ChIP-Seq preparation and sequencing
To obtain an overview of the chromatin state and regulatory element
activity in V. cardui females during a critical time point of the
oogenesis-flight syndrome progression, we focused on head and antennae.
The rationale behind this selection was that nervous tissue is highly
concentrated to the head of butterflies and environmental cues are
perceived and processed by the sensory organs. Analysing head tissue was
therefore a logical first step to understand potential differential
regulatory element activities related to the propensity for migration.
Heads, including antennae, of the snap frozen individuals were ground to
fine powder in liquid nitrogen. Ice cold PBS with a Protease Inhibitor
Cocktail (PIC) was added before cross-linking with 1% formaldehyde for
seven minutes at room temperature. The reaction was halted with 125 mM
glycine and incubated at room temperature for five minutes. Samples were
then centrifuged for five minutes at 500 rpm in 4°C and subsequently
washed twice with ice-cold PBS + PIC. The samples were continuously
flash frozen at -80°C until all samples had been processed.
For the chromatin immunoprecipitation we used the SimpleChIP® Enzymatic
Chromatin IP Kit (Cell Signaling Technology Inc.) with some
modifications (see below). The chromatin was prepared by homogenizing
the tissue with a pestle in eppendorf tubes and then pooling four
samples (three pools per treatment/replicate). The cells were lysed with
DTT lysis buffer according to the manufacturer’s instructions. The
chromatin was fragmented by adding 0.5 µl Micrococcal Nuclease to each
pool and incubation at 37°C for 10 minutes. The nuclear membrane was
lysed with a Bioruptor Pico sonicator using six cycles of 30 second (s)
pulses and 30 s pauses. The samples from each treatment were then pooled
to get a single sample per replicate. The fragment size distribution of
the chromatin was quantified with a 2100 Bioanalyzer (Agilent Inc.)
after extracting DNA from a subset of the sample product. The
immunoprecipitation was performed according to the manufacturer’s
protocol with some minor modifications (see below). A small aliquot
(10µl) of the digested chromatin was set aside before adding antibodies
as an input control sample. We used specific antibodies against the
histone tail modifications H3K27ac and H3K4me3, which are associated to
active enhancers and promoters. 2.5 µg of Rabbit monoclonal H3K27ac,
H3K4me3 or negative control IgG was added to one aliquot from each
replicate, respectively. Each aliquot contained approximately 3 µg of
digested chromatin. The reactions were incubated overnight and
antibody-chromatin complexes were extracted with magnetic beads
according to the manufacturer’s protocol (except the extended 2 hours at
65°C with rotation and vortexing five times to eluate the chromatin from
the beads). Reverse cross-linking was performed with Proteinase K lysis
for 2 h at 56°C, and finally, DNA purification was performed with
columns provided in the kit. The input control sample was extracted
together with the immunoprecipitated samples. The purity and fragment
distribution of the DNA was assessed with Nanodrop (Thermo Scientific
Inc.) and a 2100 Bioanalyzer (Agilent Inc.). A total number of 12
libraries were prepared (2 treatments x 2 replicates x 3 extractions
including two precipitations and the input sample for each pool) with
SMARTer ThruPLEX DNA-seq and sequencing of 2x150 bp reads was performed
on a single Illumina NovaSeq6000 S4-300 lane at the National Genomics
Infrastructure (NGI, see acknowledgements) in Stockholm.