Sep 02, 2024
Preferential Lysis of S. rosetta for Total RNA (VERSION 2)
- 1University of California, San Francisco
Protocol Citation: David Booth 2024. Preferential Lysis of S. rosetta for Total RNA (VERSION 2). protocols.io https://dx.doi.org/10.17504/protocols.io.261ge4bojv47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 18, 2021
Last Modified: September 02, 2024
Protocol Integer ID: 52478
Keywords: choanoflagellate, mRNA, RNA-seq
Abstract
Sterol-based detergents, like digitonin more effectively disrupt membranes with sterols, like those of eukaryotes. This protocol leverages the different membrane compositions of eukaryotes and bacteria to preferentially lyses the eukaryotic membranes of the choanoflagellate species, S. rosetta, from a co-culture with bacteria. The lysis buffer includes RNase inhibitors to preserve RNAs for RNA purification or cDNA synthesis, and the included protease inhibitors make this protocol suitable for protein extractions for western blots.
Prepare Lysis Buffer
Prepare Lysis Buffer
Combine the following components for the lysis buffer:
| A | B | C | D | E | |
| Chemical | [Final] | [Stock] | Final Vol | Chemical Vol | |
| Water | 10 ml | 0.41 ml | |||
| Tris-HCl, pH 8.0 | 20 mM | 1 M | 200 µl | ||
| KCl | 150 mM | 2 M | 750 µl | ||
| MgCl2 | 5 mM | 1 M | 50 µl | ||
| Sucrose | 250 mM | 1.75 M (60% w/v) | 1ml 420 uL | ||
| Cycloheximide | 100 ug/ml | 100 mg/ml | 10 ul | ||
| Protease inhibitor tablet | 2 mini tablet / 10 ml | 1 ml | |||
| Digitonin | 10 mM | 20 mM | 5 ml | ||
| Sodium Heparin | 1 mg/ml | 100 mg/ml | 100 µl | ||
| Pefabloc SC | 1 mM | 200 mM | 50 µl | ||
| DTT | 1 mM | 1 M | 10 µl | ||
| Turbo DNase | 0.1 U/ml | 2 U/µl | 500 µl | ||
| SUPERaseIN | 1 U/ml | 20 U/µl | 500 µl |
Notes:
1. Prepare the buffer ahead of time by combining all but the italicized reagents (DTT, Turbo DNase, and SUPERaseIn), splitting into 449.5 µl aliquots, and storing at -20°C.
2. Just before use, thaw the prepared lysis buffer on ice and then add 0.5 µl of 1 M DTT, 25 µl of Turbo DNase, and 25 µl SUPERaseIn to the 449.5 µl aliquot for a total volume of 500 µl.
Count Cells
Count Cells
Determine the total number of cells in the culture that will be harvested using a hemocytometer.
Fix 200 µl of cells with 2 µl of 37% formaldeyde and vortex well.
Pipet up and down to homogenize cells, and pipet 12 µl of cells into the chamber of a hemocytometer.
Count the number of cells ( ) in the four corner quandrants of a Neubauer, bright-line hemocytometer.
Calculate the cell concentration ( cells/ml) according to this equation:
cells/ml
Determine the volume of the culture ( ) and then calculate the total number of cells in the culture ( ) according to this equation:
cells
Calculate the volume of lysis buffer ( µl) to add to the cells for lysis:
µl
Harvest Cells
Harvest Cells
Harvest cells of S. rosetta.
Centrifuge the cells in 50 ml conical tubes at 2400 x g, 4°C, 00:05:00
Gently remove supernatant with a serological pipette, leaving a small amount of liquid of the pellet.
Gently remove the remaining supernatant with a fine-tip transfer pipet.
Lyse Cells
Lyse Cells
Lyse cells in preferential lysis buffer.
Resuspend the cell pellet (go to step #3.3 ) in the calculated volume of (go to step #2.6 ).
Pipet the cells gently up and down and then incubate On ice for 00:10:00 .
Homogenize the lysate by passing 5 times through a 30G needle attached to a luer lock syringe.
Clarify the lysate by centrifugation 6000 x g, 4°C, 00:10:00
10m
Separate the S. rosetta lysate from the bacterial pellet by using a gel-loading tip to gently transfer the supernatant into a new tube. Pay attention not to disturb the pellet.
Storage
Storage
Flash freeze lysate in liquid nitrogen and store at -80°C for long term storage.