Attempting TagSeq Protocol Again (Partial) on 4 RNA Samples from Holobiont Project, Doing Each Library Twice with Either New DTT or New SmartScibe and Buffer

Goal Generate more yield and get 1-2ng/ul library after the cDNA step (what it says you should have in the UTGSAF protocol at this step). Only going to this step, this also only takes 1 day
Results No difference between the two prep styles and not able to get 1-2ng/ul Takeaways Need to troubleshoot more. Next ideas are increasing concentration of S-ILL, 3-ILL, and 5-ILL primers, and using KAPA amp mix

Library prep followed almost exactly the protocol from UT Austin (where applicable), except stopped after the cDNA cleanup. Adapter and template switching oligo sequences are from the original Lohman protocol.

Everything besides the Qubit and TapeStation was done on the RNA bench

Sample Information

RNA from holobiont project was used as to not freeze-thaw Ariana’s RNA. To be able to repeat these samples 4 times I had to pick samples with a high RNA concentration

Sample/Plug ID	species	ng/ul	Library Attempt Number
1445	Pacuta	83.3	9
1653	Pacuta	114	10
1059	Pacuta	73.2	11
2493	Pacuta	73.7	12

Ordered new SmartScribe with FS buffer and pre-made 0.1M DTT from Thermo to try.

RNA Fragmentation and RT Primer Annealing

• Cleaned bench, pipettes, and racks with RNaseZap
• Thawed RNA samples on ice
• Made 8 new strip tubes for sample dilution. Labeled both with 9-12 A or B.
• A samples get new smartscibe and buffer, B samples get new DTT
• Samples diluted to 500ng in 10ul total
• Vortexed and spun down RNA before aliquoting on ice

Sample	ul ultrapure water	ul RNA
9 A and B	4	6
10 A and B	5.62	4.38
11 A and B	3.17	6.83
12 A and B	3.22	6.78

• Turned on themocycler and started program 95 so the themocycler warmed up and the block was at 95 C (95C hold, 95C 2.5min)
• Prepared RNA fragmentation/RT master mix (RFRT) A
  • 1ul dntps (10uM) * 4.4 = 4.4ul
  • 2ul 0.1M OLD DTT * 4.4 = 8.8ul
  • 4ul 5x NEW FS buffer * 4.4 = 17.6ul
  • 1ul 3iLL-30TV (10uM) *4.4 = 4.4ul
• Prepared RNA fragmentation/RT master mix (RFRT) B
  • 1ul dntps (10uM) * 4.4 = 4.4ul
  • 2ul 0.1M NEW DTT * 4.4 = 8.8ul
  • 4ul 5x OLD FS buffer * 4.4 = 17.6ul
  • 1ul 3iLL-30TV (10uM) *4.4 = 4.4ul
• Pipette mix and spin down RFRT
• Added 8ul RFRT to each RNA strip tube with the 500ng aliquots, making sure A tubes get A mix, and B tubes get B mix
• Pipette mixed strip tubes and spun down
• Placed strip tubes in warmed up thermocycler and pressed enter on program
• Took tubes out at the 2.5 min mark and put on the ice bucket for 2 minutes (at least, going into next step)
• Noted here that sample tube 9A had a crack in it and had lost a lot of volume, decided to not continue with that sample/tube
• Took out 1.5ul from each sample to save for RNA qubit
• Made 1X FS buffer to add back in volume: A
  • 2ul new FS buffer
  • 8ul ultra pure H2O
• Made 1x FS buffer to add back in volume: B
  • 2ul old FS buffer
  • 8ul ultrapure H20
• Added 1.5 1X FS buffer back into tubes, either A or B

1st Strand cDNA Synthesis

• Made 1st strand master mix (FSMM) A
  • 1ul SiLL - SWMW (10uM) * 4.4 = 4.4ul
  • 1ul NEW SmartScribe RT * 4.4 = 4.4ul
• Made 1st strand master mix (FSMM) B
  • 1ul SiLL - SWMW (10uM) * 4.4 = 4.4ul
  • 1ul OLD SmartScribe RT * 4.4 = 4.4ul
• Pipette mixed FSMM and keep on ice
• Added 2ul FSMM to each strip tube, either A to A or B to B
• Pipette mixed with 15ul and spun down strip tubes
• Turned on themocycler and started 1st Strand cDNA program, once the block was at 42 degrees, put the strip tubes in the machine and pressed enter (42 degrees C hold, 42 degrees C 60 min, 65 degrees C 15 min, 4 degree hold). Program is 1 hour 15 min long

