title: ATAC_Microglia_Ki67Positive_Ki67rfp_Male_PND8_rep1

city: Québec

first: David

last: Gosselin

state: Quebec

address: CRCHUL - 2705 Boulevard Laurier, Bloc T - 2-34

department: Molecular Medicine

institute: Université Laval

data_processing:
Alignment: FASTQ files from sequencing experiments were mapped to the mouse mm10 reference genome. STAR (2.7.0f) with default parameters was used to map RNA-seq experiments. Bowtie2 (2.3.4.1) with default parameters was used to map ChIP-seq and ATAC-seq experiments. HOMER was used to convert aligned reads into “tag directories” for further analyses.
RNA-seq: For differential expression analyses, a table read count including relevant samples was first created using the analyzeRepeats.pl program of HOMER (v4.11; http://homer.ucsd.edu/homer/) with the following parameters: rna -noadj -condenseGenes -count exons -pc 3. Non-coding RNA and pseudogenes were then removed to retain only protein-coding mRNAs and the resulting dataset was used as input file for HOMER’s getDiffExpression.pl program, which leveraged R/DESeq2 for to assess differential expression. For data interpretation, only genes with 16 or more normalized reads were considered. Alternatively, Transcript Per Million-normalized datasets were generated using the analyzeRepeats.pl program of HOMER with the following parameters: rna -TPM -condenseGenes -count exons -pc 3. The base-2 logarithm of the TPM values was taken after adding a pseudocount of 1 TPM to each gene.
ChIP-seq: H3K4me3, H3K27ac ChIP-seq regions calling and differential analyses-For each condition, the repertoire of genomic regions positive for presence of H3K4me3 or H3K27ac were first generated using HOMER’s getDiffPeakReplicate.pl program, using all replicates and related input of a condition of interest. Parameters specified for H3K4me3 were as follow: -style histone. For H3K27ac, parameters were: -genome mm10 -region -size 500 -min Dist 1000 -L1. For differential enrichment assessments, relevant regions files were then first merged together with HOMER’s mergedPeaks.pl program with -size given parameter, and tag counts of each replicate annotated to each region using HOMER’s annotatePeak.pl program with mm10 -raw parameters used. For H3K27ac, data matrix was then used as input for HOMER’s getDiffExpression.pl program, which leveraged R/DESeq2 to assess differential enrichment. For read count tables output, each experiment was normalized to 10 million total reads (-simpleNorm default parameter specified in getDiffExpression.pl). For data interpretation, only regions with 16 or more normalized reads were considered.  Rpb2 ChIP-seq peak calling and analyses-Rpb2 peaks were first identified using HOMER’s getDiffPeakReplicate.pl program with the parameter -style factor specified and using two independent biological replicates of Rpb2 ChIP-seq experiments and associated inputs generated from Adult-Basal microglia. Rpb2 peaks from PND8-Ki67+ microglia (n=1) were identified using findPeaks.pl program with the -style factor parameter, using ChIP-seq and linked input data. To identify C1 and C2 promoter regions for which Rpb2 is present or absent, tables of genomic coordinates of C1 and C2 promoters were first generated by annotating C1 and C2 NCBI genes using HOMER’s annotatePeaks.pl program in tss mode using as input the list of NM RefSeq gene identifiers and specifying the following parameters: -list <relevant NM list.txt> -size -1000,500. Output was then curated to retain only gene regions included in the original NM list .txt file. Following this, C1 and C2 promoter regions were intersected with Rpb2 peaks files using BEDOPS (3.1.1 v2.4.39) with --ec --element-of-1 parameters specified.
ATAC-seq: ATAC-seq accessible chromatin region identification-Regions of accessible chromatin were called using HOMER’s findPeaks.pl program with the -style factor parameter.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Matrix tables with raw gene counts (Microglia_RNA_dUTP_rawdata.txt) or TPM-normalized values (Microglia_RNA_dUTP_TPMnormalization.txt) for every protein-coding genes gene and every RNA-seq samples.
Supplementary_files_format_and_content: Tab-delimited text files include peak coordinates (ChIP, ATAC) and enrichment over input (ChIP).

