#Chip seq from flashfrozen cells serial#

There should be two equal cell pellets for each 200 mL culture. At this point the cell pellets may be flash frozen using a dry iceethanol or liquid nitrogen bath and stored at 80 C. Pellet cells at 2,000 rpm for 5 minutes at 4C. Remove the supernatant without disturbing the cell pellet. Scrape the cells off the plate with a cell scraper and transfer into 15-ml conical tubes on ice. Aspirate the PBS and add 5-8 ml cold (4C) Farnham lysis buffer. Our results suggest that GtaC regulates multiple physiological processes as Dictyostelium transitions from a group of unicellular amoebae to an integrated multicellular organism. Remove media from plates and wash cells with equal volume cold (4C) 1X PBS. We identify direct GtaC targets and observe cotemporaneous GtaC-binding and developmental expression regulation. We find that GtaC exhibits temporally distinctive DNA-binding patterns concordant with each developmental stage. GtaC is essential for developmental progression, decoding extracellular cAMP pulses during early development and may play a role in mediating cell-type differentiation at later stages.

#Chip seq from flashfrozen cells serial#

Here we report the developmental regulatory dynamics of GtaC, a GATA-type zinc-finger transcription factor, through the analyses of serial ChIP- and RNA-sequencing data. The final cell pellets were flash frozen in liquid nitrogen and stored at -80. However, efforts to examine sequential genome-wide changes of gene regulation in developmental processes have been fairly limited. ChIP libraries were prepared for sequencing with the Illumina ChIP-seq. In many systems, including the social amoeba Dictyostelium discoideum, development is often marked by dynamic morphological and transcriptional changes orchestrated by key transcription factors.