GSM1479212: HA-AUTS2 293TREx ChIPseq; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
AUTS2
Cell type
Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
source_name
293T-Rex cells expressing FLAG/HA-tagged AUTS2
mouse strain
Human embryonic kidney cells stably expressing the tetracycline repressor protein
Stage
15-20
antibody
HA (abcam, ab9110, lot# GR20600-2)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ChIP-seq in mouse brain, the whole brains were homogenized with a glass douncing homogenizer. The cell homogenate was fixed with 1% paraformaldehyde for eight min at room temperature and quenched with 0.125 M glycine for five min at room temperature, followed by centrifugation through a 29% iodixanol cushion. The nuclei pellet was resuspended and sonicated using a Diagenode Bioruptor to an average size of ~250 bp. After pre-clearing with BSA-blocked protein G Sepharose, chromatin was incubated with antibodies at 4°C overnight. The chromatin immunocomplexes were recovered with the same BSA-blocked protein G beads.For ChIP-seq in 393T-REx cells, cells were crosslinked with 1% formaldehyde for eight min at room temperature and the nuclei were then isolated. As described above, following sonication and pre-clearing, chromatin IP was performed to isolate associated DNA. For ChIP-seq library construction, 5-10 ng of DNA extracted from the chromatin immunocomplexes. Libraries were prepared according to manufacturer’s instructions (Illumina). Immunoprecipitated DNA was first end-repaired using End-It Repair Kit (Epicenter), tailed with an A using Klenow exo minus (NEB M0212), and ligated to custom adapters with LigaFast (Promega, city, state #M8225). Fragments of 350±50 bp were size-selected and subjected to ligation-mediated PCR amplification (LM-PCR), using Phusion DNA polymerase (NEB M0530).