title: H3K23ac +TSA Input rep2

status: Public on Jan 15 2017

data_processing:
library strategy: Paired-end MNase ChIP-seq
Fastq files were aligned to saccer3 using BWA
Samtools was used to filter for mapped reads with a mapping quality score of at least 10
Bedtools and AWK were used to convert the BAM files into bed files of mapped DNA fragments
Fragments smaller than 500bp were selected for using AWK
Read coverage wig files were generated using the java-genomics-toolkit base.align.count function
Genome_build: saccer3
Supplementary_files_format_and_content: wig files, coverage in 1bp intervals

source_name: yeast_H3K23ac +TSA Input

growth_protocol:
Yeast strains were arrested in G1 with alpha factor, treated with 25 μM TSA for 15 minutes, and crosslinked with 1% formaldehyde for 15 minutes
Cells were grown in YPD

extract_protocol:
Cell pellets were resuspended in lysis buffer (50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 2% Tritox X-100, 0.2% Na-deoxycholate, 1X roche protease inhibitor cocktail, 1mM PMSF) and bead beat for 25 minutes. Cell lysates were spun down at 15,000g for 30 minutes. The chromatin pellets were re-suspended in 1000 μl of NP-S buffer (0.5 mM spermidine, 1 mM β-ME, 0.075% NP-40, 50 mM NaCl, 10 mM Tris pH 7.4, 5 mM MgCl2, 1 mM CaCl2) and digested with microccocal nuclease at 37°C for 10 minutes to obtain predominantly mono-nucleosomal DNA. Reactions were stopped with the addition of 10 mM EDTA and digested lysates were clarified by centrifugation at 9000g for 10 minutes. To extract insoluble chromatin, pellets were re-suspended in 300 μl of lysis buffer with 0.2% SDS, and sonicated in a Diagenode Bioruptor at high output for 30 seconds on/30 seconds off four cycles. Extracts were then re-clarified by centrifugation at 9000g for 10 minutes, and the supernatant pooled with the pre-existing extract. The buffer composition of the lysate was adjusted to that of the original lysate buffer (50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 2% Tritox X-100, 0.2% Na-deoxycholate, 1X roche protease inhibitor cocktail, 1mM PMSF), and 10% was set aside as input. The supernatant was pre-cleared by incubation with magnetic protein-G Dynabeads (Life Technologies) for 1 hour at 4°C. Pre-cleared lysates were incubated with αHA (Roche, cat no. 12013819001), αH3K4me3 (Abcam, cat no. ab1012), αH3K23ac (Active Motif, cat no. 39131), or αIgG (Millipore, cat no. PP64) antibodies at 4°C overnight. Immune complexes were precipitated by incubation with magnetic protein-G Dynabeads for one hour at 4°C. Beads were washed twice with lysis buffer, high salt wash buffer (50mM HEPES pH 7.5, 640mM NaCl, 1mM EDTA, 2% Tritox X-100, 0.2% Na-deoxycholate), LiCl was buffer (10mM Tris-HCL pH 8.0, 250mM LiCl, 0.6% NP-40, 0.5% Na-deoxycholate, 1mM EDTA), and once with TE. The immunoprecipitated DNA was subjected to Illumina HiSeq paired-end sequencing.
Library construction for the Illumina platform was performed using a custom procedure for paired-end sequencing. Briefly, 2–10 ng of ChIP material was end-repaired and A-tailed before being ligated to TruSeq PE adaptors. In between each reaction, the material was purified using phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation. The resulting material was then amplified in the Phusion HF master mix (NEB) using TruSeq PE PCR primer 1.0 and custom indexed multiplexing primers [5′ AAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC 3′, where “NNNNNN” corresponds to unique hexamer barcodes]. PCR amplification was performed as follows: denaturation at 98 °C for 60 s; eight cycles of (98 °C, 30 s; 65 °C, 30 s; 72 °C, 30 s), and a final extension at 72 °C for 5 min. Amplified libraries were purified using 0.8 (vol) Agencourt AMPure XP solid phase reversible immobilization paramagnetic beads and eluted in 10 mM Tris⋅HCl pH 8.5. An aliquot of each library was run on an Agilent High Sensitivity chip to check the size distribution and molarity of the PCR products. Equimolar amounts of indexed, amplified libraries were pooled, and fragments in the 200–600 bp size range were selected on an 8% (wt/vol) Novex TBE PAGE gel (Invitrogen). An aliquot (1 μL) of the library pool was run on an Agilent High Sensitivity chip to confirm proper size selection and measure DNA concentration. The pooled libraries were diluted to 15 nM and their concentration was confirmed using the Quant-iT dsDNA HS assay kit and Qubit fluorometer (Invitrogen).

