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Comparative profiling of chromatin state maps and transcription factor occupancy during human fetal and adult erythropoiesis[Histone Modification]
External Reference: GSE36985    
Measurement Type: Histone Modification (ChIP-Seq) Factors: Immunoprecipitation Antibody
Contact(s): Stuart Orkin
Curator(s): Emily Merrill
Submitted Date: 01/21/2015

summary

Examination of various histone modifications by ChIP-seq in fetal and adult proerythroblasts.


Growth Protocol

Primary maturing fetal or adult erythroblasts were generated ex vivo using a serum-free two-phase liquid culture system.

Treatment Protocol


Extraction Protocol

For ChIP-seq analysis using the HeliScope™ Single Molecule Sequencer, ChIP DNA was processed for 3’ polyA tailing followed by 3’ ddATP-blocking. Processing samples by ligation, amplification and size selection are not required by Helicos sequencing. Briefly, 6-9 ng of ChIP DNA or input DNA was used in the 3’ polyA tailing reaction in 14.8 ul containing 2 ul of 2.5 mM CoCl2, 2 ul of 10 x terminal transferase buffer and nuclease-free water. The reaction mixture was denatured in 95C for 5 min, followed by rapid cooling in ice water slurry. Then 1 U of terminal transferase, 4 ul of 50 uM dATP and 0.2 ul of NEB BSA were added to the mixture. Samples were incubated in a thermocycler at 37C for 1 h, 70C 10 min, followed by denaturing at 95C for 5 min and rapid cooling in ice water slurry. For 3’ ddATP-blocking, 0.5 ul of 200 uM ddATP, 1 ul of 10 x terminal transferase, 1 ul of 2.5 mM CoCl2, 1 U of terminal transferase, and 6.5 ul of nuclear-free water were added to the above reaction. Samples were incubated in a thermocycler at 37C for 1 h and 70C for 20 min. Then 2 picomoles of carrier oligonucleotide were added to the reaction, and samples were hybridized to the Helicos flow cells.

Name

Input


Organism

Homo sapiens


Cell type

CD34-positive proerythroblast


Disease state


Platform

Helicos HeliScope Single Molecule Sequencer



citations

Combinatorial assembly of developmental stage-specific enhancers controls gene expression programs during human erythropoiesis. Xu J, Shao Z, Glass K, Bauer DE, Pinello L, Van Handel B, Hou S, Stamatoyannopoulos JA, Mikkola HKA, Yuan G-C, et al. Dev Cell.