Extractions for A. pulchra IGA and CGA Moorea 2022

This protocol is based on Maggie’s coral egg/larvae extraction protocol and Kevin’s coral larvae extraction protocol. This post details the info about the extraction steps for the samples that were collected in Moorea 2022 for D. Becker’s experiment on A. pulchra intra- and cross-generational acclimatization in response to marine heatwaves. The github for that project is linked here.

Samples

The samples run today were both adult and larval samples. The larval samples were 248, 249, 250, 260, 261, and 261. The larval samples had ~50 larvae in them and were preserved in ~600 uL of DNA/RNA Shield. The adult samples were ACR_5_5_20220418, ACR_8_5_20220418, ACR_12_5_20220418, ACR_16_5_20220418, ACR_20_5_20220418, and ACR_24_5_20220418. I abbreviated the adult sample names to 5, 8, 12, 16, 20, and 24. The adult samples had ~0.5 cm diameter tissue pieces and were preserved in ~1 mL of DNA/RNA Shield. This photo is post-bead beating:

Materials

• Zymo Quick-DNA/RNA Miniprep Plus Kit HERE Protocol Booklet
• Tris-Ethylenediaminetetraacetic acid (EDTA) 1X buffer for DNA elution
• Heating block capable of heating to 70ºC
• Centrifuge and rotor capable of spinning at 15,000 rcf
• Plastics
  • 3 1.5 mL microcentrifuge tubes per sample
  • 2 PCR tubes per sample
  • 2 Qubit tubes per sample
  • 1 5 mL tube per sample

Protocol

Protocol was followed according to this post. A few differences:

• I added 400 uL of DNA/RNA shield to the larval samples after thawing them so that the volume in the tubes was 1 mL of shield. I aliquoted out 500 uL for extractions and saved the remaining fraction in case we need to re-extract.
• In the RNA extraction, I added another wash step with 400 uL of wash buffer after the DNase incubation (i.e., after the incubation, I did 400 uL of prep buffer, 700 uL of wash buffer, 400 uL of wash buffer, and another 400 uL of wash buffer).
• I eluted in 80 uL of either Tris (DNA) or DNA/RNA free water (RNA) instead of 100 uL.
• Since we may send these samples out for small RNA sequencing (in addition to regular RNA-Seq), I split the RNA volume into two separate tubes (ie after QC, there is 35 uL of RNA in each tube).

QC

Qubit results

tube.num	type	DNA1	DNA2	DNA_avg	RNA1	RNA2	RNA_avg
260	LARVAE	12.6	12.6	12.6	20	19.8	19.9
261	LARVAE	11.8	11.8	11.8	17.4	17.4	17.4
262	LARVAE	12.2	12.1	12.15	19.2	19.4	19.3
248	LARVAE	14.5	14.7	14.6	23.2	23.2	23.2
249	LARVAE	18.2	18.3	18.25	23.8	24	23.9
250	LARVAE	23	23	23	30	30.2	30.1
5	ADULT	41.6	41.8	41.7	14.2	14.6	14.4
8	ADULT	33	33	33	11.8	11.8	11.8
12	ADULT	37.2	37.6	37.4	11.8	11.8	11.8
16	ADULT	37.6	37.8	37.7	15	15	15
20	ADULT	102	102	102	24	24	24
24	ADULT	22.6	22.8	22.7	12.8	12.6	12.7

Gel