Download We cultured Caenorhabditis monodelphis sp. n. strain JU1677 at 20

Nucleic acid isolation from Caenorhabditis monodelphis sp. n strain JU1677
Nematode culture
We cultured Caenorhabditis monodelphis sp. n. strain JU1677 at 20°C on NGM medium
plates (enriched in agarose and bacto-peptone) seeded with E. coli OP50. Nematodes were
harvested with M9 (supplemented with 0.01% Tween20) when plates are nearly starved and
then pelleted at 5000 rpm for 5 min. The pellet was washed twice more in M9+Tween and
excess buffer removed. The pellet was frozen at -80°C.
DNA extraction
We followed a DNA extraction protocol developed for the Caenorhabditis Genomes Project
by M.-A. Felix, A. Richaud, D. Laetsch, M. Blaxter and colleagues. In brief, 600 µl of Cell
Lysis Solution and 5 µl of proteinase K were added to 100-200 µl of frozen nematode pellet
and allowed to thaw before vortexing. The sample was incubated overnight at 65°C with
gentle shaking. 10 µl of RNAse A was added and mixed by inversion, before incubation at
37°C for 1 hr. 200 µl of Protein Precipitation Solution was added and vortexed. The mixture
was centrifuged at 13000 rpm (4°C) and the supernatant collected. 600 µl of isopropanol
was added and mixed by inversion. The sample was incubated at -20°C overnight before
being centrifuged for 30 min at 13000 rpm (4°C) and the supernatant discarded. The
remaining pellet was washed in 600 µl of 70% ethanol, before being centrifuged for 10 min
at 13000 rpm (4°C). The supernatant was removed and the pellet was allowed to dry. The
dried pellet was dissolved in 50 µl of TE and 5 µl of Riboshredder was added, mixed by
inversion, and incubated at 37°C for 2 hr. 300 µl of DNA Binding Buffer (ZymoResearch
Genomic DNA Clean & Concentrator kit) was added and mixed by inversion. The mixture
was transferred to a Zymo-Spin IC-XL Column in a Collection Tube and centrifuged at top
speed for 30 seconds with the liquid that flowed through discarded. The column was washed
twice using 200 µl of Zymo DNA Wash Buffer, each time discarding the liquid. Finally, 20 µl
of elution buffer was added to the column and centrifuged to elute the DNA. DNA was
quantified using a Qubit fluorimeter.
RNA extraction
We followed an RNA extraction protocol developed for the Caenorhabditis Genomes Project
by M.-A. Felix, A. Richaud, D. Laetsch, M. Blaxter and colleagues. In brief, 500 ul of Trizol
was added to 100 ul of nematode pellet and vortexed. The Trizol suspension was frozen in
liquid nitrogen and transferred to a 37°C water bath to thaw. This freeze/thaw process was
repeated 4 times, before 5 cycles of 30 s of vortexing and 30 s of resting. 100 µl of
chloroform was added and mixed by shaking. The mixture was centrifuged for 15 min at
13000 rpm (4°C). The aqueous phase was transferred to a new tube and 250 µl of
isopropanol added. The mixture was centrifuged for 15 min at 13000 rpm (4°C) and the
supernatant discarded. The remaining pellet was washed with 500 µl of 75% ethanol and
centrifuged for 5 min at 13000 rpm (4°C) and the supernatant discarded. The pellet was
allowed to air-dry before being dissolved in 100 µl of RNAse-free water.