title: GSM3317248: Kc167_rps6_Rep2; Drosophila melanogaster; OTHER
library_construction_protocol: Cells (500 ml at ~9x106 cells/ml) were spun down (400g, 4°C, 10 minutes) two times and resuspended in 30 ml PBS both times. Protein-DNA complexes were cross-linked by adding 37% formaldehyde (final concentration 1%) to the cells and incubating at room temperature for 10 minutes. Formaldehyde was quenched by adding 1.25 M glycine (final concentration 125 mM). The cells were washed with 30 ml PBS, resuspended in 10 ml lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP40), and incubated on ice for 15 minutes. The cells were dounced 15 times on ice with a B pestle, incubated on ice for another 15 minutes, then dounced 15 times again. The nuclei were pelleted at 2000g at 4°C for 5 minutes. Depending on the viewpoint, 5x108 nuclei were resuspended in 500 µl DpnII (scs', hts, snf) or NlaIII (RpS6) restriction enzyme buffer and distributed into 50 µl aliquots in 10 tubes. Nuclei were lysed by adding 10% SDS (final concentration: 0.3%) and shaking at 37°C overnight. SDS was diluted to 0.1% with DpnII or NlaIII restriction enzyme buffer, and the SDS was quenched by adding 20% Triton X-100 (final concentration 1.1%) and shaking at 37°C overnight. The following day 22.5 units of DpnII or 15 units of NlaIII (New England Biolabs) was added to each of the appropriate 10 tubes and shaken at 37°C overnight. The same amount of restriction enzyme was added the next day and shaken again at 37°C overnight. Restriction digestion efficiency was determined by isolating DNA from 5% of the digested samples to determine the DNA concentration and perform PCR using primers spanning viewpoint restriction sites (S1 Table), as described [34], and found to be approximately 90% cut. Restriction digestions were stopped by adding SDS to 1.6% and heating at 65°C for 30 minutes. Samples cut with the same restriction enzyme were pooled, 30 µg was diluted to 2 ng/µl with ligation buffer, and 20% Triton X-100 was added to a final concentration of 1.1%. Samples were incubated at 37°C for 4 hours, then 6000 units T4 DNA ligase (New England Biolabs) was added and incubated at 4°C for 72 hours. After ligation, Proteinase-K was added to 0.1 mg/ml and incubated overnight at 65°C. Next samples were treated with 0.22 ng/ml RNAse A at 37°C for one hour, and DNA was extracted using phenol chloroform and ethanol precipitated. The DNA pellet was resuspended in 100 µl TE buffer and quantified by Qubit (Thermo-Fisher). We then set up second restriction digestions, using 3 µg per viewpoint. For DNA first cut with DpnII, we used CviQI (scs') or NlaIII (snf and hts); for DNA first cut with NlaIII we used DpnII (RpS6). After the second digestion samples were phenol chloroform extracted, ethanol precipitated, resuspended, and 1 µg was ligated at 0.8 ng/µl at 16°C overnight. The final ligated DNA was again extracted, precipitated and quantified by Qubit. This DNA was the input for making 4C libraries. The four 4C libraries were made by inverse PCR using Phusion polymerase (New England Biolabs) and primers with Ion Torrent PGM barcodes and adapters incorporated into the primer sequences (S1 Table). Two libraries were made for each of the four viewpoints, one for each end of a viewpoint. PCR products were size selected on a 2% agarose gel (100 – 500 bp). DNA from the gel was extracted with a Wizard PCR clean-up kit (Promega) and submitted to the LSU Genomics Facility for Ion Torrent sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3317248