Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Chromatin immunoprecipitation (ChIP): Briefly, dejellied X. tropicalis embryos were treated with 1% formaldehyde (in 1% Marc's Modified Ringer's, MMR) for 15-45 min at room temperature to cross-link chromatin proteins to nearby genomic DNA. Duration of fixation was determined empirically and depended mainly on the developmental stage and antibody epitopes as described in Gentsch and Smith (2017). Efficient Preparation of High-Complexity ChIP-Seq Profiles from Early Xenopus Embryos. Methods in Molecular Biology, 1507, 23-42. Fixation was terminated by rinsing embryos three times with ice-cold 1% MMR. If required, post-fixation embryos were dissected to select specific anatomical regions in ice-cold 1% MMR. Fixed embryos were homogenized in CEWB1 (10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.1% SDS) supplemented with 0.5 mM DTT, protease inhibitors and, if using phospho-specific antibodies, phosphatase blockers (0.5 mM orthovanadate and 2.5 mM NaF). To solubilise yolk platelets and separate them from the nuclei, the homogenate was left on ice for 5 min and then centrifuged (1000 g) for 5 min at 4ºC. Homogenization and centrifugation was repeated once before resuspending the nuclei containing pellet in 1-3 ml CEWB1. Nuclear chromatin was solubilized and fragmented by isothermal focused or microtip-mediated sonication. The solution of fragmented chromatin was cleared by centrifuging (15,000 g) for 5 min at 4ºC. If required, ~1% of the cleared chromatin extract was set aside for the input sample (negative control for ChIP). ChIP-grade antibodies were used to recognize specific chromatin features and to enrich these by coupling the antibody-chromatin complex to protein G magnetic beads and extensive washing. These steps were carried out at 4ºC. The beads were washed twice in CEWB1, twice in WB2 (10 mM Tris pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.1% SDS), twice in WB3 (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Igepal CA-630 and 1% sodium deoxycholate) and once in TEN (10 mM Tris pH 8.0, 150 mM NaCl and 1 mM EDTA). ChIP was eluted off the beads twice with 100 µl SDS elution buffer (50 mM Tris pH 8.0, 1 mM EDTA and 1% SDS) at 65ºC. ChIP eluates were pooled before reversing DNA-protein cross-links. Input (filled up to 200 µl with SDS elution buffer) and ChIP samples were supplemented with 10 µl 5 M NaCl and incubated for 6-16 h at 65ºC in a hybridization oven. Samples were treated with proteinase K and RNase to remove any proteins and RNA from the co-immunoprecipitated DNA fragments. The DNA was purified with phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.9) using phase-lock gel heavy microcentrifuge tubes for phase separation and precipitated with 1/70 volume of 5 M NaCl, 2 volumes of absolute ethanol and 15 µg blue glycogen (GlycoBlue). After centrifugation, the DNA pellet was air-dried and dissolved in 11 µl elution buffer (10 mM Tris-HCl, pH 8.5). The DNA concentration was determined on a fluorometer using high-sensitivity reagents for double-stranded DNA (10 pg/µl to 100 ng/µl). DNase-probed chromatin accessibility: The assay to probe chromatin accessibility with DNase was adapted to early X. tropicalis embryos using a novel approach. Ultracentrifugation- or gel electrophoresis-mediated size selection was replaced by two rounds of solid phase reverse immobilization (SPRI) to remove high molecular weight (HMW) DNA from the informative, short DNA fragments. Wide-bore pipette tips were used for the resuspensions and the transfers of biological samples from the second homogenization step until after SPRI to avoid the shearing of HMW DNA. About 250 dejellied mid-blastula embryos (stage 8+) were collected in 2-ml round-bottom microcentrifuge tubes and homogenized in 2 ml ice-cold LB-DNase buffer (15 mM Tris-HCl pH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM and 0.5 mM spermidine) supplemented