T-Cell Receptor Sequencing Using RNA-Seq Protocol

RNA Isolation and Quality Assessment

1. Ensure a correct setup of the chip priming station.
2. The following kit components need to be prepared before first time use according to the manufacturer's instructions:
(a) RNA ladder aliquots, stable at -70 °C for extended time periods.
(b) Agilent RNA 6000 Nano gel matrix aliquots (65 μl), can be stored at 4 °C for 1 month (protect from light during use).
3. To prepare the gel-dye matrix, allow one aliquot Agilent RNA 6000 Nano gel matrix (65 μl) and RNA 6000 Nano dye concentrate to come to room temperature for 30 min.
4. Vortex the dye concentrates for 10 s and spin down.
5. Pipette 1 μl of dye concentrate to 65 μl of gel matrix (one aliquot) in a microcentrifuge tube.
6. Vortex thoroughly and check for proper mixing of gel and dye.
7. Spin at room temperature for 10 min at 13,000 ×g, protect from light, and use within 1 day. Store at 4 °C if not used immediately.
8. To load the gel-dye matrix, check proper setup of the chip priming station, and place an unused RNA 6000 Nano chip on the chip priming station.
9. Pipette 9.0 μl of the gel-dye mix to the bottom of the well "G" with black background.
10. Position the plunger of the syringe in the chip priming station to 1 ml; then close the chip priming station and pressurize quickly by pressing down the plunger. Keep for exactly 30 s.
11. Release the plunger and pull back gently to the 1 ml mark.
12. Open the chip priming station and pipette 9.0 μl gel-dye matrix to the bottom of the two wells marked "G" without background.
13. To load ladder and sample to the chip, pipette 5 μl of RNA marker to all sample wells and the well that is marked with the ladder symbol.
14. Add 1 μl of ladder to the well that is marked with the ladder symbol.
15. Add 1 μl of RNA of interest to all sample wells. If there are less than 12 samples to measure, put 1 μl of RNA marker to wells that are not used.
16. Vortex the chip horizontally in the IKA vortex (1 min, 2400 rpm), and insert to the Bioanalyzer 2100 station within 5 min to perform analysis using 2100 Expert Software.

Library Preparation

Purification and Fragmentation of mRNA

1. Bring 0.1–1.0 μg of total RNA to 50 μl volume using nucleasefree ultrapure water and transfer to the individual wells of the RNA bead plate.
2. Vortex RNA purification beads and transfer 50 μl to each well.
3. Seal the plate with Microseal "B" and shake plate (1 min, 1000 rpm).
4. Heat the plate in the microheating system (5 min, 65 °C, lid closed), and cool down on ice (1 min).
5. Incubate the plate at room temperature (5 min). Meanwhile bring the microheating system to 80 °C.
6. For magnetic pull-down, remove the seal and put the plate on the magnetic stand. Inspect visually until the liquid is clear.
7. Remove the supernatant from all wells. Then remove the plate from the stand.
8. Pipette 200 μl Bead Washing Buffer to all wells; seal the plate and mix by shaking (1 min, 1000 rpm).
9. Repeat steps 6 and 7.
10. Pipette 50 μl Elution Buffer to all wells; seal the plate and mix by shaking (1 min, 1000 rpm).
11. Heat the plate in the microheating system (2 min, 80 °C, lid closed), and cool down on ice (1 min).
12. Place the plate at the bench and remove the seal.
13. To prepare for RNA fragmentation, pipette 50 μl Bead Binding Buffer to all wells; seal the plate and mix by shaking (1 min, 1000 rpm).
14. Incubate the plate at room temperature (5 min).
15. Repeat steps 6–9 (magnetic pull-down, bead washing, another magnetic pull-down).
16. Pipette 19.5 μl Fragment, Prime, Finish Mix to all wells, seal the plate, and mix by shaking (1 min, 1000 rpm).
17. Remove the seal and transfer the samples well by well to the RNA fragmentation plate.
18. Seal the plate and run the following program on the thermocycler with preheated lid:
(a) 94 °C for 8 min
(b) Hold at 4 °C.
19. Quickly spin down.

First Strand cDNA Synthesis

Purified RNA fragments are reverse transcribed to first strand cDNA using random hexamer primers.
1. For magnetic pull-down, put the plate on the magnetic stand. Inspect visually until the liquid is clear.
2. Remove the seal and transfer 17 μl supernatant well by well to the cDNA plate.
3. Spin down the First Strand Synthesis Act D Mix (5 s, 600 ×g).
4. Mix SuperScript II reverse transcriptase and the First Strand Synthesis Act D Mix at a 1:10 ratio (1 μl SuperScript II reverse transcriptase plus 9 μl First Strand Synthesis Act D Mix (can be stored for extended time periods at -20 °C).
5. Pipette 8 μl of this mix to each well of the cDNA plate and mix by shaking (20 s, 1600 rpm).
6. Spin down by centrifugation (1 min, 280 ×g).
7. Run the following program on the thermocycler with the lid preheated to 100 °C:
(a) 25 °C for 10 min.
(b) 42 °C for 15 min.
(c) 70 °C for 15 min.
(d) Hold at 4 °C.

