & data generation.