Stem Cell Laboratory

Laboratory Protocols

Neural stem cells Cromatin Immunoprecipitation

ChIP protocol for NS cells

Chromatin Preparation

  1. Aspirate the medium from a confluent 60mm plate and wash cells with PBS. Add 400μl Accutase and incubate 2-3min at 37oC. Collect the cells by adding medium and centrifuge at 1200rpm for 5min to pellet. Wash pellet once with PBS.
  2. Resuspend cells in 900μl PBS and add 100μl Formaldehyde 10% (CF = 1%) to fix the cells. Rotate at RT for 10min.
  3. Add 100μl glycine to a final concentration of 0.125M to stop fixation. Incubate 5min on ice and centrifuge at 2000rpm to pellet the cells. From that step work on ice.
  4. Remove supernatant and resuspend the pellet in cold PBS/PMSF and centrifuge at 2500rpm for 5min at 4o C. Repeat for two more times.
  5. Remove supernatant and resuspend pellet in Lysis buffer (1ml per 107 cells). Homogenize manually and incubate for 30min at 4oC rotating. Prepare aliquots of 300μl for sonication.
  6. Sonicate in Diagenode Bioraptor for 45 cycles (30sec ON/30sec OFF) settings: High.
  7. Centrifuge immediately at full speed for 15min. at 4oC. If necessary take the supernatant and centrifuge it again at full speed for 15min. Your supernatant has the sheared chromatin.
  8. Measure chromatin at nanodrop and dilute samples in Lysis buffer at final concentration of 1μg/μl. Make aliquots of 20μg. Aliquots can be kept at -800C until chromatin precipitation.

 

Beads preparation

The day before or 5-6h before precipitation magnetic beads should be pre-blocked and conjugated with antibody as follows:

  1. Add 50μl Dynabeads-Protein A per each IP to a microfuge tube.
  2. Remove supernatant and add 1ml 0.5% BSA in PBS.
  3. Collect beads and wash two more times for 10min (2x10min) with 0.5% BSA/PBS.
  4. Resuspend beads in 250μl 0.5% BSA/PBS and add 2-15μg antibody
  5. Incubate at least 5 hours or overnight in rotating platform at 4o C.
  6. Wash beads before adding extract 3x10min with 0.5% BSA/PBS.

Chromatin Precipitation and DNA extraction

Note:Each wash includes 10min constant rotation of the tubes at 4oC

  1. Before precipitation take out one 20μg sample and let it thaw on ice. Adjust the volume at 1ml with 0.5% BSA/PBS. From this 1ml sample keep 5% for input (50μl).
  2. Transfer the extract in the tube with the antibody-conjugated beads and incubate for 16h at 4oC rotating.
  3. Collect the beads using a magnetic stand and wash 2x5min with Lysis buffer.
  4. Wash sequentially with Wash Buffer A and Wash buffer B.
  5. Transfer Wash buffer B into clean tubes and wash last with TE buffer.
  6. Add 150μl Elution buffer to the beads and incubate at 65oC for 10min. Vortex well and collect the beads with a magnetic stand. Transfer the supernatant into a clean tube and repeat the elution step. Combine the two supernatants (V=300μl) and add 180μl of H2O and NaCl to a final concentration of 110mM. Now the total volume of the sample is 500μl.
  7. In parallel thaw the input sample (50μl) and supplement with 240μl elution buffer, 110μl H2O and NaCl.
  8. Incubate at 65oC for at least 5h or overnight to de-crosslink.
  9. Add 1μl RNAse A from a 10mg/ml,DNAse-free stock and incubate at 37oC for 1h.Transfer immediately in ice and incubate for 5min.
  10. Add 4μl EDTA from a 0.5M stock and 2μg of ProteinaseK and incubate at 42oC for 2h.
  11. Add same volume of PCI (V=500μl) and centrifuge in maximal speed for 30min. Transfer the aqueous phase to a fresh tube and repeat extraction with chloroform.
  12. For precipitation of DNA add 20μg Glycogen, 50μl NaOAc from a 3M stock and 900μl isopropanol. Vortex well and leave to precipitate at -20oC for 1h.
  13. Centrifuge at full speed for 30min and remove the supernatant.
  14. Wash the pellet with 70% EtOH and centrifuge again at full speed.
  15. Remove the supernatant and leave the pellet to dry completely.
  16. Resuspend in 50μl wfi.

Protocol was kindly provided by Dr. Diogo Castro.

A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets., Castro et al., Genes and Development 2011.

This work was supported by Aristeia II, GEMCCTR “Self-renewal and differentiation decisions in neural stem cells: Geminin, cell cycle control and transcriptional regulation”

 

Buffers

Lysis Buffer

SDS 1%, EDTA 10mM

Tris 50mM pH8         

Protease Inhibitor (PI) 1%

 

Wash Buffer A

HEPES 50mM pH7.9

NaCl 500mM

EDTA 1mM

TritonX 1%

Na-Deoxycolate 0.1%

SDS 0.1%

PMSF 0.5mM

PI 1%

 

Wash Buffer B

Tris 20mM pH8

EDTA 1mM

LiCl 250mM

NP40 0.5%

Na-Deoxycolate 0.5%

PMSF 0.5mM

PI 1%

 

Elution Buffer

SDS 1%, EDTA 10mM

Tris 50mM pH8

NaHCO3 50mM        

Protease Inhibitor (PI) 1%