ChIP-Seq 샘플 조건

Requirements for DNA samples

Sample condition: DNA samples

Sample quantity (for a single library preparation): ≥ 15 ng, the more, the better

Sample concentration: ≥ 5 ng/μl

Sample purity: OD260/280= 1.8～2.0

Gel electrophoresis analysis result should be provided to make sure that the size of DNA fragments is between 100bp to 500bp, and the size of most fragments should be about 250 bp.

Please provide a detailed sample information sheet and the result of qPCR which is designed to test the reliability of ChIP enrichment.

Notes:

   1) The final concentration is determined by QUBIT result after the samples are received.
   2) A backup DNA sample will be saved if the DNA sample is more than required for experiment.
   3) The rest DNA sample won’t be saved after project is finished.

Requirements for Cell Samples for ChIP Enrichment

Sample condition: cell-line;
Cell numbers (for a ChIP enrichment):5×10⁷ (more than 5×10⁶ cells for the species with genome size similar to human, 5×10⁵ cells, with the same treatment).
Pretreatment of cells: we set up two different ChIP enrichment platforms for cell-line samples, and the pretreatment of cells for two platforms are as follows:

a) Cross-linking Chromatin Immunoprecipitation (X-Chip)

1) At least 5×10⁷ (more than 5×10⁶cells for the species with genome size similar to human) cells are required for one ChIP enrichment

experiment. Preparation of an extra tube is necessary (5×10⁵ cells, with the same treatment)

2) Preheat fresh 1% formaldehyde solution at 37℃ in a water bath.

3) Add preheated formaldehyde solution to cell culture plate, and incubate at 37℃ for 10min.

4) For suspension cells, centrifuge at 800 g for 1 min to remove supernatant.

5) Remove formaldehyde solution, and then rinse twice with pre-chilled 1x PBS+BSA (5 mg/ml) and 1x PBS respectively.

6) Scraps 500ul pre-chilled PBS (with Protein Inhibitor Cocktail) to the culture plate

and transfer all the cells from culture plate to 1.5 ml EP-tube.

7) Centrifuge at 800g for 1min. Remove supernatant and store at -80℃.

b) Native Chromatin Immunoprecipitation (N-ChIP)

1) At least 5×10⁷ (more than 5×10⁶ cells for the species with genome size similar to human) cells are required for one ChIP enrichment

experiment. Preparation of an extra tube is necessary (5×10⁵cells, with the same treatment)

2) Remove cell culture medium

3) Rinse cells with pre-chilled 1xPBS+BSA (5 mg/ml)

4) Rinse cells with pre-chilled 1xPBS

5) For suspension cells, centrifuge at 800 g for 1 min to remove supernatant.

6) Scraps 500μl pre-chilled PBS (with Protein Inhibitor Cocktail) to the culture plate

and transfer all the cells from culture plate to 1.5 ml EP-tube.

7) Centrifuge at 800g for 1min. Remove supernatant and store at -80℃