TITLE: Transcriptomics
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The potato root secretes root exudates, a mixture of organic molecules out of which solanine is found in the highest concentration (range of ug/ml). Also, solanine is a molecule specifically found in the root exudate of potato plants, which makes it a good candidate for a dependecy molecule which will ensure that </span><span style = "font-style:italic;">Bacillus mycoides</span><span> doesn't escape the designated action area. Studies have shown that there are soil bacteria able to metabolize solanine as a carbon source. Unfortunately, we weren't able to find an already described solanine-inducing promoter.  Because the natural habitat of </span><span style = "font-style:italic;">B. mycoides</span><span> is the potato rhizosphere, we have strong reasons to believe that if such a promoter may already be present in its genome. </span></div><div class = "text-block">In order to find this promoter, we designed a CHIP-Seq experiment. B. mycoides will be incubated with a different range of solanine concentrations. The DNA will be extracted and the CHIP-Seq technique will be used to detect potential binding sites of solanine operators using monoclonal anti-solanine antibodies. </div></div>

[STEPS]
?. [Sample preparation]
Dilute an overnight culture of B. mycoides to OD600=0.05 in LB. Divide the culture in 2 parts.Supplement one of them with solanine (concentration range from   to ) . Keep the other one as a negative control.
?. [Sample preparation]
Culture the cells for
30 33
?. [Sample preparation]
Collect cells by spinning down at
Centrifuge: 500 34, 5 min, 4 10
?. [Sample preparation]
Wash cells with PBS at room temperature.
?. [Sample preparation]
Remove the PBS and add freshly made basic cell culture media  containing formaldehyde to cross-link the DNA-protein complexes.
?. [Sample preparation]
Incubate for  with gentle agitation on a rocking platform.
0 Room temperature
?. [Sample preparation]
Remove the fixation solution by spinning down at .
Centrifuge: 500 34, 5 min, 4 10
?. [Sample preparation]
To quench the reaction, for cell count of 1-5 million, add  glycine solution. (!glycine solution concentration dependent on cell count)
3 mL
?. [Sample preparation]
Incubate at   with gentle agitation on a rocking platform.
0 Room temperature
?. [Sample preparation]
Remove the glycine solution by  . Discard thesupernatant. The pellet can be frozen at    after adding   of Protease Inhibitor Cocktail (PIC).
Centrifuge: 500 34, 5 min, 4 10
-80 °C
1 µl
?. [Sample preparation]
To lyse the cells, for cell count of 1-5 million, add  of Hypotonic Buffer; Resuspend the cells andincubate at .
0.4 mL
4 °C
?. [Sample preparation]
Centrifuge the hypotonic slurries at    to collect the nuclei.
Centrifuge: 5000 34, 5 min, 4 10
?. [Sample preparation]
For cell count of 1-5 million, add   of Digestion Buffer to the nuclei, immediately followed byadding   of PIC to each sample.
0.3 mL
2 µl
?. [Sample preparation]
Add Micrococcal Nuclease to each sample to digest the DNA. Mix by inverting the tube several timesand incubate at  . Mix by inversion every 3-5 minutes. The amount and incubationtime of Micrococcal Nuclease required to digest the genomic DNA to an optimal 150 900 bp lengthmay need to be determined empirically for individual cell types.
37 °C
?. [Sample preparation]
Stop digestion by adding   of  EDTA per sample and place the sample on ice.
10 µl
?. [Sample preparation]
Pellet nuclei by centrifugation at  . Discard the supernatant.
Centrifuge: 12000 34, 1 min, 4 10
?. [Sample preparation]
Resuspend nuclear pellet in Lysis Buffer and incubate the sample  to lyse thenuclei. Alternatively, sonicate to shear the DNA. The time and strength for sonication may need to bedetermined empirically.
on ice
?. [Sample preparation]
Centrifuge the sample at  .Transfer the supernatant to a clean dry microcentrifuge tube.