value: his3D200 leu2D1 lys2-128d ura3-52 trp1D63 bar1::KAN

tag: treated with

value: 25 μM TSA for 15 min


title: H3K23ac +TSA IP rep2

type: SRA

city: Vancouver

first: Benjamin

last: Martin

state: BC

address: 2350 Health Sciences Mall

department: Biochemistry

country: Canada

institute: UBC

library_source: genomic

molecule: genomic DNA

organism: Saccharomyces cerevisiae

source_name: yeast_H3K23ac +TSA IP

treatment_protocol: 1% formaldehyde crosslinking

tag: genotype/variation

tag: chip antibody

value: H3K23ac (Active Motif, 39131, Lot:104)

tag: growth stage


title: H3K23ac Input rep2

source_name: yeast_H3K23ac Input

growth_protocol:
Yeast strains were arrested in G1 with alpha factor, and crosslinked with 1% formaldehyde for 15 minutes
Cells were grown in YPD


title: H3K23ac IP rep2

source_name: yeast_H3K23ac IP


title: CHD7-13 whole blood CHD7 LOF genomic DNA

status: Public on May 03 2017

type: genomic

city: Toronto

first: Rosanna

last: Weksberg

state: Ontario

address: 555 University Ave.

department: Genetics and Genome Biology

institute: The Hospital for Sick Children

hyb_protocol: Bisulphite converted DNA was amplified, fragmented and hybridized to Illumina Infinium Human Methylation450 Beadchip using standard Illumina protocol

scan_protocol: Using an Illumina HiScan

data_processing: Background subtraction and normalization to internal array controls were performed on raw intensity data using Illumina GenomeStudio v2011.1 Methylation Module 1.9 software. Probes containing single nucleotide polymorphisms (SNPs), cross-reactive probes with sequences aligned to multiple positions with ≥ 90% identity, and non-specific chromosome probes were removed.

label: Cy5 and Cy3

organism: Homo sapiens

source_name: whole blood genomic DNA

label_protocol: Standard Illumina Protocol

extract_protocol: All genomic DNA were extracted from whole blood using standard procedures. One microgram of genomic DNA was sodium bisulfite modified using the Qiagen EZ DNA Methylation kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Modified genomic DNA was then processed and analyzed on the Infinium HumanMethylation450 BeadChip from Illumina according to the manufacturer’s protocol.

tag: gender

value: male

tag: age (years)

tag: sample type

value: CHD7 LOF discovery cohort

tag: disease state

value: CHARGE

tag: tissue

value: whole blood


title: H3K23ac Input rep1


title: ICRH80 29406

status: Public on Jul 05 2016

city: Philadelphia

first: Jonathan

last: Schug

state: Pennsylvania

address: 12-156 Smilow Ctr Trans Research

department: School of Medicine

country: USA

institute: University of Pennsylvania

library_source: transcriptomic

data_processing:
Poor quality sequence was trimmed from FASTQ files.
FASTQ files were aligned to human genome hg19 and transcripts using RUM (Grant et al Bioinformatics 2011)
A best representation of each gene was selected using the highest expressing transcript, converted to RPM, and normalized to the average expression of the 10 most deeply sequenced cells.
Differential expression, where reported was performed using EdgeR or DESeq2 on raw read counts.
Genome_build: hg19
Supplementary_files_format_and_content: Normalized expression values for RefSeq transcripts

molecule: polyA RNA

source_name: pancreas

growth_protocol: islets were cultured in the standand prodo islet media (PIM-S001GMP, PIM-ABS001GMP, PIM-G001GMP) at 37oc with 5% CO2.

