Feb 10, 2023
Version 1
RNAi Library screen V.1
- 1London Institute of Medical Sciences
Protocol Citation: e.warren Warren 2023. RNAi Library screen. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqje5ovk5/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: February 08, 2023
Last Modified: February 10, 2023
Protocol Integer ID: 76666
Abstract
Protocol for screening a RNAi knockdown library in for behavioural and phenotypic effects in C. elegans
Materials
NGM agar plates
M9 Buffer
Tracking plates (UNIPLATE 650,96,PS,CLEAR:WHAT7701-1651)
Tracking plate lids (LID,POLYSTYRENE,CLEAR,UNIVERSAL PK100 : 512-1093)
96 well culture plates (Life technologies 268200)
Boekel pin replicator (140500 boekel)
Integra VIAFILL
Intrgra VIAFLO
Tips for VIAFLO (12.5 µl GRIPTIP, Sterile, LONG - 5 XYZ Racks of 384 Tips: 6404)
IPTG: 1M stock (dissolved in H20)
Nystatin: 5000x
Carbenicillin: 100 mg/ml (1000x) (dissolved in H20)
Tetracycline: 50mg/ml (5000x) (dissolved in 70% EtOH)
FUDR: 1g/10 ml (1000x) (dissolved in H20)
Perparing C. elegans
Perparing C. elegans
Prepare in advance
Prepare ten 90 mm NGM agar plates for each replicate of the screen for worm maintenance.
To prepare NGM follow the steps in the protocol for "Making normal NGM" and pour 35 ml per plate.
Protocol
NAME
Making normal NGM
CREATED BY
Bonnie Evans
Seed each NGM agar plate with 1 ml of OP50 bacteria, and allow to dry.
11 days before tracking
Chunk worms onto ten seeded 90 mm NGM agar plates.
Prepare tracking plates
Prepare tracking plates
11 days before tracking
Prepare 75 tracking plates (WHAT7701-1651, PK100 : 512-1093) containing NGM agar, prepared as above, with the addition of 1 mM IPTG, 100 µg/ml carbenicillin, 10 µg/ml tetracycline, 1x nystatin (60 Units/ml).
Dispense 200 µl per well using the protocol "Dispensing agar into multiwell plates".
Protocol
NAME
Dispensing agar into multiwell platesCREATED BY
Ida Barlow
Dry the tracking plates in a drying oven (with lids removed) until they lose 2.5 to 3.5% of their total weight.
Bleach synchronising C. elegans
Bleach synchronising C. elegans
8 days before tracking
Bleach synchronise worms from ten 90 mm plates according to the protocol "Bleach synchronisation of C. elegans".
Leave the eggs in M9 buffer in 15 ml faclon tubes on a rotator at 20 dgerees for two days.
Protocol
NAME
Bleach synchronisation of C. elegansCREATED BY
Ida Barlow
Grow up RNAi library
Grow up RNAi library
We are using a version of the Ahringer RNAi library containing 6198 C. elegans genes with orthologues in humans.
This library is stored as frozen glycerol stocks in seventy three 96 well plates.
8 days before tracking
Grow up the RNAi library.
CITATION
CITATION
Dispense LB broth (containing 100 µg/ml Carbenicillin, and 10 µg/ml Tetracycline), into seventy four 96 well plates (numbered 1-74) using the Integra VIAFILL.
Replicate all wells of the RNAi library into corresponsing plates of LB (with carbenicillin and tetrecycline).
Use a metal 96 pin replicator (Boekel) to transfer glycerol stock from stock plates to the LB culture plates.
To do this remove plates a few at a time from the -80 on dry ice.
Allow each plate around 1 minute to deforst slightly.
Firmly press the replicator into the stock plate, making sure to pick up bacteria from each well.
Transfer replicator to corresponding LB culture plate.
Between replicating each plate, the replicator is dipped in 100% ethanol and flame sterilised. This process is repeated twice to ensure full sterilisation.
One negative control plate is inncoluated after double flame sterilisation of the replicator.
Safety information
Take caution when flame sterilising the replicator.
Do not place flaming replicator back into the ethanol bath.
Work in a clear area - remove any flammable items, papers, tissue, card board boxes etc
Familiarise yourself with nearest safety blanket.
Place the LB plates containing the replicated library into large plastic boxes containing moistened paper towel, and incubate for 15 hours at 37 degrees.
Record OD600 values of 10 of the library plates using a plate reader (Tecan, Spark) to evaluate library growth.
Visually check the whole library and manually record any wells which have not grown.
Seeding tracking plates
Seeding tracking plates
7 days before tracking
Seed the tracking plates with the bacterial cultures using the Integra VIAFLO.
Using the Integra VIAFLO with GRIPTIPs mix each well of the library by pipetting 12.5 µl up and down 6 times, before transferring 10 µl from the library to the tracking plates twice.
Note
This double transfer is required to pipette a total of 20 µl of bacterial culture to each well of the tracking plates since the max volume of this VIAFLO is 12.5 µl.
Dry the tracking plates in a biosafety cabinet with lids removed for 3 hours.
Stack the tracking plates plates (upside down) in a sealed plastic box containing damp paper towel and incubate at 20 degrees.
Dispensing C. elegans to tracking plates
Dispensing C. elegans to tracking plates
6 days before tracking
Dispense C. elegans onto the tracking plates using the protocol "Dispensing C. elegans to 96 well tracking plate using Integra VIAFILL".
We aimed to dispense an average of 4 worms per well, but will have some unavoidable variation between wells.
Note
This step should be carried out at the same time for each replicate, such that FUDR can be added 53 hours after dispensing worms.
For example: We dispensed worms at 4pm on Wednesday and add FUDR at 9 am on the following Friday.
Protocol
NAME
Dispensing C. elegans to 96 well tracking plate using Integra VIAFILLCREATED BY
e.warren Warren
Leave plates to dry for 1 hour in a biosafety cabinet with lids removed.
Stack the tracking plates plates (upside down) in a sealed plastic box containing damp paper towel and incubate at 20 degrees.
Addition of FUDR
Addition of FUDR
4 days before tracking,
53 hours after dispensing worms, add FUDR to each well of the tracking plates to prevent hatching of progeny.
DIspense 10 µl of 20X FUDR to each well using the Integra VIAFILL with the small 8 channel cassette.
Leave plates to dry for 1 hour in a biosafety cabinet with lids removed.
Note
Check that plates are visibly dry.
Stack the tracking plates plates (upside down) in a sealed plastic box containing damp paper towel and incubate at 20 degrees.
Tracking using Hydra rigs
Tracking using Hydra rigs
On the day of tracking
Move tracking plates into the tracking room at 12 pm, one hour before tracking commences to aclimatise to temerature and humidity.
Record worm behaviour using the syngenta script (5 mins prestim; 6 mins bluelight with 60 sec OFF, [10sec ON, 90sec OFF] x 3 times; 5 mins postsim).
Run the following script
~/scripts$ python run_syngenta_experiment_v2.py -f 291122_rnaifullscreen_run1 – r 01 02 03 04 05
-f : date_file_ run number :
-r : specifies which rigs to start recording on
Note
Do not use blank spaces in file name
Include the run number for each run of tracking
Move all files to the NAS and then transfer files from the NAS to the group drive on the network for processing.
Citations
Step 6
Hernando-Rodríguez, B., Erinjeri, A.P., Rodríguez-Palero, M.J. et al.. Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans.
https://doi.org/10.1186/s12915-018-0496-5Step 6
Kamath, R., Fraser, A., Dong, Y. et al.. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi
https://doi.org/10.1038/nature01278