with 0.05% Igepal CA-630. The homogenate was left on ice for 3 min before centrifuging (1,000 g) for 2 min at 4ºC. The pellet was gently resuspended in 2 ml ice-cold LB-DNase buffer (without Igepal CA-630) before centrifuging (1,000 g) again for 2 min at 4ºC. The pellet was resuspended in 600 µl of room temperature LB-DNase buffer supplemented with 6 mM CaCl2. The sample was distributed equally to two 1.5-ml microcentrifuge tubes. Approximately 0.1 U DNase I was added to one aliquot while leaving the other aliquot untreated (internal control). Both samples were incubated for 8 min at 37ºC before adding 300 µl STOP buffer (50 mM Tris pH8.0, 100 mM NaCl, 100 mM EDTA, 0.1% SDS, 80 µg RNase A, 333 nM spermine and 1 µM spermidine). The samples were incubated for 15 min at 55ºC. Next, they were digested with 200 µg proteinase K for 2 h at 55ºC. The digests were transferred to phase-lock gel heavy microcentrifuge tubes for phenol:chloroform:isoamylalcohol (25:24:1) purification. The DNA was precipitated with 1/20 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of absolute ethanol. The DNA pellets were dissolved in 27 µl elution buffer (10 mM Tris pH8.5). To remove any remaining RNA (i.e. not digested in a first round of RNase treatment), 10 µg RNase A were added to the DNA samples. 5 µl (an equivalent of 20 mid-blastula embryos) from the internal control was digested with 0.3 U DNase I for 5 min at 37ºC to generate a negative control profile (i.e. DNase-treated naked genomic DNA) for chromatin accessibility. The purification and precipitation of negative control DNA was carried out along with the chromatin digest after SPRI-mediated size selection. The DNA sample from the chromatin digest was further processed by two rounds of SPRI. 22.5 µl SPRI beads were added to 25 µl DNA sample without pipetting up and down. After 3 min, by which time HMW DNA caused beads to coalesce, the tubes were clipped into a magnetic stand for microcentrifuge tubes. After 3 min, the supernatant was transferred to a 96-well microplate. 47.5 µl elution buffer and 43 µl SPRI beads were added sequentially and mixed gently by slowly pipetting up and down. After 3 min, the plate was transferred to a magnetic stand for 96-well plates. Once the beads have settled to bottom of the well, the supernatant and the digest of naked genomic DNA (see above) were transferred to separate 1.5-ml phase-lock gel heavy microcentrifuge tubes and purified with phenol:chloroform:isoamylalcohol (25:24:1, pH7.9). The DNA fragments were precipitated with 1/20 volume of 3 M sodium acetate (pH 5.2) and 3 volumes of absolute ethanol. After centrifugation, the DNA pellets were dissolved in 12 µl elution buffer. The DNA concentrations were determined on a fluorometer using high-sensitivity reagents for double-stranded DNA (10 pg/µl to 100 ng/µl). Chromatin conformation capture (3C): About 500 dejellied mid-blastula embryos (stage 8+) were fixed with 1% formaldehyde (in 1% Marc's Modified Ringer's, MMR) for 40 min at room temperature. The fixation reaction was terminated by rinsing the embryos three times with ice-cold 1% MMR. The embryos were aliquoted equally into two 2-ml round-bottom microcentrifuge tubes and homogenized in 2 ml ice-cold CEB-3C (10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.2% SDS) supplemented with protease inhibitors and 0.05 mM DTT (=CEB-3C*). The homogenates were kept on ice for 5 min before centrifuging (1,000 g) for 2 min at 4ºC. The pellet was resuspended in 0.5 ml ice-cold CEB-3C*. The resuspensions were pooled and then equally divided to two 50-ml conical tubes and each filled up with CEB-3C to 50 ml. The embryonic extracts were incubated at 37ºC for 1 h in a hybridization oven with the tubes rotating inside a hybridization bottle. The tubes were then centrifuged (1,000 g) for 5 min at room temperature. The pellets were resuspended in 50 ml double distilled water. The tubes were centrifuged (1,000 g) agai