extract_protocol:
Islets were dissociated to single cell by 0.05% Trypsin, 9 mins at 37oC. Cells (250-500 cells/μl) were subsequently loaded on Fluidigm C1 IFC with either 5-10 μM or 10-17 μM capture size.
Smarter-seq V2 kit was utilized for library construction per manufacturer's protocol to generate cDNA.  Prepration of Illumina libraries from cDNA was done with the NexteraXT kit per Fluidigm’s instructions.

value: pancreatic islets

tag: age

value: child

tag: health

value: control

tag: curated-cell-type

value: dropped


title: ICRH80 29405

value: mesenchyme


title: CHD7-26 whole blood CHD7 VUS genomic DNA

value: CHD7 sequence variant

value: CHD7 variant


title: CHD7-17 whole blood CHD7 LOF genomic DNA


title: CHD7-6 whole blood CHD7 LOF genomic DNA


title: ICRH80 29400

value: beta


title: ICRH80 29390


title: ICRH80 29393


title: ICRH80 29394

value: duct


title: ICRH80 29392


title: ICRH80 29391


title: ICRH80 29388


title: ICRH80 29389


title: CHD7-15 whole blood CHD7 LOF genomic DNA


title: CHD7-9 whole blood CHD7 LOF genomic DNA


title: CHD7-11 whole blood CHD7 LOF genomic DNA


title: ICRH80 29385


title: CHD7-4 whole blood CHD7 LOF genomic DNA


title: ICRH80 29386

value: alpha


title: ICRH80 29384


title: CHD7-5 whole blood CHD7 LOF genomic DNA


title: CHD7-12 whole blood CHD7 LOF genomic DNA


title: CHD7-16 whole blood CHD7 LOF genomic DNA


title: ICRH80 29381


title: CHD7-7 whole blood CHD7 LOF genomic DNA


title: CHD7-1 whole blood CHD7 LOF genomic DNA

value: female


title: CHD7-3 whole blood CHD7 LOF genomic DNA


title: ICRH80 29380


title: CHD7-10 whole blood CHD7 LOF genomic DNA


title: ICRH80 29376


title: CHD7-8 whole blood CHD7 LOF genomic DNA


title: ICRH80 29374


title: ICRH80 29372


title: ICRH80 29383


title: ICRH80 29364


title: H3K23ac +TSA Input rep1

value: his3D200 leu2D1 lys2-128d ura3-52 trp1D63 bar1::KAN

value: 25 μM TSA for 15 min


title: CHD7-2 whole blood CHD7 LOF genomic DNA

value: male


title: CHD7-14 whole blood CHD7 LOF genomic DNA

value: CHD7 LOF discovery cohort


title: ICRH80 29367


title: ICRH80 29362


title: ICRH80 29361


title: ICRH80 29360


title: ICRH80 29356


title: ICRH80 29358


title: ICRH80 29357


title: ICRH80 29354


title: ICRH80 29352


title: ICRH80 29353


title: ICRH80 29350


title: ICRH80 29351


title: ICRH80 29349


title: ICRH80 29347


title: ICRH80 29348


title: ICRH80 29345


title: ICRH80 29344


title: ICRH80 29341


title: ICRH80 29340


title: ICRH80 29339


title: ICRH80 29337


title: ICRH80 29333


title: ICRH80 29334


title: ICRH80 29330


title: ICRH80 29329


title: H3K23ac IP rep1

value: H3K23ac (Active Motif, 39131, Lot:104)


