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Sample GSM4990688

Query DataSets for GSM4990688

Status

Public on Apr 08, 2021

Title

MEOX1-HA_Unstim_Rep3_input

Sample type

SRA

Source name

Cardiac fibroblasts

Organism

Mus musculus

Characteristics

cell type: Cardiac fibroblasts
treatment: Vehicle
type of experiment: ChIP seq

Growth protocol

A Tcf21MCM mouse was crossed with a Rosa26-Ai6 mouse (Jackson Laboratory stock# 007906). At 10 weeks of age, intraperitoneal injection of Tamoxifen (75 mg tamoxifen/kg) was done for 5 days (once a day). Tamoxifen was prepared following Jackson Laboratory guidelines https://www.jax.org/research-and-faculty/resources/cre-repository/tamoxifen#). After 5 days of injection, the mouse was sacrificed and the non-CM cells isolated as described in a previous section of this method. 100k ZsGreen positive cells were sorted with BD AriaII sorter and cultured for 3 days at 37C in a humidified incubator with 5% CO2 and maintained in FB medium: high glucose DMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Hyclone, GE Healthcare), 1x Non-Essential Amino Acid (NEAA), 10U/ml penicillin/streptomycin and 1mM sodium pyruvate (all from Life Technologies). For immortalization, FB were seeded at 105 per 100 mm plate in the afternoon. Next morning, 2 hours before infection, fresh media was added on plates. The SV40 T Antigen Cell Immortalization Kit (Alstem, #CILV01) was used. 5 µl/well lentivirus supernatant (vials have 20ul) in the presence of Polybrene (added to a final concentration of 5 µg/ml). The next day, remove viral supernatant and switch again to FM medium. After 72 hours incubation, subculture the cells into 2 × 100 mm dishes and add the appropriate amount of puromycin for stable cell-line generation (1ug/ml for mouse cardiac FBs). After 10 days after puromycin selection, multiple clones were picked for expansion. For daily cell culture, FBs were split every 3-4 days and the media was changed every other day. For the FB TGF-β stimulation, FBs were seeded at 1x105/well of 6 well plate at day 1. On day 2, the media was changed to same basal media with 0.5% FBS. On the day 3, TGF-1(Peprotech) was added into the media at concentration of 10ng/ml. Cells were collected on day 5 for RNA extraction and subsequent gene expression analysis with real time PCR.

Extracted molecule

genomic DNA

Extraction protocol

The pHR-HAtag-mMeox1 vector was constructed by PCR amplifying the HA tag-mMeox1 fragment from the vector HA-tag-MEOX1 mouse (Twist Biosciences). For generating the pHR-HAtag-mMEOX1, we replaced the KRAB and dCas9 cassettes from the pHR-SFFV-KRAB-dCas9-mCherry vector with the HA-tag-MEOX1 cassette using Cold-fusion cloning kit (SBI System BioSciences, Palo Alto, CA, USA) following the instruction provided by the manufacturer. The construct was sequencing verified. For generating the lentiviral particles, the same procedure described in the section “CRISPR interference (CRISPRi) for sequence-specific repression” was used. The obtained supernatant was then used for performing transduction on cardiac FBs with the lentiviral vector that express HA-tag-MEOX1 and mCherry. Pure polyclonal population of HA-tag-MEOX1 FBs were sorted by flow cytometry (BD Arial II) for stable mCherry expression. For ChIPseq experiment, cells were treated as follows: 10x106 HA-tag-MEOX1 FBs (either in Unstimulated or TGFb-treated conditions) were pelleted and suspended in 10ml DMEM and cross-linked in 1% formalin solution (Thermo Fisher Scientific) by rocking in room temperature for 10 minutes. Then glycine (final concentration 0.125M) was added to quench the cross-link for 5minutes. Samples were centrifuged at 1000 rcf for 5minutes at 4C. Cells were washed with 10ml of cold 1x PBS supplemented with proteinase inhibitors and phosphatase inhibitors (Roche # 4693132001) and the pellets were snap frozen in liquid nitrogen. All samples were stored at -80C until use. When ready, cell pellets were incubated in cell lysis buffer (20 mM Tris-HCl, pH 8, 85 mM KCl, 0.5% NP-40, protease inhibitors for 10 min on a rotator at 4C. Nuclei were isolated by centrifugation (2,500 x g, 5 min, 4C), resuspended in nuclear lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, pH 8, 1% SDS, protease inhibitors) and incubated on a rotator for 30 min at 4C. Chromatin was sheared using a Covaris S2 sonicator (Covaris Inc) for 20 min (60 s cycles, 20% duty cycle, 200 cycles/burst, intensity = 5) until DNA was in the 200–700 base-pair range. Chromatin was diluted 3-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2mMEDTA, 16.7mMTris-HCl, pH 8, 167 mM NaCl, protease inhibitors) and incubated with 3ul of anti-HA antibody (Abcam #9110) at 4C overnight under rotation. Antibody-protein complexes were immunoprecipitated using Pierce Protein A/G magnetic beads at 4C for 2 h under rotation. Beads were washed five times (2-min/wash under rotation) with cold RIPA buffer (50 mM HEPES- KOH, pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate), followed by one wash in cold final wash buffer (1xTE, 50 mM NaCl). Immunoprecipitated chromatin was eluted at 65C with agitation for 30 min in elution buffer (50mMTris-HCl pH 8.0, 10mMEDTA, 1% SDS). High-salt buffer (250mM Tris-HCl, pH 7.5, 32.5 mM EDTA, pH 8, 1.25M NaCl) and Proteinase K (New England Biolabs Inc (NEB)) were added and crosslinks were reversed overnight at 65C. Samples were treated with RNase A, and DNA was purified with AMPure XP beads (Beckman Coulter cat #A63881).
Fragmented ChIP and input DNA were end-repaired, 5’-phosphorylated and dA-tailed with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645). Samples were ligated to adaptor oligos for multiplex sequencing (NEB, E7335), PCR amplified, and sequenced on an Illumina NextSeq 500 at the Gladstone Institutes.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Data processing

Cluster generation, sequencing, and base calling was performed on Illumina NextSeq 500.
Trimming of known adapters and low-quality regions of reads was performed using Fastq-mcf. Sequence quality control was assessed using FastQC.
Alignment to the mm10 reference genome was performed using Bowtie 2.2.4. Peaks were called using GEM.
Genome_build: Mm10
Supplementary_files_format_and_content: We provide BigWig files for PROseq We provide bed files and bigWig files for ChIPseq.

Submission date

Dec 28, 2020

Last update date

Apr 08, 2021

Contact name

Pawel Przytycki

E-mail(s)

pawel.przytycki@gladstone.ucsf.edu

Organization name

Gladstone Institutes

Department

GIDB