E5 Time Series RNA and DNA Re-Extractions, August 1, 2024

Protocol Link

Extraction of 8 POC, POR, and ACR samples from Moorea from the E5 Time Series (Time Points 1-4, sampled January-November 2020)

Samples

Absolutely need to be clipped (no shield tube):

• POC-53, TP4 (Nov)
  • subset bag in freezer: blue (M1)
• POR-262, TP2 (Mar)
  • subset bag in freezer: yellow (M2)

Can clip or can try to extract from shield tube:

• ACR-173, TP4 (Nov)
  • subset bag in freezer: blue (M1)
  • shield tube: 775
  • extraction attempts:
    • 20210902 - no RNA
    • 20220211 - no RNA
• ACR-186, TP1 (Jan)
  • subset bag in freezer: blue (M1); orange (M2)
  • shield tube: 249
  • extraction attempts:
    • 20211005 - no RNA
    • 20220221 - no RNA
• POC-219, TP3 (Sep)
  • subset bag in freezer: GREEN - 2 M2 frags?
  • shield tube: 521
  • extraction attempts:
    • 20211004 - degraded, and molecular sheet doesn’t match notebook
• POR-245, TP4 (Nov)
  • subset bag in freezer: pink (M1)
  • shield tube: 903
  • extraction attempts:
    • 20211116 - no RNA
    • 20220208 - no RNA
• POR-74, TP2 (Mar)
  • subset bag in freezer: green (M1) + yellow (M2)
  • shield tube: 479
  • extraction attempts:
    • 20211020 - nothing on Qubit but gel and nanodrop look okay???
    • 20220208 - nothing on Qubit; but gel looks okay
• POR-83, TP1 (Jan)
  • subset bag in freezer: orange (M1) + blue (M2)
  • shield tube: 215
  • extraction attempts:
    • 20211130 - no RNA
    • 20220217 - no RNA
• ACR-150, TP1 (Jan)
  • subset bag in freezer: blue (M1) + orange (M2)
  • shield tube: 211
  • extraction attempts:
    • 20211130
• ACR-145, TP1 (Jan)
  • subset bag in freezer: blue (M1) + orange (M2)
  • shield tube: 187
  • extraction attempts:
    • 20211115
• ACR-173, TP1 (Jan)
  • subset bag in freezer: blue (M1) + orange (M2)
  • shield tube: 203
  • extraction attempts:
    • 20211130
• POC-52, TP1 (Jan)
  • subset bag in freezer: orange (M1) + blue (M2)
  • shield tube: 213
  • extraction attempts:
    • 20220221
    • 20220203

Clipping list (all samples)

• POC-53, TP4 (Nov)
  • subset bag in freezer: blue
• POR-262, TP2 (Mar)
  • subset bag in freezer: yellow (M2)
• ACR-173, TP4 (Nov)
  • subset bag in freezer: blue
• ACR-186, TP1 (Jan)
  • subset bag in freezer: blue
• POC-219, TP3 (Sep)
  • subset bag in freezer: GREEN - 2 M2 frags?
• POR-245, TP4 (Nov)
  • subset bag in freezer: pink (M1)
• POR-74, TP2 (Mar)
  • subset bag in freezer: green (M1) + yellow (M2)
• POR-83, TP1 (Jan)
  • subset bag in freezer: blue
• ACR-150, TP1 (Jan)
  • subset bag in freezer: blue
• ACR-145, TP1 (Jan)
  • subset bag in freezer: blue
• ACR-173, TP1 (Jan)
  • subset bag in freezer: blue
• POC-52, TP1 (Jan)
  • subset bag in freezer: blue

Protocol

• We will clip them following this protocol into 2.0 screw cap tubes with 1000 uL of DNA/RNA shield and 0.25 mL of 0.5mm glass beads. These were kept on dry ice during clipping and immediately put into the -80 ºC freezer.

• On extraction day, follow protocol with adjustments:
  1. Bead beat for 1-1.5 minutes on the vortex at max speed. I started off with one minute for all samples but added a minute of bead-beating for samples if the liquid still looked very light-colored after one minute of homogenization.
  2. Briefly spin down and remove 400 uL of supernatant into a clean tube. Spin for 3 mins at 9,000 rcf.
  3. Put original samples and bead tubes back in -80 ºC freezer.
  4. Remove 300 uL of the supernatant into a new tube and continue on with the protocol below (from the Pro K step) as written.
• Other than the adjustments mentioned above, I followed protocol exactly.