Centrifuge: 16000 34, 10 min, 4 10
?. [Sample preparation]
Add    of PIC to each sample and mix.
1 µl
?. [Sample preparation]
Chromatin sample is now ready for ChIP Assay. If the sample is not to be used immediately, store at  . It is recommended that the shearing efficiency is analyzed at this stage to ensure that 150-900bp fragments are obtained during shearing the DNA to increase the ChIP efficiency.
-80 °C
?. [Sample preparation]
Mix the Chromatin Sample, Protease Inhibitor Cocktail (PIC), optimal quantity of BioLegend’s Go-ChIP-GradeTM Purified antibodies and add Column Conditioning Buffer to make final   slurries.Gently rotate at   for **incubation can also be extended  .
1 mL
4 °C
?. [Chromatin Immunoprecipitation Assay:]
Prepare the high-throughput (HT) Protein A or G 96 well plate or Spin Column by adding   ofColumn Conditioning Buffer in each well or column and allow it to flow through via gravity (~ ).
600 µl
?. [Chromatin Immunoprecipitation Assay:]
Discard the flow-through and repeat steps 18 and 19.
?. [Chromatin Immunoprecipitation Assay:]
Remove the slurries from the rotator following   incubation and briefly spin down to removeresidual liquid from the caps.
4 °C
?. [Chromatin Immunoprecipitation Assay:]
Load the entire   slurries and allow to flow completely through the high-throughput (HT) ProteinA or G 96 well plate or Spin Column at room temperature (approximate ).
1 mL
?. [Chromatin Immunoprecipitation Assay:]
Add   of Wash Buffer 1 to each well or column and centrifuge at  for plate, orfor column, for at . Discard the flow through and repeat once.
600 µl
Centrifuge: 2.000 34
Centrifuge: 4.000 34
0 Room temperature
?. [Chromatin Immunoprecipitation Assay:]
Add of Wash Buffer 2 to each well or column and centrifuge at  for plate, or for column, for  at  . Discard the flow through and repeat once.
600 µl
Centrifuge: 2.000 34
Centrifuge: 4.000 34
0 Room temperature
?. [Chromatin Immunoprecipitation Assay:]
Add of Wash Buffer 3 to each well or column and centrifuge at  for plate, or for column, for  at  . Discard the flow through and repeat once.
600 µl
Centrifuge: 2.000 34
Centrifuge: 4.000 34
0 Room temperature
?. [Chromatin Immunoprecipitation Assay:]
Spin dry at for the plate or  for the column for one minute at room temperature toremove any remaining liquid from the membrane of the plate or column. Place a clean 96 wellcollection plate or a collection tube beneath the plate or column.
Centrifuge: 4.000 34
Centrifuge: 16.000 34
?. [Chromatin Immunoprecipitation Assay:]
Add  of Elution Buffer to each well or column. Incubate at room temperature for .
50 µl
?. [Chromatin Immunoprecipitation Assay:]
Centrifuge the plate or column at for the plate, or  for the column, for at  to collect the eluted chromatin-protein complex.
Centrifuge: 4.000 34
Centrifuge: 16.000 34
0 Room temperature
?. [Chromatin Immunoprecipitation Assay:]
To each eluted sample, add  of 1M NaHCO3, NaCl and  of Distilled Water. Mix thoroughly and incubate at  on the heat block for two hours or the incubation time can be extended to overnight.
5 µl
5 µl
50 µl
65 °C
?. [Chromatin Immunoprecipitation Assay:]
Remove the sample from the heat block, add  of Proteinase K to each sample, vortex briefly andperform a short spin down. Incubate at  for .
1 µl
37 °C
?. [Chromatin Immunoprecipitation Assay:]
Add  of Proteinase K Stop Solution to each sample, vortex briefly and perform a short spin down.
2 µl
?. [Chromatin Immunoprecipitation Assay:]
Purify the DNA by QIAquick™ PCR purification kit, according to manufacturer’s manual. Purified DNAis ready for downstream real-time qPCR analysis.