A | B | |
*Fragmentation Master Mix (1x) | ||
Reagent (lilac tubes) | Volume (uL per sample) | |
ERCC (stock concentration 50pg/uL) | 0.5 | |
First Strand Synthesis Reaction Buffer | 0.4 | |
Random Primers | 0.1 | |
Total Reaction Volume | 1.0 | |
First Strand Synthesis Master Mix (1x) | ||
Reagent (lilac tubes) | Volume (uL per sample) | |
Fragmentation Reaction Volume | 1.0 | |
NEBNext First Strand Synthesis Enzyme Mix | 0.2 | |
Nuclease Free Water | 0.8 | |
Total Reaction Volume | 2.0 | |
Second Strand Synthesis Master Mix (1x) | ||
Reagent (orange tubes) | Volume (uL per sample) | |
First Strand Reaction Volume | 2.0 | |
Second Strand Synthesis Reaction Buffer | 0.8 | |
Second Strand Synthesis Enzyme Mix | 0.4 | |
Nuclease Free Water | 4.8 | |
Total Reaction Volume | 8.0 |
A | B | |
RNA FRAGMENTATION (BP) | ||
1 rxn | ||
Reagent | vol (uL) | |
RNA (USE ERCC @ 50pg/uL) | 0.5 | |
(pink) First SS Reaction Buffer 5x | 0.4 | |
(pink) Random Primers | 0.1 | |
rRNA + Globin FastSelect (1:10) | 0.1 | |
Total volume | 1.1 | |
uL/rxn |
A | |
FRAGMENTATION INCUBATION (heated lid set to 105ºC) | |
1 minutes at 94° | |
2 minutes at 75°C | |
2 minutes at 70C | |
2 minutes at 65°C | |
2 minutes at 60°C | |
2 minutes at 55°C | |
5 minutes at 37°C | |
5 minutes at 25°C |
First Strand Synthesis Incubation | ||
Heated Lid: 105 °C | ||
Temperature (°C) | Time (mins) | |
25 | 10 | |
42 | 15 | |
70 | 15 | |
4 | Hold |
Second Strand Synthesis Incubation | ||
Heated Lid: Off | ||
Temperature (°C) | Time (mins) | |
16 | 60 | |
10 | Hold |
A | |
Prepare 80% ethanol (fresh, 50 mL reservoir) and beads (50 mL reservoir, left out at RT for 30 min prior to use) | |
• Add 12 uL nuclease-free water to sample wells to bring up to 20 uL. Adding water to increase overall volume helps when eluting beads. | |
• Add 28 uL beads to sample plate, mix, and incubate 5 minutes (visually confirm sufficient mixing) | |
• Move sample plate to magnet and incubate 5 minutes | |
• Remove 48 uL supernatant and transfer to waste | |
• Wash with 150uL 80% ethanol, pause 30 seconds, remove and move to waste | |
• Repeat ethanol wash step. Ensure wells are free of ethanol using a p20—this is crucial | |
• Air dry beads for 10 minutes *do not overdry and visually ensure no ethanol is present* | |
• Move sample plate off magnet | |
• Resuspend beads in 6uL eluent (water), mix, and incubate 5 minutes (visually confirm sufficient mixing) | |
• Move sample plate to magnet and incubate 3-5 minutes | |
• Aspirate 5uL of supernatant (containing cDNA) and transfer into a new clean PCR plate | |
• Seal final plate and place in -20 until day 2. |
A | B | |
End Prep Master Mix (1x) | ||
Reagent(green tubes) | Volume per sample(uL) | |
Post-Second Strand Synthsis Bead Clean Volume | 5.0 | |
Ultra II End Prep Reaction Buffer | 0.7 | |
Ultra II End Prep Enzyme Mix | 0.3 | |
Total Volume | 6.0 | |
Adaptor Ligation Master Mix (1x) | ||
Reagent(red tubes) | Volume per sample(uL) | |
End Prep Reaciton Volume | 6.0 | |
NEBNext Ultra II Ligation Master Mix | 3.0 | |
NEBNext Ligation Enhancer | 0.1 | |
Total Volume | 9.1 | |
Adaptor Master Mix (1x) | ||
Reagent(red tube) | Volume per sample(uL) | |
Diluted Adaptor (1:100) | 0.25 | |
