Post detailing the protocol for RNA extraction of the multispecies coelomocyte samples.

Sample Info

The samples are coelomocytes pelleted and preserved in 300ul of DNA/RNA Shield from Zymo Research and stored in -80C.

Here’s the protocol we used to sample and preserve the coelomocytes at Marrowstone - samples were later transferred to Roberts Lab -80 at SAFS:

Supply needs

  • 29G 3/10cc, 1/2” insulin needles - for juvenile star collection
  • Sterile, DNAse/RNAse-free microfuge tubes
  • Labeled PSC ### for coelomocytes
  • Labeled PSCS ### for coelomocyte supernatant
  • Centrifuge
  • DNA/RNA Shield
  • P1000 and sterile tips
  • Sample boxes - one for PSC samples, and one for PSCS samples
  • -80C storage space
  • Ice bucket with ice
  • 70% ETOH spray bottle
  • Paper towel

Protocol

  1. Collect coelomic fluid from the armpit using an insulin needle, wipe the needle with a paper towel sprayed with ETOH, and place in labeled microfuge tube
    i. If need to do multiple sites, use a new needle every time
  2. Collect as much volume as possible, with a max of 1mL (highly unlikely to collect this much)
  3. Place immediately on ice
  4. Bring to the dry lab
  5. Centrifuge for 5 minutes at 1200 rpm to separate the coelomocytes
  6. Remove the fluid using P1000 Pipette and tips and place in a labeled microfuge tube and store in -80C box for PSCS samples i. Note: will not be able to see a pellet, so act as though there is one at bottom of tube when removing supernatant
  7. Add 300 ul of DNA/RNA Shield to the pellet containing coelomocytes.

Store in -80C box for PSC samples

RNA Extraction

Kit: Zymo Research, Quick DNA/RNA Microprep Plus Kit (Cat D7005)

Extraction Preparations - labeling, etc

Let thaw:

  1. Proteinase K
  2. DNAse I

Label:

  1. 1 set of nuclease-free snap cap tube for each sample
  2. 1 set of Zymo-Spin IC-XM Column (yellow) plus the collection tube
  3. 1 set of Zymo=Spin IC Coolumn (clear) plus collection tube
  4. 1 nuclease-free tube for DNAse treatment
  5. 1 set of final labelled nuclease-free tube for eluted RNA.

I. Sample Preparation

Get samples from -80, and let thaw on wet ice, Mix well by vortex.

  1. Add 15ul Proteinase K and 30ul PK Digestion Buffer to each sample.
  2. Pipet to mix and let thaw at room temp for 30mins or longer.
  3. Vortex the sample after incubation and centrifuge at max speed for 2 minutes to pellet debris. Transfer 300ul of the cleared supernatant to a new nuclease-free tube (labeled).
  4. Add a 1:1 ratio (300ul) of DNA/RNA Lysis Buffer to the supernatant and mix well by pipetting.

II. DNA/RNA Purification

Perform all steps at 16,000g for 30sec unless specified.

  1. Transfer the sample (~600ul) to a Zymo-Spin IC-XM Column (yellow) in a collection tube and centrifuge at 16,000g for 30sec. i. FLOW-THROUGH HAS RNA!!! FILTER HAS DNA –> PUT IN -20C ii. Put DNA filter into a new collection tube and place in -20C freezer in FTR 213
  2. Add 1 volume (600ul) of 95-100% EtOH to the flow-through and mix by pipetting. i. Transfer the sample into a Zymo-Spin IC Column (clear) with collection tube ii. Discard flow-through
  3. DNase I Treatment i. Add 400ul DNA/RNA Wash Buffer to the column and centrifuge at 16,000g for 30 sec. Discard flow-through
    ii. Prepare DNase I Reaction Mix in labelled nuclease-free tube a. 5ul of DNase I : 35ul of DNA Digestion Buffer x ___ reps b. invert gently to mix iii. Add 40ul DNase I Reaction Mix to each sample and let incubate at room temp for 15 minutes
  4. Add 400ul DNA/RNA Prep Buffer and centrifuge at 16,000g 30s. Discard flow-through
  5. Add 700ul DNA/RNA Wash Buffer and centrifuge at 16,000g 30s. Discard flow-through
  6. Add 400ul DNA/RNA Wash Buffer and centrifuge at 16,000g for 2 minutes. Discard flow-through and transfer column to a new, labelled nuclease-free tube.
  7. Add 15ul Room Temp DNAse/RNAse-Free Water directly to column and centrifuge 16,000g 30s.

Can now store at -80C or immediately run 1ul on Qubit.

Quantify RNA (Qubit)

Example ratios if had 4 samples to run:

4 samples, 2 standards, 1 extra prep = 4+2+1= 7 preps
Mix the Working solution: 7 x 1ul RNA HS Dye = 7 ul RNA HS dye 7 x 199ul RNA HS Buffer = 1393 ul RNA HS Buffer

Standards:
10ul S1 + 190ul working solution
10ul S2 + 190ul working solution

Samples:
1ul of sample + 199ul working solution.

Vortex well 5 sec.

Incubate RT 2 mins.

Run on Qubit with RNA HS, record data

Extraction Plan/Schedule

I currently have RNAseq data for Day 12 from 6 exposed bins. Each bin has 3 RNAseq libraries associated with it, one for P. helianthoides, one for P. ochraceus, and one for D. imbricata.

See figure showing the timepoint (red line) where current data is from:
img

I have funding to send out 54 samples, with the following breakdown for the multi-species project:

Day 12 Controls n=18 libraries
n=6 P. helianthoides
n=6 P. ochraceus
n=6 D. imbricata

Day 6 Exposed
n=18 libraries
n=6 P. helianthoides
n=6 P. ochraceus
n=6 D. imbricata

Day 6 Controls
n=18 libraries
n=6 P. helianthoides
n=6 P. ochraceus
n=6 D. imbricata

Total libraries = 18x3 = 54 libraries.

Extraction plan

Notes:

  • I’m working remotely June 18-24 for a wedding and family time during which I’ll work on Day 12 exposed Multi-species data
  • RNA samples need to be shipped early in the week to ensure that there’s no shipping issues to New Jersey… so I am aiming to send them out June 30th, July 1st, or July 2nd

Additionally, I’ll aim to extract in sets of 18, with one round of 4 samples to review the extraction before I jump into the main samples.

Schedule:
Note: I have one full box of 50 preps, plus 32 extra preps, so 82 total preps.

Tuesday, June 24th (I land at 2pm)–> afternoon:

  • Do a practice extraction with 4 samples
  • Run 1ul on Qubit

Wednesday, June 25th:

  • Extract RNA from 18 samples
  • Run 1ul on Qubit

Thursday, June 26th:

  • Extract RNA from 18 samples
  • Run 1ul on Qubit

Friday, June 27th:

  • Extract RNA from 18 samples
  • Run 1ul on Qubit

NOTE: Randomize the 54 samples across the three days so I’m not extracting RNA from each group.

Monday, June 30th:

  • Prepare samples for shipping

If I can’t finish the extractions in time for June 30th, I can shift a little and do extraction of 18 samples on Monday June 30th and/or Tuesday July 1st. With shipping Tuesday July 1 or Wednesday July 2.