title: ICRH80 29326


title: ICRH80 29324


title: ICRH80 29343


title: ICRH80 29323


title: ICRH80 29325


title: ICRH80 29320


title: ICRH80 29319


title: ICRH80 29321


title: ICRH80 29317


title: ICRH80 29318


title: ICRH80 29315


title: ICRH80 29314


title: ICRH80 29316


title: ICRH80 29327


title: HP-15085-01T2D::fresh 28185

value: adult


title: HP-15085-01T2D::fresh 28184

value: pancreatic islets


title: HP-15085-01T2D::fresh 28183


title: ICRH80 29375

value: child


title: HP-15085-01T2D::fresh 28182

value: acinar


title: HP-15085-01T2D::fresh 28180

value: pp


title: HP-15085-01T2D::fresh 28181

value: dropped


title: HP-15085-01T2D::fresh 28179


title: HP-15085-01T2D::fresh 28175


title: ICRH76 26132

value: control


title: ICRH76 26131

value: delta


title: ICRH76 26130


title: ICRH76 26129

value: beta


title: ICRH76 26125


title: ICRH76 26126


title: ICRH76 26127


title: ICRH80 29365

value: mesenchyme


title: ICRH76 26124


title: ICRH76 26122


title: ICRH80 29312


title: ICRH80 29328


title: H3K23ac +TSA IP rep1


title: ICRH76 26120


title: ICRH76 26117


title: ICRH76 26119


title: ICRH80 29379


title: ICRH76 26114

value: alpha


title: ICRH76 26111


title: ICRH76 26110


title: ICRH76 26112


title: ICRH80 29371


title: ICRH76 26109


title: ICRH76 26107

value: duct


title: ICRH76 26106


title: ICRH76 26104


title: ICRH76 26102


title: ICRH76 26105


title: ICRH76 26103


title: ICRH76 26123


title: ICRH76 26100


title: ICRH76 26099


title: ICRH76 26101


title: ICRH76 26097


title: ICRH76 26095


title: ICRH76 26116


title: ICRH76 26094


title: ICRH76 26091


title: ICRH76 26092


title: ICRH80 29377


title: ICRH76 26088


title: ICRH76 26090


title: ICRH76 26096


title: ICRH76 26093


title: ICRH76 26087


title: HP-15085-01T2D::8dcult 26084


title: ICRH76 26086


title: HP-15085-01T2D::8dcult 26083


title: ICRH80 29382


title: HP-15085-01T2D::8dcult 26082


title: HP-15085-01T2D::8dcult 26080


title: HP-15085-01T2D::8dcult 26078


title: HP-15085-01T2D::8dcult 26081


title: HP-15085-01T2D::8dcult 26079


title: HP-15085-01T2D::8dcult 26077


title: ICRH76 26098


title: HP-15085-01T2D::8dcult 26076


title: HP-15085-01T2D::8dcult 26075


title: HP-15085-01T2D::8dcult 26074


title: HP-15085-01T2D::8dcult 26071


title: HP-15085-01T2D::8dcult 26070


title: HP-15085-01T2D::8dcult 26066


title: HP-15085-01T2D::8dcult 26073


title: HP-15085-01T2D::8dcult 26067


title: HP-15085-01T2D::fresh 26065


title: HP-15085-01T2D::8dcult 26068


title: HP-15085-01T2D::8dcult 26064


title: HP-15085-01T2D::8dcult 26060


title: HP-15085-01T2D::fresh 26058


title: HP-15085-01T2D::8dcult 26063


title: HP-15085-01T2D::8dcult 26057


title: HP-15085-01T2D::8dcult 26055


title: HP-15085-01T2D::8dcult 26054


title: ICRH80 29370


title: HP-15085-01T2D::8dcult 26053


title: HP-15085-01T2D::8dcult 26050


title: HP-15085-01T2D::8dcult 26045


title: HP-15085-01T2D::8dcult 26046


title: HP-15085-01T2D::8dcult 26049


title: ICRH80 29336


title: HP-15085-01T2D::fresh 26051


title: HP-15085-01T2D::8dcult 26069


title: HP-15085-01T2D::8dcult 26052


title: HP-15085-01T2D::8dcult 26047


title: HP-15085-01T2D::8dcult 26043


title: HP-15085-01T2D::fresh 26044


title: HP-15085-01T2D::8dcult 26048


title: HP-15085-01T2D::8dcult 26038


title: HP-15085-01T2D::8dcult 26039


title: HP-15085-01T2D::8dcult 26040


title: HP-15085-01T2D::8dcult 26041


title: HP-15085-01T2D::8dcult 26056


title: HP-15085-01T2D::8dcult 26033


title: HP-15085-01T2D::8dcult 26032


title: HP-15085-01T2D::8dcult 26059


title: HP-15085-01T2D::8dcult 26062


title: ICRH80 29359


title: HP-15085-01T2D::fresh 26037


title: HP-15085-01T2D::8dcult 26061


title: HP-15085-01T2D::8dcult 26035


title: HP-15085-01T2D::8dcult 26036


title: HP-15085-01T2D::8dcult 26034


title: HP-15085-01T2D::8dcult 26042


title: HP-15085-01T2D::fresh 26029


title: HP-15085-01T2D::fresh 26028


title: HP-15085-01T2D::fresh 26026


title: HP-15085-01T2D::fresh 26030


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title: HP-15085-01T2D::fresh 26025


title: HP-15085-01T2D::8dcult 26031


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title: HP-15085-01T2D::fresh 26018


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title: HP-15085-01T2D::fresh 26011


