title: NextSeq 500 paired end sequencing; In situ Hi-C study of chromatin conformation during early Drosophila development
design: In situ Hi-C study of chromatin conformation during early Drosophila development
library_construction_protocol: 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. We followed the general procedure for in situ Hi-C as described in Rao et al. (doi: 10.1016/j.cell.2014.11.021), which we adapted for use with fly embryos. The frozen embryos were placed on ice and suspended in 500 μl of ice-cold lysis buffer (10 mM Tris-Cl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630, cOmplete Ultra protease inhibitors). Using a tight-fitting metal pestle (Carl-Roth P985.1) the embryos were ground until a homogenous suspension was achieved. After 15 min incubation on ice, the nuclei were spun down at 1000 g, 4°C for 5 min. The supernatant was removed and the nuclei pellet resuspended in 500 μl of lysis buffer. Following another spin, the supernatant was discarded and the pellet resuspended in 100 μl 0.5% SDS. The nuclei were permeabilised by incubating at 65°C for 10 min. The SDS was then quenched by adding 50 μl of 10% Triton TX-100 and incubating at 37°C for 15 min. Chromatin was digested by adding 25 μl of CutSmart buffer and 50 U of MboI (New England Biolabs), followed by three hours of incubation at 37°C. The enzyme was added in two instalments of 25 U in the beginning and after 90 min of incubation. MboI was heat-inactivated by incubating at 62°C for 20 min. The overhangs left by MboI were filled in by adding 18.75 μl of 0.4 mM biotin-14-dATP (Life Technologies), 2.25 μl of a 3.3 mM dCTP/dGTP/dTTP mixture and 40 U of DNA Polymerase I Klenow Fragment (New England Biolabs), followed by incubation at 37°C for 90 min. The DNA fragments were then ligated by adding 657 μl of water, 120 μl of 10x T4 DNA Ligase buffer (Thermo Fisher Scientific), 100 μl of 10% Triton X-100, 6 μl of 20 mg/ml BSA and 25 Weiss U of T4 DNA Ligase (Thermo Fisher Scientific). The mixture was rotated slowly at room temperature for four hours. A second instalment of 25 Weiss U of T4 DNA Ligase was added after two hours. The nuclei were then spun down at 2500 g for 5 min, the supernatant discarded and the pellet resuspended in 500 μl of extraction buffer (50 mM Tris-Cl pH 8.0, 50 mM NaCl, 1 mM EDTA, 1% SDS). To digest the protein, 20 μl of 20 mg/ml Proteinase K (Applichem) was added and the mixture incubated at 55°C, 1000 rpm shaking for 30 min. 130 μl of 5 M sodium chloride was added and the mixture was incubated at 68°C, 1000 rpm shaking for at least 8 hours. For DNA precipitation, 63 μl of 3 M sodium acetate pH 5.2, 2 μl of 15 mg/ml GlycoBlue (Life Technologies) and finally 1008 μl of absolute ethanol were mixed with the sample, followed by incubation at -80°C for 15 min. The sample was then spun at 20,000 g, 4°C for one hour. The supernatant was removed and the DNA pellet washed two times using 800 μl of 70% ethanol each. All traces of remaining supernatant were removed; the pellet air-dried for 2 min and then solubilized in 50 μl of 10 mM Tris-Cl pH 8.0. RNA was digested by adding 1 μl of 20 mg/ml RNase A and incubating at 37°C for 15 min. Biotin-14-dATP was removed from the ends of unligated fragments by mixing 50 μl of the Hi-C library with 12 μl of 10x NEB2 buffer (New England Biolabs), 3 μl of 1 mM dATP, 3 μl of 1 mM dGTP, 0.6 μl of 20 mg/ml BSA, 15 U of T4 DNA Polymerase (New England Biolabs) and water to 120 μl total. The mixture was incubated at 20°C for 30 min and then stopped by adding 3 μl of 0.5 M EDTA. The DNA was sheared to ~200-400 bp with a Covaris S2 sonicator using the following program: 2 cycles á 50 seconds, 10% duty, intensity 5, 200 cycles/burst. For the preparation of magnetic beads for biotin pull-down, 30 μl of Dynabeads MyOne Streptavidin C1 (Life Technologies) were separated on a magnetic stand. The supernatant was discarded. The beads were washed once and then resuspended in 120 μl of 1x B&W buffer (2x stock: 10 mM Tris-Cl pH 7.4, 1 mM EDTA, 2 M NaCl). 120 μl of the sheared Hi-C sample was mixed with 120 μl of the prepared magnetic beads and the suspension was rotated at room temperature for 15 min. The beads were washed twice with 600 μl each of 1x B&W buffer + 0.1% Triton X-100 by incubating at 55°C, 1000 rpm shaking for 2 min. This was followed by a wash with 10 mM Tris-Cl pH 8.0. The beads were then resuspended in 50 μl of the same solution. Next, library preparation for Illumina sequencing was performed using components from the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). 3 μl of End Prep Enzyme Mix and 7 μl of End Prep Reaction buffer were combined in a 0.2 ml PCR strip tube. The bead suspension in 50 μl Tris-Cl pH 8.0 was mixed into the End Prep mixture and 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 (1:10 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. The beads were separated on a magnetic stand and the supernatant was discarded. The beads were washed twice using 1x B&W + 0.1% Triton X-100 and transferred to a 1.5 ml tube. The beads containing the final adapter-ligated Hi-C library were washed once with 10 mM Tris-Cl pH 8.0 and then resuspended in 50 μl of the same solution.
attributes.tag: Experimental Factor: nuclear cycle 14
attributes.value: developmental stage
attributes.tag: Experimental Factor: None
attributes.value: compound
attributes.tag: Experimental Factor: S-phase
attributes.value: cell cycle phase
attributes.tag: Experimental Factor: sh zld
attributes.value: genetic modification
attributes.tag: Experimental Factor: NA
attributes.value: immunoprecipitate