Appendix
Library Construction for NGS
Example Protocol: TruSeq Stranded Total RNA LT Sample Preparation Kit
(Illumina)
1. Isolate total RNA
• Determine quality and quantity of the isolated RNA.
• RIN 9–10.
2. Preparations.
• Use 1μg of Total RNA to initiate protocol (0.1–1.0μg starting range recommended).
• Thaw frozen rRNA Binding Buffer [1], rRNA Removal Mix [2], rRNA Removal
Beads & Elute Prime Fragment High Mix [7]. Place Resuspension Buffer at 4
C for
subsequent experiments.
• Prepare 80% Ethanol [4] for AMPure XP bead washes.
• Remove Elution Buffer [6], rRNA Removal Beads [3], and AMPure XP Beads
[5] from 4
C and bring to RT.
• Rotate beads at 6 rpm.
3. Ribo-Zero Deplete and Fragment RNA (bind rRNA, rRNA removal, RNA clean-up,
Depleted RNA Fragmentation)
• Dilute 1ug of total RNA to 10μl using nuclease free H 2 O in new 0.5 ml PCR tubes.
• Add 5μl rRNA Binding Buffer to each sample.
• Add 5μl of rRNA removal Mix.
• Store rRNA Binding Buffer and rRNA Removal Mix at À20
C.
• Gently pipette the entire volume up and down to mix thoroughly.
• Incubate in thermal cycler using the following profile to denature RNA:
# Springer Nature Switzerland AG 2021
M. Kappelmann-Fenzl (ed.), Next Generation Sequencing and Data Analysis, Learning
Materials in Biosciences, https://doi.org/10.1007/978-3-030-62490-3
193
Library Construction for NGS
Example Protocol: TruSeq Stranded Total RNA LT Sample Preparation Kit
(Illumina)
1. Isolate total RNA
• Determine quality and quantity of the isolated RNA.
• RIN 9–10.
2. Preparations.
• Use 1μg of Total RNA to initiate protocol (0.1–1.0μg starting range recommended).
• Thaw frozen rRNA Binding Buffer [1], rRNA Removal Mix [2], rRNA Removal
Beads & Elute Prime Fragment High Mix [7]. Place Resuspension Buffer at 4
C for
subsequent experiments.
• Prepare 80% Ethanol [4] for AMPure XP bead washes.
• Remove Elution Buffer [6], rRNA Removal Beads [3], and AMPure XP Beads
[5] from 4
C and bring to RT.
• Rotate beads at 6 rpm.
3. Ribo-Zero Deplete and Fragment RNA (bind rRNA, rRNA removal, RNA clean-up,
Depleted RNA Fragmentation)
• Dilute 1ug of total RNA to 10μl using nuclease free H 2 O in new 0.5 ml PCR tubes.
• Add 5μl rRNA Binding Buffer to each sample.
• Add 5μl of rRNA removal Mix.
• Store rRNA Binding Buffer and rRNA Removal Mix at À20
C.
• Gently pipette the entire volume up and down to mix thoroughly.
• Incubate in thermal cycler using the following profile to denature RNA:
# Springer Nature Switzerland AG 2021
M. Kappelmann-Fenzl (ed.), Next Generation Sequencing and Data Analysis, Learning
Materials in Biosciences, https://doi.org/10.1007/978-3-030-62490-3
193