Note: Adaptor should be diluted based on approximate sample input and should not be added to adaptor ligation master mix to avoid adaptor-dimers. | ||
USER/PCR Master Mix (1x) | ||
Reagent(USER- white tube; Q5- blue tube) | Volume per sample(uL) | |
Adaptor Ligation Reaction Volume | 5.0 | |
Nuclease Free Water | 2.5 | |
NEB USER Enzyme | 0.9 | |
NEBNext Ultra II Q5 Master Mix | 7.5 | |
Total Volume | 15.9 |
End Prep Incubation | ||
Heated Lid: >75 °C | ||
Temperature (°C) | Time (mins) | |
20 | 30 | |
65 | 30 | |
10 | Hold |
Adaptor Ligation | ||
Heated Lid: Off | ||
Temperature (°C) | Time (mins) | |
20 | 15 | |
10 | Hold |
A | |
Prepare 80% ethanol (fresh, 50 mL reservoir) and beads (50 mL reservoir) | |
• Add 25.65 uL nuclease-free water to sample wells to bring up to 35 uL. Adding water to the sample to increase volume helps when eluting beads. | |
• Add 28 uL beads to sample plate, mix, and incubate for 5 minutes (visually confirm sufficient mixing) | |
• Move sample plate to magnet and incubate 5 minutes | |
• Remove 64 uL supernatant and transfer to waste | |
• Wash with 150uL 80% ethanol, pause 30 seconds, remove and move to waste | |
• Repeat ethanol wash step. Remove remaining ethanol with a p20—this is crucial for proper drying. | |
• Air dry beads for 10 minutes *do not overdry but ensure wells are free of ethanol* | |
• Move sample plate off magnet | |
• Resuspend beads in 6uL eluent (barcodes), mix, and incubate 5 minutes (visually confirm sufficient mixing) | |
• Move sample plate to magnet and incubate 5 minutes | |
• Aspirate 5uL of supernatant (containing adaptor-ligated cDNA and barcodes) and transfer into the final plate prepared with USER/PCR master mix | |
• Gently vortex and quick-spin plate to mix |
A | B | C | |
USER/PCR INCUBATION (heated lid set to 105C) | |||
cycle # | |||
37ºC for 15 mins | 1 | ||
98ºC for 30s | 1 | ||
98ºC for 10s | 12-18* | *NOTE: number of PCR cycles should be chosen based on approximate RNA sample input. PAXgene and NP: 14; BAL: 16-18; Plasma: 16 | |
65ºC for 75s | |||
65ºC for 5mins | 1 | ||
Hold at 4ºC | ∞ |
A | |
*The following bead clean should be done on the bench or anywhere BUT the pre-PCR hood | |
Prepare 80% ethanol (fresh, 50 mL reservoir) and beads (50 mL reservoir) | |
• Add 12.72 uL beads to sample plate, mix, and incubate 5 minutes (visually confirm sufficient mixing) | |
• Move sample plate to magnet and incubate 5 minutes | |
• Remove 27.5 uL supernatant and transfer to waste | |
• Wash with 150uL 80% ethanol, pause 30 seconds, remove and move to waste | |
• Repeat ethanol wash step | |
• Air dry beads for 10 minutes *do not overdry* | |
• Move sample plate off magnet | |
• Resuspend beads in 33uL eluent (water), mix, and incubate 5 minutes (visually confirm sufficient mixing) | |
• Move sample plate to magnet and incubate 5 minutes | |
• Aspirate 31uL of supernatant (containing cDNA) and transfer into a new clean Echo source plate | |
• Seal final plate and freeze for downstream use - using an Echo plate for the final plate means pooling can be done with the Echo | |
• use a regular 384PP source plate rather than an LDV plate - LDV plates seem to bind DNA and cause loss of library | |
• Remaining 2uL in the bead plate can be recovered and used for QuBit and Tapestation (to test several samples for successful library prep) |