PSC samples from 2023 are pelleted cells stored in 300ul of DNA/RNA Shield Based on what I did for the Day 12 exposed PSC samples from the Multi-Species work. # 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 3. Collect as much volume as possible, with a max of 1mL (highly unlikely to collect this much) 4. Place immediately on ice 5. Bring to the dry lab 6. Centrifuge for 5 minutes at 1200 rpm to separate the coelomocytes 7. 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 8. 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)[https://www.zymoresearch.com/products/quick-dna-rna-microprep-plus-kit?srsltid=AfmBOoqbz4OOia6Gt3N81ntbBEpzRDfNY9JF21Ez587mK4QaWbmDZBTT]) ## 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. 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 in a collection tube and centrifuge at 16,000g for 30sec. i. FLOW-THROUGH HAS RNA!!! FILTER HAS DNA --> PUT IN FRIDGE ii. Put DNA filter into a new collection tube and place in fridge in FTR 213 2. Add 1 volume (**600ul**) of 95-100% EtOH 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. ii. Prepare **DNase I Reaction Mix** 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. 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