Illumina MiSeq paired end sequencing; In situ Hi-C study of chromatin conformation during early Drosophila development
In situ Hi-C study of chromatin conformation during early Drosophila development
Following a pre-collection period of at least one hour, fly embryos were collected on yeasted 0.4% acetic acid agar plates at 25°C. After 30 min of collection, the embryos on the plate were incubated at 25°C until they reached the desired developmental stage. Embryos were dechorionated for 2 min in 2.5% sodium hypochlorite and rinsed with water. Embryos were suspended in 2 ml of PBS, 0.5% Triton X-100 and 6 ml of heptane. Cross-linking was initiated by adding 100 μl of 37% formaldehyde, followed by vigorous shaking. 10 min later, samples were spun at 500 g for 1 min and embryos were collected from the lower aqueous layer. 15 min after the start of fixation, 5 ml of PBS, 0.5% Triton X-100, 125 mM glycine was added to the embryos, followed by vigorous shaking for 1 min. The embryos were rinsed 3 times with PBS, 0.5% Triton X-100. Crosslinking and chromatin preparation from nuclear cycle 14 embryos was carried out as described in (Blythe and Wieschaus, 2015). After injection and storage at -80°C, 150 to 200 embryos where mechanically homogenized in 700 μl of RIPA (150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) + protease inhibitors (Sigma 04693159001) using a pellet pestle. After centrifugation for 5 min at 16000 g and 4°C, the supernatant was discarded taking care of pipetting out lipids at the surface and not touching the pellet. The pellet was then resuspended in 130 μl of fresh RIPA + protease inhibitors and sonicated on a Covaris S2 sonicator at 5% duty cycle for 10 minutes. Following sonication, the samples were centrifuged at 16000 g for 5 min at 4°C. The supernatant (~ 120 μl), containing the chromatin, was finally carefully removed and split in 2x 50 μl aliquots. The remaining 20 μl were decrosslinked overnight at 61°C with proteinase K 1 mg/ml final and loaded on a 1.5% agarose gel with 1X GelRed (Biotium #41003), migrated for 1.5 hours at 65V, and imaged to control that the size of the fragments was between 150 and 500 bp. During the whole procedure, the samples were constantly kept on ice to minimize protein degradation. Chromatin immunoprecipitation was performed using commercial kits (17-10085 Millipore) in 1.5 ml DNA LoBind Eppendorf tubes (Eppendorf) as follows. For each ChIP, 450 μl of dilution buffer was supplemented with 2.25 μl of protease inhibitor cocktail, and added to a 50 μl chromatin aliquot. For each sample, 5 μl (corresponding to 1% of the total volume) is set apart and stored at 4°C for later processing. These aliquot contain non-immunoprecipitated chromatin, which will serve as input during the processing of the ChIP-Seq and the analysis of the qPCR. To each 500 μl reaction volume, 20 μl of magnetic beads were added. The antibodies (anti-RNA Pol II pSer5: ab5408, Abcam; anti-pan RNA Pol II: ab103968, Abcam) were respectively used at a final dilution of 6:1000 and 18:1000 (3 μl or 9 μl in a 500 μl volume, respectively). The samples were then incubated overnight at 4°C on a rotating wheel. The next day, the beads were washed twice with the low salt buffer, twice with the high salt buffer, once with the LiCl buffer, and once with TE. These more stringent washes allow for better ChIP enrichment. Both the beads and the 5 μl previously set aside were finally resuspended in 100 μl of elution buffer + proteinase K 1 mg / ml final and incubated overnight at 61°C. The following day, the volume of the samples were adjusted to 400 μl with TE and DNA purified with an equal volume of phenol:chloroform:isoamyl alcohol 25:24:1 (Sigma) in a phase-lock-gel tube (5prime). After centrifugation at 12000 g for 5 min at RT, the supernatant (400 μl) was transferred to a new tube. The DNA was precipitated by addition of 40 μl of sodium acetate 3M pH 5, 2 μl of glycogen 20 mg/ml and 640 ml of -20°C ethanol 100%. The samples were then incubated for 1h at -20°C and centrifuged at 16000 g for 5 min at 4°C. Finally, the DNA pellets were washed twice with 800 μl -20°C ethanol 70% and resuspended in 20 μl TE. ChIP-Seq libraries were prepared directly from immunoprecipitated material using NEBNext Ultra II DNA Library Prep Kits (E7645, NEB) as follows. The immunoprecipitated material volume was adjusted to 50 μl with Tris-Cl pH 8.0 and transferred to a 0.2 ml PCR strip tube. To the reaction volume, 3 μl of End Prep Enzyme Mix and 7 μl of End Prep Reaction buffer were added. The mixture was incubated in a thermocycler running the following program: 20°C for 30 min, 65°C for 30 min, 4°C hold. 30 μl of Ligation Master Mix, 2.5 μl Adaptor for Illumina (1:15 diluted from stock) and 1 μl of Ligation enhancer were added to the sample and the mixture was incubated in a thermocycler at 20°C for 15 min. Next, 3 μl of USER enzyme was added and the mixture incubated at 37°C for 15 min in a thermocycler. Because of the low amount of initial material (2-5 ng),  as recommended by the manufacturer, a size-selection step was not performed and fragments were cleaned up to eliminate unligated adaptors by adding 87 μl of Ampure XP beads (Agencourt) and mixing it with the reaction volume. After a 5 min incubation at RT, the beads were separated on a magnetic stand and the supernatant containing the short fragments was discarded. The beads were then washed twice with 200 μl of ethanol 80%, air dried for 5 min and resuspended in 15 μl of Tris-Cl pH 8.0. After separating the beads on a magnetic stand, the supernatant was transferred to a new 0.2 ml PCR. For each library, a PCR reaction was prepared as follows: 25 μl of 2x NEBNext Ultra II Q5 Master Mix, 5 μl of 25 μM Universal PCR primer, 5 μl of 25 μM Indexed PCR primer and finally 15 μl of the ChIP-Seq library. The PCR reactions were run using the following program: 98°C for 30 sec, (98°C for 10 s, 65°C for 75 s, ramping 1.5°C/s) repeated 12 times, 65°C for 5 min, 4°C hold. The PCR product was size-selected using Ampure XP beads (Beckman Coulter). The PCR reactions were combined and adjusted to exactly 110 μl volume with water. 110 μl of beads were mixed with the sample and incubated at room temperature for 5 min. After separation on a magnetic stand, the supernatant was moved to a new tube and the beads discarded. Another 40 μl of beads was added to the sample and incubated at room temperature for 5 min. After separation, the supernatant was discarded and the beads washed twice with 700 μl of 80% ethanol, each time incubating for 30 seconds. Following complete removal of the ethanol, the beads were resuspended in 100 μl of 10 mM Tris-Cl pH 8.0 and incubated at room temperature for 1 min. The following second round of selection removes all traces of primers and possible primer dimers. Without separating, another 80 μl of fresh beads was added and the suspension was incubated at room temperature for 5 min. After separation of the beads, the supernatant was discarded and the beads were washed twice with 80% ethanol, as above. After all traces of ethanol were removed, the beads were resuspended in 20 μl of 10 mM Tris-Cl pH 8.0 and incubated at RT for 5 min. Finally, the supernatant, containing the final library was transferred to a 1.5 ml DNA LoBind Eppendorf tube.
Experimental Factor: nuclear cycle 14
developmental stage
Experimental Factor: alpha-amanitin
compound
Experimental Factor: S-phase
cell cycle phase
Experimental Factor: wild type
genetic modification
Experimental Factor: RNA polymerase II CTD Panel (RNA pol II CTD, phospho S2, phospho S5)
immunoprecipitate