title: HP-15085-01T2D::fresh 26027


title: HP-15085-01T2D::fresh 25991


title: ICRH80 29338


title: HP-15085-01T2D::fresh 26019


title: HP-15085-01T2D::fresh 25994


title: HP-15085-01T2D::fresh 25990


title: HP-15085-01T2D::fresh 25988


title: HP-15085-01T2D::fresh 26015


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title: ICRH80 29373


title: HP-15085-01T2D::fresh 26008


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title: ACAP236 22304


title: HP-15041 22918


title: HP-15041 22909


title: ACAP236 22303


title: ACAP236 22302


title: HP-15041 22914


title: HP-15041 22912


title: ACAP236 22298


title: ACAP236 22299


title: ACAP236 22297


title: ACAP236 22300


title: HP-15041 22916


title: ACAP236 22295


title: ACAP236 22293


title: ACAP236 22292


title: ACAP236 22290


title: ACAP236 22288


title: ACAP236 22284


title: ACAP236 22282


title: ACAP236 22283


title: ACAP236 22281


title: ACAP236 22301


title: ACAP236 22280


title: ACAP236 22279


title: ACAP236 22278


title: ACAP236 22275


title: ACAP236 22277


title: ACAP236 22274


title: ACAP236 22272


title: ACAP236 22273


title: ACAP236 22294


title: ACAP236 22268


title: ACAP236 22267


title: ACAP236 22269


title: ACAP236 22291


title: ACAP236 22289


title: ACAP236 22266


title: ACAP236 22287


title: ACAP236 22270


title: ACAP236 22285


title: ACAP236 22286


title: ACAP236 22264


title: ACAP236 22265


title: ACAP236 22260


title: ACAP236 22262


title: ACAP236 22263


title: ACAP236 22259


title: ACAP236 22276


title: ACAP236 22261


title: ACAP236 22256


title: ACAP236 22257


title: ACAP236 22255


title: ACAP236 22258


title: ACAP236 22252


title: ACAP236 22251


title: ACAP236 22254


title: ACAP236 22248


title: ACAP236 22247


title: ACAP236 22244


title: ACAP236 22243


title: ACAP236 22240


title: ACAP236 22238


title: ACAP236 22236


title: ACAP236 22237


title: ACAP236 22239


title: ACAP236 22235


title: ACAP236 22253


title: ACAP236 22234


title: ACAP236 22233


title: ACAP236 22232


title: ACAP236 22250


title: ACAP236 22231


title: ACAP236 22229


title: ACAP236 22230


title: ACAP236 22227


title: ACAP236 22225


title: ACAP236 22228


title: ACAP236 22246


title: ACAP236 22245


title: ACAP236 22242


title: ACAP236 22223


title: ACAP236 22224


title: HP-15041 22944


title: ACAP236 22221


title: ACAP236 22220


title: ACAP236 22217


title: ACAP236 22219


title: ACAP236 22218


title: ACAP236 22213


title: ACAP236 22214


title: ACAP236 22216


title: ACAP236 22215


title: ACAP236 22226


title: ACAP236 22210


title: ACAP236 22212


title: AAJF122 13789


title: AAJF122 13794


title: ACAP236 22222


title: AAJF122 13785


title: ACAP236 22211


title: AAJF122 13780


title: ACAP236 22209


title: AAJF122 13791


title: AAJF122 13796


title: AAJF122 13784


title: AAJF122 13788


title: PSU_ChipSeq_G1E-ER4_Input_diffProtD_24hr_Timecourse

status: Public on Aug 31 2012

city: Stanford

first: ENCODE

last: DCC

state: CA

address: 300 Pasteur Dr

institute: ENCODE DCC

organism: Mus musculus

tag: lab

value: PSU-m

tag: lab description

value: Hardison - Penn State University

tag: datatype

value: ChipSeq

tag: datatype description

value: Chromatin IP Sequencing

tag: cell

tag: cell organism

value: mouse

tag: cell description

