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Chromatin immunoprecipitation of Drosophila Schneider cells using antibodies against HP1a

ABSTRACT: Preparation of nuclear extracts

D. melanogaster Schneider cells were prepared as previously described by Andrews and Faller (1991). Briefly, in a typical preparation, 8x106 cells (grown in Schneider medium plus 10% fetal calf serum; GIBCO-BRL) were pelleted and resuspended in 1,5 mL cold PBS1X; the cell suspension is then tranferred to a microfuge tube. Cells are pelleted for 10 seconds and resuspended in 400 M-5L cold Buffer A (10mM HEPES-KOH pH 7.9 at 4M-0C, 1.5mM MgCl2, 10mM KCl, 0.5mM duthiothreitol, 0.2mM PMSF, 0.2U/microL RNasin). The cells are allowed to swell on ice for 10 minutes, and then vortexed for 10 seconds. Samples are centrifuged for 10 seconds, and the supernatant fraction is discarded. The pellet is resuspended in 100 M-5L of cold Buffer C (20mM HEPES-KOH pH 7.9, 25% glycerol, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.5mM dithiothreitol, 0.2mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris is removed by centrifugation for 2 min at 4M-0C and the supernatant fraction is stored at M-^V70M-0C. All buffers were prepared from double-distilled autoclaved water that had been treated with 0.1% DEPC (Sigma).

RNA immunoprecipitation and Reverse Transcription

For RNA immunopreciptation, the nuclear extract was incubated whit 50M-5g of monoclonal C1A9 anti HP1 antibody and the mixture was kept at 4M-0C overnight under continuous gentle movement. One-hundred microliters ofprotein G-Sepharose (Sigma) suspension (50% packed Sepharose in Buffer C) was added and the incubation was continued overnight as described.The beads were pelleted by 2 min centrifugation at 240 g at 4M-0C; the pellet was washed briefly three times whit each 1mL of IP Wash Solution (150mM NaCl, 50mM Tris pH 7.5, 0.5% NP40) and the Sepharose was transferred for eluition into a fresh plastic tube , pelleted again and then the supernatant was removed completely. To elute the immunocomplexes for protein analysis, an aliquot of beads were suspended in 50M-5L SDS-PAGE sample buffer and incubated for 10 min at 90M-0C; following centrifugation the supernatant was removed and used for Western blot for testing the presence of HP1 protein.
To elute the immunoprecipitated RNAs, the pelleted beads were boiled in 200M-5L of DEPC wather for 5 min, spun, and the supernatant recovered; 1mL of Trizol (Invitrogen) was added to 200 M-5L of supernatant and mixed well, followed by the addition of 200M-5L of chloroform. This mixture was incubated at 4M-0C for 5 min and then centrifuged at 12000 g for 15 min; the RNAs in the aqueous phase was precipitated whit half volume of isopropanol; after precipitation, the RNAs was resuspend in 10M-5L of DEPC wather. Contaminating DNA was digested whit RNase-free DNase I (Sigma). The RNA purified from the previous step is used as a template to synthesize cDNA using oligo dT , random hexamers and SuperScript reverse transcriptase III (Invitrogen) according to the manufacturer's protocol.

cDNA amplification and labeling

The cDNA was used as template for a two-step random PCR amplification; in Round A, Sequenase is used to extend randomly annealed primers (Primer A) to generate templates for subsequent PCR; during Round B, the specific primer B is used to aplify the templates previously generated and finally round C consist of additional PCR cycles to inorporate the amino allyl dUTP nucleotide.
About 25 ng of each cDNA sample was used for two 8 min extension with 2,7 mM Round A primer (5M-^R-GTT TCC CAG TCA CGA TCN NNN NNN NN-3M-^R, N being a mixture of all four nucleotides with 60% A+T and 40% G+C) at 37M-0C with 267U/ml Sequenase version 2.0 (usb). DNA was denatured at 94M-0C for 2 min and cooled to 10M-0C , and Sequenase 2.0 was added between extensions. The resulting products were used as template for 25 cycles of PCR using 1U/100M-5l Taq polymerase (Platinum Taq Invitrogen) and 10mM Round B primer (5M-^R-GTT TCC CAG TCA CGA TC-3M-^R). Finally this DNA was used as template for 25 cycles PCR to inorporate the amino allyl dUTP nucleotides to which the fluorescent dye may then be attached. To remove Tris buffer which interferes with the indirect coupling, the aminoallyl-cDNA samples were desalted by filtering through a Microcon M-^V30 and then mixed with the succinimidyl esters of the Cy3 or Cy5 dyes (Amersham Biosciences) in 0,1M sodium bicarbonate buffer (pH 9); the coupling reaction was icubated overnight in the dark at room temperature.
Each dye labeled sample was purified by AutoSeq MicroSpin G-50 columns (Amersham Biosciences) following the manufacterers directions.

Microarrays Hybridization and washing

The dried labeled cDNAs pellet were resuspended in 80M-5l of DIG Easy Hyb (Roche) hybridization buffer containg 0,5 mg/ml yeast tRNa and 0,5 mg/ml salmon sperm DNA. Finally the DNA probe was denaturated by incubation at 65M-0C for 10 min and, after a brief centrifugation to spin down the drops, the mixture was pipetted onto a microarray; a coverslip was applied and the slide was placed in a microarray hybridation chamber (BioRad) and incubated overnight at 37M-0C.
After hybridization, the slide was submerged in 0,01% SDS, 1%SSC until the coverslip slipped off the surface; The slide was transferred to a solution of 1%SSC and shaken at 50M-0C for 10 min, then washed once more by shaking in 0,1%SSC.
Slide was dried by centrifugation for 3 min at 550 rpm and scanned with an ScanArray Lite Microarray Scanner (Packard Bioscience) with laser intensities chosen to maximize signals while avoiding pixel saturation.
ScanArray express softwere was used to quantify hybridation signals; bad spots were flagged automatically by softwere and subsequently each slide was inspected manually.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Lucia Piacentini

PROVIDER: E-MEXP-1556 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Chromatin immunoprecipitation of Drosophila Schneider cells using antibodies against HP1a

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