Extractions for Mcap developmental time series Hawaii 2023

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 M. capitata developmental time series experiment in Hawaii 2023. The github for that project is linked here.

Samples

Today I am re-extracting M8, M9, M13, M14, M26, M28, M37, M39, M51, M52, M62, M63, M74, M75, M87, and M88. I am only extracting RNA for these samples!

The samples extracted today were ones that I have previously extracted and sequenced. However, the smRNA sequencing failed for almost all of the samples, but Steven has offered to pay to resequenced them!! Very kind. I am re-extracting the RNA for the smRNA sequencing so that the facility can get fresh RNA that hasn’t been thawed/frozen several times I used 300uL as my input volume instead of 500uL. These samples had already been bead beaten from previous extractions.

Forgot to take sample picture :’(

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
  • 1 PCR tubes per sample
  • 1 Qubit tubes per sample

Protocol

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

• 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 DNA/RNA free water (RNA) instead of 100 uL.

QC

Qubit results

Used RNA BR Qubit kit.

Tube ID	Extraction Date	QC Date	Qubit RNA1 (ng/uL)	Qubit RNA2 (ng/uL)	Qubit RNA Average (ng/uL)
M8	20250309	20250309	21	21	21
M9	20250309	20250309	NA	NA	NA
M13	20250309	20250309	28.4	28.4	28.4
M14	20250309	20250309	29.4	29.6	29.5
M26	20250309	20250309	27.4	28	27.7
M28	20250309	20250309	NA	NA	NA
M37	20250309	20250309	26	26	26
M39	20250309	20250309	14	13.8	13.9
M51	20250309	20250309	62.6	62.8	62.7
M52	20250309	20250309	17	17	17
M62	20250309	20250309	33	33.2	33.1
M63	20250309	20250309	37	37	37
M74	20250309	20250309	15.8	15.8	15.8
M75	20250309	20250309	15	14.6	14.8
M87	20250309	20250309	48.2	48.2	48.2
M88	20250309	20250309	63.2	63.2	63.2