value: Gata1 restored erythroid cells, differentiation induced by estradiol (E2)

tag: cell sex

value: M

tag: antibody

value: Input

tag: antibody description

value: Control signal which may be subtracted from experimental raw signal before peaks are called.

tag: treatment

tag: treatment description

value: 24 hours differentiation of G1E-ER4 cells with 10 nM beta-estradiol (Hardison)

tag: labexpid

value: Timecourse

tag: age description

value: Embryonic day 0 (stem cell)

tag: control

tag: control description

tag: controlid

tag: replicate

tag: strain

tag: strain description

value: Strain 129, has widely available embryonic stem cells


title: hPGC male week 7 rep2

status: Public on Dec 26 2014

city: St Louis

first: Sabine

last: Dietmann

state: MO

address: 4565 McKinley Avenue

institute: Washington University School of Medicine

description: Primary cells

data_processing:
Low quality bases (q < 20) from the 3' end and adapter sequences were removed with the TrimGalore wrapper scripts (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore).
Reads were mapped to the human (GRCh37/hg19) reference genome with Tophat 2.0.12 (http://ccb.jhu.edu/software/tophat)
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Processed data files contain read counts per RefSeq transcripts generated by using the HTSeq 0.6.1 python package (http://www-huber.embl.de/users/anders/HTSeq)

molecule: total RNA

source_name: hPGC isolated from developmental week 7 genital ridge

extract_protocol:
Total RNA was extracted with the PicoPure RNA Isolation Kit. Total RNA (0.5-2 ng) was reverse transcribed and amplified into cDNA using Ovation RNA-Seq System V2 (Nugen) according to manufacturer’s instructions.
Amplified cDNA was sonicated into 250 bp by Covaris S220 Focused-ultrasonicators. Subsequently, RNASeq library was generated with 500 ng of fragmented cDNA using Ovation Ultralow DR Multiplex System (Nugen). Library was quantified by KAPA Library Quantification Kit (Kapa Biosystems) using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems).

tag: cell type

tag: facs

value: TNAP and cKIT+

tag: gender

value: Male


title: hPGC male week 7 rep1

description: Primary cells

value: hPGC isolated from developmental week 7 genital ridge


title: AAJF122 13781

tag: tissue

value: pancreatic islets

tag: age

value: adult

tag: health

value: control

tag: curated-cell-type

value: alpha


title: PSU_ChipSeq_G1E-ER4_Input

status: Public on Jun 11 2012

tag: lab

value: PSU-m

tag: lab description

value: Hardison - Penn State University

tag: datatype

value: ChipSeq

tag: datatype description

value: Chromatin IP Sequencing

tag: cell

tag: cell organism

tag: cell description

tag: cell sex

tag: antibody

tag: antibody description


title: AAJF122 13782


title: AAJF122 13783


title: AAJF122 13779


title: AAJF122 13778

value: dropped


title: HP-15041 22943


title: Differentiated mouse podocytes

status: Public on Jan 01 2008

type: RNA

city: Seattle

first: Shreeram

last: Akilesh

state: Washington

address: 1959 NE Pacific St, Box 356100

institute: University of Washington

description: In vitro differentiated immortalized mouse podocytes

scan_protocol: Standard scan conditions

data_processing: dChip software

label: Biotin

source_name: in vitro differentiated podocytes - 37*C for 10 days in the absence of IFNg

