GSM2948736: LUHMES differentiated ChIP-seq CTCF Sample 2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Unclassified
Antigen
Unclassified
Cell type
Cell type Class
Neural
Cell type
LUHMES
Tissue
Mesencephalon
Attributes by original data submitter
Sample
source_name
Lund human mesencephalic cells (LUHMES)
cell line
Lund human mesencephalic cells (LUHMES)
Stage
dopaminergic neurons, differentiated
Sex
female
passages
3-10
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ChIP experiments, we used previously published protocols (Rhie et al., 2014). Briefly, about 1 x 107 cells were fixed by adding fresh formaldehyde directly to the culture medium at a final concentration of 1%. The reaction was quenched with 10X (1.15 M) glycine for 5 min at room temperature. Chromatin from fixed cells was sonicated using a Bioruptor Pico (Diagenode, Cat # B01060001) with 30 seconds on and 30 seconds off cycles to produce fragments between 200 and 500 base pairs. For immunoprecipitation, 100 μg of sonicated chromatin was used and 10 μg (10%) was saved as an input control. To probe for active enhancers in both undifferentiated and differentiated cells, samples were incubated at 4 °C overnight with an H3K27ac primary antibody (Active Motif, Cat # 39133) or an IgG control (Sigma, Cat # R9133). For CTCF sites, CTCF(D31H2) XP (Cell Signaling Technology, cat # 3418) was used as primary antibody. For secondary antibody, A/G magnetic beads (Pierce, Cat # 88802) were added to the samples prior to an additional incubation for 2 h at 4 °C. The beads were then washed with a series of salt buffers before elution. The immunoprecipitated and input control DNA was purified using A QIAprep Spin Miniprep Kit (Qiagen, Cat # 27104). Libraries for Input and IP samples were prepared by the Van Andel Genomics Core from 10 ng of input material and all available IP material using the KAPA Hyper Prep Kit (v5.16) (Kapa Biosystems, Wilmington, MA USA). Prior to PCR amplification, end-repaired and A-tailed DNA fragments were ligated to Bio Scientific NEXTflex Adapters (Bioo Scientific, Austin, TX, USA). The quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor dsDNA System (Promega Corp., Madison, WI, USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). Sequencing (75 bp, single end) was performed on an Illumina NextSeq 500 sequencer using a 75-bp sequencing kit (v2) (Illumina Inc., San Diego, CA, USA). Base calling used Illumina NextSeq Control Software (NCS) v2.0, and the output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.