CIRI-long Nanopore Library Preparation

1. Total RNA Extraction & Ribosomal RNA Depletion

2. Poly(A) Tailing & RNase R Treatment

Then, additional poly(A) tails are added to the linear transcripts to increase RNase R digestion efficiency.

2.1 Add Poly(A) Tails To Linear RNAs

E.coli Poly(A) Polymerase (NEBNext) is used to add poly(A) tails to the 3’ end of linear RNAs, which can increase the RNase R digestion ability to RNAs with secondary structures.

  • Add the following components in the order specified:

Component Volume
rRNA-depleted RNA 15 uL
10X E.coli Poly(A) Polymerase Reaction Buffer 2uL
ATP (10mM) 2 uL
E.coli Poly(A) Polymerase (5 U/uL) 1 uL
Total Volume: 20 uL
  • Ribosomal-depleted total RNA is incubated at 37ºC with 1uL of E.coli Poly(A) Polymerase (NEBNext) for 30min.

  • Stop the reaction by proceeding to the cleanup step.

2.2 Purification After Poly(A) Treatment

Agencourt RNAClean XP kit (Beckman) is used to remove contamination after poly(A) treatment.

Component Volume
Agencourt RNAClean XP 44 uL
Polyadenylated RNA 20 uL
Total Volume: 64 uL
  • Add 2.2uL of Agencourt RNAClean XP per 1.0 uL of sample.

  • Wash beads + RNA fragments twice with 75% Ethanol to remove contaminants.

  • Elute purified RNA from beads with 20uL H2O.

2.3 RNase R Treatment To Effectively Digest Linear RNAs

Polyadenylted RNA is treated using RNase R (Epicentre) to remove linear RNAs.

Component Volume
Polyadenylated RNA 17.5 uL
RNase R Buffer 2 uL
RNase R (20 U/uL) 0.5 uL
Total 20 uL
  • Polyadenylated RNA was incubated with RNase R at 37ºC for 15 min.

2.4 Purification after RNase R Treatment

2.2x bead-based cleanup is used to remove contamination after RNase R treatment as described above (See Part 2.2).

  • Add 2.2uL of Agencourt RNAClean XP per 1.0 uL of sample.

  • Wash beads + RNA fragments twice with 75% Ethanol to remove contaminants.

  • Elute purified RNA with 5uL H2O.

3. SMARTer Reverse Transcription

Then, RNase R-treated RNA is reverse transcribed using random hexamers and SMARTer cDNA synthesis kit (Takara Bio) according to the manufacturer’s instructions. The 3’ SMART CDS Primer II A 5'-AAGCAGTGGTATCAACGCAGAGTACT(30)N-1N-3' was replaced with 5'-AAGCAGTGGTATCAACGCAGAGTACNNNNNN-3' to amplify circular RNAs without poly(A) sequences.

  • Prepare reaction as follows:

Component Volume
RNase R treated RNA 3.5 uL
SMARTer CDS random primer (12 uM) 1 uL
Total Volume: 4.5 uL
  • Incubated at 72ºC for 3min, 25ºC for 10min, hold at 42ºC.

  • Add the following mixture:

Component Volume
5x First Strand Buffer (RNase-Free) 2 uL
Dithiothreitol (DTT; 100 mM) 0.25 uL
dNTP Mix (10 mM) 1 uL
SMARTer II A Oligonucleotide (12 uM) 1 uL
RNase Inhibitor (40 U/uL) 0.25 uL
SMARTScribe Reverse Transcriptase (100 U/uL) 1 uL
Total Volume: 5.5 uL
  • Incubation at 42ºC for 90 min.

  • Denatured at 70ºC for 10 min.

4. cDNA PCR Amplification

To obtain sufficient cDNA products for sequencing, PCR amplification is performed using 2uL of cDNA with NEBNext LongAmp Taq 2x master mix and SMARTer primers under the following conditions:

STEP TEMP TIME
Initial Denaturation 95ºC 30 s
19-20 Cycles 95ºC
62ºC
65ºC
15 s
15 s
2 min
Final Extension 65ºC 2 min
Hold 4-10ºC

5. Fragment Size Selection

Afterward, 0.5x Agencourt AMPure XP magnetic beads (Beckman) is used for size selection of the cDNA fragments:

  • Add 0.5uL of AMPure XP per 1.0 uL of sample.

  • Wash beads + cDNA fragments twice with 75% Ethanol to remove contaminants.

  • Elute purified cDNA with 20-30uL H2O.

6. Nanopore Sequencing

Finally, cDNA libraries are prepared according to the ONT protocol SQK-LSK109 and barcoded with EXP-NBD104 / EXP-NBD114 kits, and nanopore sequencing is performed using the MinION (MN26543) platform with a FLOW-MIN106 flow cell. Please refer to the Nanopore Community for detailed instructions.