extract_protocol: Conditionally immortalized podocytes from a wildtype mouse (carrying the Immorto transgene) were cultured on collagen I coated plates at 33°C in a growth-permissive medium of RPMI supplemented with 10% FBS and 100 U/ml of recombinant mouse IFNg. For differentiation, cells were shifted to 37°C and the medium was changed to RPMI supplemented with 5% FBS without rIFNg. Under these conditions, the cells growth arrested and acquired a flatter morphology with extensive processes. Cells were incubated for a minimum of 10 days at 37°C before trypsinization, disaggregation and collection for genechip analysis. Total RNA was isolated using an Ambion RNAqueous 4 PCR kit. Biotinylated cRNA target was generated and hybridized to an Affymetrix 430v2 Murine Genome Array. Microarray data were analysed using dChip software.

tag: cell type

value: conditionally-immortalized podocytes


title: Undifferentiated mouse podocytes

status: Public on Aug 28 2010

description: Gene expression data from undifferentiated, conditionally immortalized mouse podocyte cell line

hyb_protocol: Per manufacturer's protocol

scan_protocol: Per manufacturer's protocol

data_processing: dCHIP software

label: biotin

source_name: 33*C undifferentiated podocytes

label_protocol: Biotinylated cRNA

growth_protocol: Routine culture of conditionally immortalized mouse podocyte cell line. Grown at 33*C in the presence of IFNy on collagen I coated dishes.

extract_protocol: Total RNA was extracted using a Qiagen RNeasy kit

treatment_protocol: No treatment/intervention


title: Primary mouse podocytes

description: FACS isolated primary mouse podocytes

source_name: FACS isolated primary mouse podocytes isolated from 3 month old wildtype C57BL/6J mice

extract_protocol: Mouse glomeruli were isolated from 3 months old wild-type C57BL/6 mice using Dynabead perfusion. Isolated glomeruli were incubated with trypsin solution containing 0.2% trypsin–EDTA (Sigma-Aldrich), 100 μg/ml heparin and 100 U/ml DNase I in PBS for 25 min at 37°C. The trypsin was inactivated with soybean trypsin inhibitor (Sigma-Aldrich) and the cell suspension sieved through a 30 μm pore size filter (BD Bioscience) as described. Cells were collected by centrifugation at 200 x g for 5 min at 4°C and re-suspended in 500ul PBS supplemented with 0.1% BSA. The single cell suspension was incubated with rabbit polyclonal antibody against the podocyte specific membrane protein podocalyxin and goat anti-rabbit Alexafluor 488 secondary antibody (Invitrogen). Fluorophore labeled podocytes were isolated by cell-sorting. RNA was extracted from 6000 positive cells obtained from 8 mice with RNeasy kit (Qiagen). Biotinylated cRNA target was generated and hybridized to an Affymetrix 430v2 Murine Genome Array. Microarray data were analysed using dChip software.

value: primary mouse podocytes


title: GLP_hippo_M1

status: Public on Dec 10 2009

city: New York

first: Anne

last: Schaefer

state: NY

address: 1230 York Avenue

institute: The Rockefeller University

hyb_protocol: Ten micrograms of labeled cRNA were hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays (http://www.affymetrix.com/products/arrays/specific/mouse430_2.affx) for 16 hours at 45ºC.  The GeneChips were washed and stained according to the manufacturer’s recommendations (Affymetrix, Santa Clara, CA) using the GeneChips Fluidics Station 450 (Affymetrix, Santa Clara, CA). This procedure included staining the chips with phycoerythrin-streptavidin, signal amplification by a second staining with biotinylated anti-streptavidin, and a third staining with phycoerythrin-streptavidin.