Second Strand cDNA Synthesis

1. Add 5 μl Resuspension buffer to each well.
2. Spin down Second Strand Marking Mix (5 s, 600 ×g), and pipette 20 μl to each well, and mix by shaking (20 s, 1600 rpm).
3. Spin down the plate (1 min, 280 ×g).
4. Place the plate on the thermocycler with lid preheated to 30 °C and run at 16 °C for 60 min. Allow to come to room temperature afterwards.
5. Pipette 90 μl of AMPure XP beads to new cDNA Clean-Up Plate, and transfer the content of the cDNA plate well by well to cDNA Clean-Up Plate. Mix by shaking (2 min, 1800 rpm).
6. Incubate at room temperature (15 min), and then spin down (1 min, 280 ×g).
7. Put the plate on the magnetic stand. Inspect visually until the liquid is clear. Remove 135 μl supernatant from each well.
8. Wash the beads with the plate retained on the magnetic stand by adding 200 μl ethanol 80% to all wells. After 30 s, remove all supernatant from each well.
9. Repeat step 8 and carefully remove all remaining ethanol (using a small-volume pipette). Let the samples air dry on the magnetic stand (15 min).
10. Remove the plate from the stand and pipette 17.5 μl Resuspension buffer to all wells. Mix by shaking (2 min, 1800 rpm). Let stand at room temperature (2 min).
11. Spin down (1 min, 280 ×g).
12. Put the plate on the magnetic stand. Inspect visually until the liquid is clear. Transfer 15 μl supernatant to the new plate (adapter ligation plate). This is the first safe stopping point, where the sealed plate can be stored at -20 °C for 1 week.

3' End Adenylation and Adapter Ligation

1. Add 2.5 μl Resuspension buffer to all wells of the adapter ligation plate and spin down (5 s, 600 ×g).
2. Pipette 12.5 μl A-Tailing Mix to each well and mix by shaking (2 min, 1800 rpm).
3. Cover plate with Microseal "B" and spin down (1 min, 280 ×g).
4. Incubate on 37 °C microheating system (30 min), then transfer to 70 °C microheating system (5 min, lid closed), and cool down on ice (1 min).
5. Spin down TruSeq RNA CD Index Plate (1 min, 280 ×g).
6. Transfer to all wells of the adapter ligation plates in this order:
(a) 2.5 μl Resuspension buffer
(b) 2.5 μl Ligation mix
(c) 2.5 μl RNA Adapters from the Index adapter Plate (to each corresponding well). And mix by shaking (2 min, 1800 rpm).
7. Spin down (1 min, 280 ×g) plate and transfer to the microheating system (10 min, 30 °C, lid closed), and then cool down on ice.
8. Spin down the Stop Ligation Buffer (5 s, 600 ×g) and add 5 μl to every well. Mix by shaking (2 min, 1800 rpm).
9. Spin down the plate (1 min, 280 ×g).
10. Add 42 μl AMPure XP beads to every well. Mix by shaking (2 min, 1800 rpm).
11. Incubate at room temperature (15 min), and then spin down the plate (1 min, 280 ×g).
12. Put the plate on the magnetic stand. Inspect visually until the liquid is clear (ca. 5 min). Remove all supernatant.
13. Wash the beads with the plate retained on the magnetic stand by adding 200 μl ethanol 80% to all wells. After 30 s remove ethanol.
14. Repeat step 13 and carefully remove all remaining ethanol (using a low-volume pipette). Let samples air dry on the magnetic stand (15 min).
15. Remove plate form stand and pipette 52.5 μl of Resuspension buffer to every well, and mix by shaking (2 min, 1800 rpm).
16. Incubate at room temperature (2 min).
17. Spin down the plate (1 min, 280 ×g) and place it on the magnetic stand. Inspect visually until the liquid is clear.
18. Transfer 50 μl supernatant well by well to a novel plate.
19. Repeat steps 10–17, but use 50 μl AMPure XP beads in step 10 and 22.5 μl Resuspension buffer in step 15.
20. Transfer 20 μl supernatant well by well to a novel plate. Sealed plate can be kept at -20 °C for 1 week (this is the second safe stopping point).

DNA Fragment Enrichment

1. On ice, pipette 5 μl PCR Primer Cocktail and 25 μl PCR Master Mix to each well, mix by shaking (20 s, 1600 rpm), and spin down (1 min, 280 ×g).
2. Transfer to thermocycler with the preheated lid set to 100 °C and run
3. Spin down plate (1 min, 280 ×g) and pipette 47.5 μl AMPure XP beads to every well. Mix by shaking (2 min, 1800 rpm).
4. Incubate at room temperature (15 min), and then spin down (1 min, 280 ×g).
5. Put the plate on the magnetic stand. Inspect visually until the liquid is clear. Remove all supernatant.
6. Wash the beads with the plate retained on the magnetic stand by adding 200 μl ethanol 80% to all wells. After 30 s, remove ethanol.
7. Repeat step 6 of Subheading 3.2.5 and carefully remove all remaining ethanol (using low-volume pipette). Let the samples air-dry on magnetic stand (15 min).
8. Resuspend the beads in 32.5 μl Resuspension buffer. Mix by shaking (2 min, 1800 rpm).
9. Incubate at room temperature (2 min), and then spin down (1 min, 280 ×g).
10. Put plate on magnetic stand. Inspect visually until the liquid is clear, and transfer 30 μl supernatant to a novel plate. Libraries can be kept at -20 °C for 1 week (this the third safe stopping point).

Sequencing

Sequencing of the libraries is performed on an Illumina sequencing system. Multiple configurations exist which provide the targeted output of 30 million reads per sample (e.g., Illumina NextSeq 550, 1000 & 2000 and NovaSeq 6000 systems). Depending on the selected sequencing system, flow cell, and read length, between 4 and 132 samples (maximum: two S4 flow cells on NovaSeq 6000 with 2x100 bp sequencing) can be multiplexed in one sequencing run.