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.