|
| Status |
Public on Jun 27, 2019 |
| Title |
replicate2 ChIP IP KO |
| Sample type |
SRA |
| |
|
| Source name |
LUHMES-derived neurons
|
| Organism |
Homo sapiens |
| Characteristics |
div: day 9
passage: 15-20
cell line: LUHMES
cell type: LUHMES-derived neurons
genotype: MeCP2 KO
chip antibody: anti- MeCP2
|
| Treatment protocol |
Cells were modified using CRISPR system for KO and infected with lentiviruses carrying MeCP2 for OE.
|
| Growth protocol |
LUHMES cells were cultured and differentiated according to published protocol (Shah et al., 2016).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
LUHMES-derived neurons at day 9 of differentiation with four levels of MeCP2: KO, WT, OE 4x and OE 11x (see Table S1) were crosslinked with 1% of Formaldehyde (Sigma) in the medium for 10 min at RT and quenched with 2.5 M Glycine (Sigma) for 2 min at RT. Cells were washed with PBS, scraped from the plate and centrifuged. Crosslinked nuclei were isolated using hypotonic buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3mM MgCl2, 0.1% [v/v] Igepal CA-630) and counted using a haemocytometer. Chromatin from ~4x106 nuclei was sonicated for 20 cycles (30 sec ON and 30 sec OFF) on the high power setting using a Bioruptor (Diagenode). Crosslinked and sonicated chromatin was mixed with 60 ng of sonicated Drosophila chromatin (Active Motif) as a spike-in and the mix was incubated overnight at 4 ºC with antibodies against MeCP2 (D4F3, Cell Signalling) plus spike-in antibody (Active Motif). After overnight incubation, magnetic Protein G coated beads (Thermo Scientific) were added and incubated further 4 hours at 4 ºC. Beads were washed and chromatin was reverse-crosslinked overnight at 65 ºC. DNA was then purified using Agencourt AMPure XP (Beckman Coulter) beads.
For ChIP-seq library preparation, IPs for each condition were pooled together to achieve 5 ng total DNA as a starting material. For example, 3-4 IPs were pooled together for the KO sample and 2 IPs were pooled for the WT sample. Libraries were prepared using NEBNext Ultra II DNA library Prep kit (NEB) for both IPs and corresponding inputs.
NEBNext Ultra II kit (NEB)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
ChIP7_IP_KOH4_13
|
| Data processing |
Pre-process reads with Trimmomatic version 0.32 ILLUMINACLIP:nextera.fa:2:40:15 MINLEN:25
Align to genome with bwa v0.7.5a-r405
Filter for unique primary alignments with Samtools 1.3.1
Remove duplicate reads with Picard v1.107
Remove mitochondrial reads and blacklisted regions with bedtools 2.27
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files of aligned reads
|
| |
|
| Submission date |
Jan 25, 2019 |
| Last update date |
Jun 27, 2019 |
| Contact name |
Shaun Michael Webb |
| E-mail(s) |
shaun.webb@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Department |
Wellcome Trust Centre for Cell Biology
|
| Lab |
Bioinformatics Core Facility
|
| Street address |
2.21 Swann Building, Kings Buildings
|
| City |
Edinburgh |
| ZIP/Postal code |
EH9 3JR |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE125659 |
Study of MeCP2 occupancy and biniding localization in the genome |
| GSE125660 |
Study of MeCP2 in LUHMES-derived neurons |
|
| Relations |
| BioSample |
SAMN10819821 |
| SRA |
SRX5291887 |
