Post-Size Selection and Clean Up of 1 Small RNA Library, Final Small RNA Library Size Selection and Cleanup, and Final Libraries TapeStations

I was concerned that the strange TapeStation results from the post-size selection libraries (peaks at ~175bp) could have been because the libraries were in the electrophoresis buffer from the Blue Pippin instead of something like TE buffer. I had done the Monarch DNA purification for the libraries that had been size selected after the TapeStation (as the protocol recommends), which can be seen here. I decided to TapeStation one sample after cleanup to see if the size of the libraries would be different in TE buffer.

20230209 TapeStation Library 3

  • I chose sample #3 because it had the highest concentration in the previous TapeStation so I thought it would be ok to used 2ul from it for this test tapestation
  • I thawed the library on ice and took 2ul down to the Genome Core
  • Took out the High Sensitivity D1000 sample buffer and D1000 screen tape from their fridge to equilibrate to room temperature
    • Note, the GSC does not purchase ladders for the HS D1000, they only use electronic ladders
  • Once the reagents were at room temp, vortexed and spun down the sample buffer
  • Got the TapeStation tube strip and strip caps
  • Added 2ul of sample buffer to 1 tube in the TapeStation tube strip
  • Added 2ul of library to the tube with sample buffer
  • Vortexed tubes for 1 minute
  • Spun down tube
  • Logged into computer and turned on TapeStation, opened software
  • Loaded sample tubes, took of the caps gently, and loaded in the screen tape
  • Selected 1 well and named them, right clicking the “ladder” position and changing it to electronic ladder so I could put a sample in the first position
  • Closed the lid and pressed start
  • Machine ran for ~5 minutes
  • A folder is created for the date, exported the results as a pdf and saved it to that folder. Then renamed the folder by adding initials and Unckless after it
  • Took out tubes, pipette tips, and screentape from the machine, put the screentape back in the fridge as there were spots left, and shut down the machine
  • Saved pdf on flashdrive
  • PDF can be seen here

This average peaks size of 157bp is basically what we were expecting originally, so this was good. It is likely that the electrophoresis buffer slightly messed up the size estimation.

I decided to size select the last sample the same way I had done previously, and then tapestation all of the libraries (except for #3) again once they were purified with the Monarch kit.

20230221 library 1 size selection, purification, and TapeStations on remaining libraries

Size Selection library 1

  • The only sample I had not size selected was library 1, because it has low concentration, I decided to use the entire 30ul of the library
  • Using BDQ3010 3% cassettes with internal marker Q3
  • Brought Marker Q3 and the electrophoresis buffer to room temp (stored in 4C), flicked to mix, and spun down
  • Thawed sample 1 on ice
  • Make a new set of strip tubes. Added 30ul of library 1 to a the strip tube
  • Mixed 10ul of Marker Q3 with the 30ul of sample, pipette mixed and spun down
  • Brought samples, pipette, tips, and cassettes to the Genome Core
  • Turned on the machine
  • Selected the 20230215-UNK-SmRNA-110-170 program (saved from previous). I tried to turn off the other lanes beside the one I was going to use, but this ended up not working because I had the reference lanes still selected (needed to be turned off as well) so voltage still ran through the rest of the cassette. That means I can’t use that cassette again, but that should be ok
  • Calibrate optics on the machine:
    • Put the calibration fixture into the machine and closed the lid
    • Pressed calibrate
  • Inspected cassette:
    • Are there any cracks or breaks in the agarose? - No
    • Are there bubbles in the optical chamber of the agarose? - No
  • Prepared cassette:
    • Dislodged bubbles from behind elution wells
    • Placed cassette in nest making sure the bubbles don’t return
    • Removed adhesive
    • Removed all buffer from elution wells, sucked up from multiple orientations
    • Replaced buffer in elution wells with 40ul of fresh electrophoresis buffer
    • Sealed elution well with tape strip
    • Checked buffer level in sample wells, made sure it is flush with the gel
  • Closed lid and performed the continuity test - Pass
  • Loading sample:
    • Re-checked buffer level in sample wells
    • Removed 40ul of buffer from first sample well - followed liquid level down with pipette tip
    • Pipette mixed the sample, then loaded it into the first sample well - followed liquid level up with pipette tip
  • Closed lid
  • Double checked that the correct protocol is loaded and then pressed start
  • Program ran for about 70 minutes, and the marker was visible so the 1st well eluted the sample
  • Removed the entire volume in the elution well and placed in a new 1.5mL tube. This was about 40ul

Library 1 post size selection Monarch Cleanup

  • Total volume out of the Blue Pippin is ~40ul, and the protocol says to use the 7:1 binding buffer to sample ratio
  • Added 252ul of DNA Cleanup Binding Buffer to the sample tube
  • Pipette mixed to mix the sample
  • Prepared a spin column
  • Added the ~300ul of sample to the spin column
  • Centrifuged the spin column at 16,000 rcf for 1 minute
  • Discarded the supernatant
  • Added 200ul DNA Wash Buffer to the column
  • Centrifuged the column at 16,000 rcf for 1 minute
  • Discarded the supernatant
  • Added another 200ul DNA Wash Buffer to the column
  • Centrifuged the column at 16,000 rcf for 1 minute
  • Discarded the supernatant
  • Centrifuged the column “dry” for 1 minute at 16,000 rcf
  • Placed the spin column in a new 1.5mL tube
  • Added 20ul of DNA Elution Buffer (TE buffer) to the column and let sit for 1 minute
  • Centrifuged column for 1 minute at 16,000 rcf

TapeStation of post size selection and cleaned up libraries 1, 2, 4, 5, and 6

  • Thawed libraries 2,4,5, and 6 on ice
  • Aliquoited 2ul from each library into new strip tubes
  • Took samples and pipettes and tips down to the genome codre
  • Took out the High Sensitivity D1000 sample buffer and D1000 screen tape from their fridge to equilibrate to room temperature
    • Note, the GSC does not purchase ladders for the HS D1000, they only use electronic ladders
  • Once the reagents were at room temp, vortexed and spun down the sample buffer
  • Got the TapeStation tube strip and strip caps
  • Added 2ul of sample buffer to each of 5 tubes in the TapeStation tubes
  • Added 2ul of library to each tube with sample buffer. I went in order of 1-6 (without 3)
  • Vortexed tubes for 1 minute
  • Spun down tube
  • Logged into computer and turned on TapeStation, opened software Loaded sample tubes, took of the caps gently, and loaded in the screen tape
  • Selected 5 wells and named them, right clicking the “ladder” position and changing it to electronic ladder so I could put a sample in the first position
  • Closed the lid and pressed start
  • Machine ran for ~5 minutes
  • A folder is created for the date, exported the results as a pdf and saved it to that folder. Then renamed the folder by adding initials and Unckless after it
  • Took out tubes, pipette tips, and screentape from the machine, put the screentape back in the fridge as there were spots left, and shut down the machine
  • Saved pdf on flashdrive
  • TapeStation PDF can be seen here

I took the average peak bp and the concentration from these TapeStations and calculated the molarity here