0.9X Bead Cleanup

• Took out KAPA pure beads 1 hour before use, stored in drawer for warm up
• Made fresh 80% ethanol
• Spun down tubes out of the thermocycler
• Added 30ul ultra pure water to each sample (total vol now 50ul)
• Added 45ul KAPA pure beads to each tube, pipette mixing 10 times for each tube
• Place tubes on the shaker for 15 min at 200rpm shaking
• After, placed tubes on the magnet stand and waited until the liquid was clear
• Removed 90ul of the clear supernatant from each tube
• Added 100ul of fresh 80% ethanol to each tube
• Removed 100ul of the clear supernatant from each tube
• Added 100ul of fresh 80% ethanol to each tube
• Removed 100ul of the clear supernatant from each tube
• Removed any remaining liquid with a p20
• Let “dry” for 3 min max
• Resuspended beads in 15ul ultra pure water
• Incubated tubes on the shaker for 5 minutes 2000rpm
• Placed on magnet and let solution go clear
• Removed 10ul in to strip tubes for continuing lib prep “c”
• Removed 5ul into strip tubes for “save” labeled S-1

cDNA Amplification

• Not two different master mixes this time
• Made cDNA master mix (CDMM):
  • 6ul ultra pure H20 * 8.8 = 52.8ul
  • 0.5ul 10uM dntps * 8.8 = 4.4ul
  • 2ul 10X PCR buffer * 8.8 = 17.6ul
  • 0.5ul 5iLL (10uM) * 8.8 = 4.4ul
  • 0.5ul 3iLL-30TV (10uM) * 8.8 = 4.4ul
  • 0.5ul Klentaq * 8.8 = 4.4ul
• Mixed by pipetting, spun down, and kept on ice
• Added 10ul CDMM to the “c” cDNA strip tubes, both A and B
• Pipette mixed and spun down
• Placed in thermocycler cDNA AMP 18 program (18 cycles recommended for less and 150ng input) (94 degrees C 1 min, 94 degrees C 1 min, 63 degrees C 2 min, 72 degrees C 2 min, 4 degrees C hold. Italics are cycled 18 times). Program runs 1 hour 45 min

0.9X Bead Cleanup

• Took out KAPA pure beads 1 hour before use, stored in drawer for warm up
• Made fresh 80% ethanol
• Spun down tubes out of the thermocycler
• Added 30ul ultra pure water to each sample (total vol now 50ul)
• Added 45ul KAPA pure beads to each tube, pipette mixing 10 times for each tube
• Place tubes on the shaker for 15 min at 200rpm shaking
• After, placed tubes on the magnet stand and waited until the liquid was clear
• Removed 90ul of the clear supernatant from each tube
• Added 100ul of fresh 80% ethanol to each tube
• Removed 100ul of the clear supernatant from each tube
• Added 100ul of fresh 80% ethanol to each tube
• Removed 100ul of the clear supernatant from each tube
• Removed any remaining liquid with a p20
• Let “dry” for 3 min max
• Resuspended beads in 22ul ultra pure water
• Incubated tubes on the shaker for 5 minutes 2000rpm
• Placed on magnet and let solution go clear
• Removed 10ul in to strip tubes for continuing lib prep “c”
• Removed 10ul into strip tubes for “save” labeled S-2
• Froze C tubes and kept S-2 tubes on ice for QC

High Sensitivity RNA Qubit Fragmentation RNA and S-1 Tubes

• Followed Qubit Protocol

Sample	Average RNA ng/ul
Standard 1	44.9 RFU
Standard 2	721 RFU
10A F	29.2
11A F	28.8
12A F	28
9B F	38.8
10B F	27
11B F	26.4
12B F	28.8
10A S-1	20.2
11A S-1	24
12A S-1	24.6
9B S-1	35.8
10B S-1	23.4
11B S-1	19.3
12B S-1	23.2

High Sensitivity DNA Qubit S-2 Tubes After cDNA

• Followed Qubit Protocol

Sample	Average RNA ng/ul
Standard 1	47 RFU
Standard 2	24467 RFU
10A S-2	too low to read
11A S-2	0.434
12A S-2	too low to read
9B S-2	too low to read
10B S-2	0.298
11B S-2	0.6
12B S-2	too low to read