- Add 8μL reverse transcriptase mix to the RNA and heat the
samples at 25C for 3 min, 42C for 5 min, 52C for 30 min;
hold at 4C. - Wash 10μL of C-1 beads per sample with 100μL Bead Binding
Buffer three times. - Resuspend beads in 5μL Bead Binding Buffer per sample. Add
5 μL bead suspension to the completed reverse transcription
reaction. - Rotate at room temperature for 30 min.
- Transfer bead suspension to new 1.5 mL tube. Let it settle on
magnet and remove supernatant. - Add 500μL Bead Wash Buffer to each sample and invert the
tubes four times to mix. - Insert the samples into a magnetic stand for 1 min, and then
remove the supernatant. - Wash 4 more times with 500μL Bead Wash Buffer and twice
with 500μL1PBS. - Resuspend beads in 50μL RNaseA/T1/H elution mix and
incubate them at 37Cfor30minat100gin a thermomixer. - Add 1μL 100% DMSO and heat at 95C for 4 min. Briefly
spin down samples and transfer eluted cDNA to a new tube. - Purify the cDNA using Zymo concentrator-5 columns.
- Elute twice with 6μL water to recover ~10μL cDNA.
3.6 cDNA
Circularization, Library
PCR, and Sequencing
- Add 4μL circularization reaction mix to the cDNA sample and
incubate them at 60C for 100 min, followed by 80C for
10 min. At the same time, warm up the lamps in the qPCR
machine by starting the program (the warm-up takes about
20 min). - Add 10.5μL qPCR reaction mix to each circularized cDNA
sample. Transfer cDNA to optical PCR tubes (each tube should
be separate so that individual tubes can be taken out of the
qPCR machine when the fluorescence signal reaches a defined
point). - Set up the following qPCR program. Choose SYBR, initial
95 C, 45 s, 10 cycles of: 95C, 15 s; 65C, 30 s; 72C, 45
s, detect fluorescence at extension step (a set of nine cycles).
Take sample out once amplification reaches exponential phase. - Transfer PCR product to 1.5 mL tube. Add 30μL Ampure XP
beads and 75μL isopropanol. - Incubate for 5 min at RT.
- Let the beads settle on the magnet for 5 min.
72 Zhipeng Lu et al.