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

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)
M6	20250308	20250308	19.4	19.8	19.6
M7	20250308	20250308	NA	10.2	10.2
M10	20250308	20250308	36.8	37.4	37.1
M11	20250308	20250308	47.4	47.8	47.6
M23	20250308	20250308	11.2	11.4	11.3
M24	20250308	20250308	30.6	30.4	30.5
M35	20250308	20250308	10.4	10.6	10.5
M36	20250308	20250308	32	32.2	32.1
M47	20250308	20250308	16.2	16.6	16.4
M48	20250308	20250308	11.2	11.4	11.3
M60	20250308	20250308	12.2	12.4	12.3
M61	20250308	20250308	56	56.4	56.2
M72	20250308	20250308	NA	NA	NA
M73	20250308	20250308	13.2	13.4	13.3
M85	20250308	20250308	12.6	12.8	12.7
M86	20250308	20250308	32.8	33	32.9

No RNA detected in M72 using the BR kit, may use the HS kit.

Gel