scan_protocol: Mouse Genome 430 2.0 arrays were scanned using the GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA) and globally scaled to 150 using the Affymetrix GeneChip Operating Software (GCOS v1.4).

data_processing: GeneChip CEL files were imported into Genespring GX 7.3.1 (Agilent Technologies, Santa Clara, CA), processed with the GC-RMA algorithm, and expression values on each chip were normalized to that chip’s 50th percentile. Statistical analysis was carried out to determine which genes are differentially expressed in the GLP and G9a mutant cortex, striatum, hippocampus and hypothalamus as compared to their respective controls. Genes were filtered for a raw expression level of > 25 and a fold change of > 1.5, followed by a Welch t-test with a p value cutoff of 0.05 and Benjamin Hochberg false discovery rate (FDR) multiple test correction. To determine which genes are differentially upregulated in the G9a deficient Drd1 or Drd2 expressing neurons, statistical analysis comparing the Drd1-Cre; Drd1-bacTRAP; G9afl/fl or Drd2-Cre; Drd2-bacTRAP; G9afl/fl to their respective controls was carried out. Genes were filtered for a raw expression level of > 25 and a fold change of > 1.5, followed by a Welch t-test with a p value cutoff of 0.05.

source_name: hippocampus of Camk2a-Cre; GLPfl/fl mice 1

label_protocol: Purified mRNA or total RNA was converted to double-stranded cDNA using the SuperScript GeneChip Expression 3’-Amplification Reagents Two-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA) and the GeneChip T7-Oligo(dT) Primer (Affymetrix, Santa Clara, CA). cDNA was used for the in vitro synthesis of cRNA using the MEGAscriptT7 Kit (Ambion, Austin, TX).  cRNA was purified using the GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA). 600ng or less of clean cRNA was used in the second-cycle cDNA synthesis reaction using the SuperScript GeneChip Expression 3’-Amplification Reagents Two- Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA) and random primers (Affymetrix, Santa Clara, CA). The cDNA was purified using the GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA). Purified cDNA was used for the in vitro synthesis of biotin- labeled cRNA using the GeneChip IVT Labeling Kit (Affymetrix, Santa Clara, CA).  cRNA was purified using the GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA) and fragmented into 35-200 base pair fragments using a magnesium acetate buffer (Affymetrix, Santa Clara, CA).

growth_protocol: All mice were raised under 12hr dark/12hr light conditions, housed ~5 mice per cage, with food and water ad libitum. All animal use protocols were approved by the Rockefeller University Institutional Animal Care and Use Committee.

extract_protocol: Total RNA from cortex, hippocampus, hypothalamus and striatum of twelve week-old Camk2a-Cre; GLPfl/fl, Camk2a-Cre; G9afl/fl, and Camk2a-Cre; GLPfl/fl; G9afl/fl mice and age and sex matched littermate controls (n=4/genotype) was extracted using the Trizol/Chloroform technique as recommended by the manufacturer (Invitrogen Corporation, Carlsbad, CA). After extraction, RNA was precipitated overnight at -80°C in isopropanol with sodium acetate and Glycoblue (Ambion, Austin, TX), washed twice with 70% ethanol, re-suspended in water, and further purified using an Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase digestion. Purified samples were analyzed using a Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE) and a Bioanalyzer (Agilent Technologies, Santa Clara, CA) in order to assess mRNA quantity and quality, as reflected by rRNA levels and integrity. Polyribosome associated mRNAs from age and sex matched Drd2-Cre; Drd2-bacTRAP; G9afl/fl and Drd2-bacTRAP; G9afl/fl, or Drd1-Cre; Drd1-bacTRAP; G9afl/fl and Drd1-bacTRAP; G9afl/fl mice (n=3x5 mice/genotype) were obtained as previously described using a mix of two monoclonal anti-GFP antibodies (19C8, 19F7) for the immunoprecipitation reaction (Doyle et al., 2008; Heiman et al., 2008).

tag: tissue type

value: hippocampus

tag: genotype

value: mutant