source_name: Microglia

extract_protocol:
Microglia isolation: Mice were first euthanized with an overdose of ketamine-xylazine administered by intra-peritoneal injection and subsequently perfused transcardially with PBS. Brain were then removed from the skull and immediately homogenized in a buffer solution (HBSS (Life Technologies, 14175-095), 1% BSA (Fisher), 1mM EDTA (Invitrogen), 1 mM sodium butyrate (Sigma B5887-1G), 1 uM Flavoperidol (Sigma F3055) by gentle mechanical dissociation on ice, using a 2 ml polytetrafluoroethylene pestle (Wheaton, 358026) as performed in Gosselin et al. 201417. Microglia were then enriched by Percoll (GE) gradient, washed and labeled with Live/Dead yellow dead stain (ThermoFisher), then incubated 1:100 with a CD16/CD32 receptor blocking antibody (1:50, eBioscience 14-0161, clone 93) for 20 min, and then incubated 1:100 with antibodies directed against cell surface proteins CD11b-BV421 (BioLegend 101251, clone M1/70), CD45-APC (BioLegend 103112, clone 30-F11), and CD44-APC-Cy7 (BioLegend 103028, clone IM7). For all ChIP-seq experiments, microglia were then further fixed with 1% PFA for 10 min at RT, permeabilized with reagents from ebioscience Foxp3 Transcription Factor staining buffer set (ThermoFisher 00-5523-00) and stained with anti-Ki67-Alexa 488 antibody (BD Pharmingen 561165, clone B56). Microglia were either analyzed with BD LSR or sorted with BD Aria II instruments (100 um nozzle). Flow cytometry results were analyzed using FlowJo software (Tree Star).
RNA-seq library preparation: Isolation and fragmentation of poly(A)   For RNA-seq, we generated 2 independent biological replicates per conditions (n=2). Following sorting, 200,000 microglia were pelleted and put into 100 ul lysis/Oligo d(T) Magnetic Beads binding buffer (100 mM Tris-HCl ph 7.5, 500 mM LiCl, 10 mM EDTA pH 8.0, 1% LiDS, 5 mM DTT, in water) and stored at -80C until processing. For human brain cortical RNA, 500 ng of RNA was diluted with proper volume of 2x lysis/ Oligo d(T) Magnetic Beads binding buffer to a final concentration of 1x lysis/Oligo d(T) Magnetic Beads binding buffer. For sequencing library preparation, samples were incubated with 20 ul Oligo d(T) Magnetic Beads (NEB, S1419S) in PCR strips for selection of polyadenylated (poly(A)) mRNA as follows: 2 min at 65 C on a PCR cycler and then 10 min at room temperature (RT). Samples were then placed on a collection magnet at RT, and washed serially with 180 ul of washing buffer 1 (RNA-WB1; 10 mM Tris-HCl pH 7.5, 0.15 M LiCl, 1 mM EDTA pH 8.0, 0.1% LiDS, 0.1% Triton X-100, in water) and then washing buffer 3 (RNA-WB3; 10 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA pH 8.0, in water). RNA was eluted from Oligo (t) beads by resuspending the beads in 50 ul of elution buffer (EB; 10 mM tris-HCl pH 7.5, 1 mM EDTA pH 8.0, in water) followed by incubation at 80C for 2 min on a PCR cycler. The samples were then placed back on the magnet, and the elution buffer supernatant containing the poly(A) RNA was carefully collected and placed on ice.   A second round of poly(A) RNA selection was then performed. Eluted Oligo d(T) beads were first washed on a magnet with 150 ul EB and then 150 ul 2X Oligo d(T) binding buffer (2x DTBB; 20 mM Tris-HCl pH 7.5, 1 M LiCl, 2 mM EDTA pH 8.0, 1% LiDS, 0.1% Triton X-100) and resuspended in 50 ul of 2x DTBB. Resuspended Oligo d(T) beads were then added to isolated RNA and incubated at 65 C and at RT as described above. Beads were then placed on a magnet and washed once with 150 ul of RNA-WB1 and then with 30 ul of 1x First-Strand buffer (made from 5x stock; Life Technologies) and transferred in a new PCR strip. Beads were collected on a magnet and resuspended in 10 ul of 2x SuperScript III buffer (Life Technologies, 1x First-Strand buffer, 10 mM DTT in water). Resuspended beads were then incubated at 94 C for 9 min to fragment poly(A) RNA. Beads were then placed back on a magnet, and the supernatant containing poly(A) RNA was carefully collected and placed on ice.  RNA-seq library preparation: Synthesis of cDNA  RNA in 2X SuperScript III buffer was incubated for 1 min at 50 C on a PCR cycler with 2.5 ul of the following mix: 1.5 ug Random Primer (Life Technologies 48190-011), 10 uM Oligo d(T) (Life Technologies 18418020), 10 units SUPERase-In (Ambion AM2696), 4 mM dNTP mix (Life Technologies 18427088), in water. Samples were then immediately placed on ice for 5 min. First-strand synthesis was then performed by incubation at 25 C for 10 min and 50 C for 50 min on a PCR cycler with 7.6 ul of the following mix: 0.2 ug actinomycin (Sigma A1410), 13.15 mM DTT (Life Technologies), 0.026% Tween-20, 100 units SuperScript III (Life Technologies kit 18080-044), in water. After incubation, RNA/DNA complexes were isolated by adding 36 ul of Agencourt RNAClean XP beads (Beckman Coulter A63987) and incubated for 10 min at RT and then 10 min on ice. Samples were then placed on a magnet and beads were washed twice with 150 ul of 75% EtOH. Following washings, beads were air-dried for 10-12 min and eluted with 10 ul of water.  Second-strand synthesis was then performed. RNA/DNA samples in 10 ul of water were incubated for 2.5 hours at 16 C with 5 ul of the following mix: 3x Blue Buffer (Enzymatics), 1.0 ul PCR mix (Affymetrix 77330), 2.0 mM dUTP (Affymetrix 77206), 1 unit RNAseH (Ezymatics Y9220L), 10 units DNA Polymerase I (Enzymatics P7050L), 0.03% Tween-20, in water). DNA was then purified by addition of 1.5 ul Sera-Mag SpeedBeads (Thermo Scientific, 651520505025), resuspended in 30 ul 20% PEG 8000/2.5 M NaCl, incubated at RT for 15 min and placed on a magnet for two rounds of bead washing with 80% EtOH. Beads were then air dried for 10-12 min and DNA was eluted from the beads by adding 40 ul of water. Supernatant was then collected on a magnet and placed on ice or stored at -20C until DNA blunting, poly(A)-tailing, and adapter ligation (see below).   RNA-seq final library preparation  Sequencing libraries were prepared from generated cDNA (RNA) by blunting, A-tailing, and adapter ligation as previously described using barcoded adapters (NextFlex, Bioo Scientific). Prior to final PCR amplification, RNA-seq libraries were digested by 30 min of incubation at 37C with Uracil DNA Glycosylase (final concentration of 0.134 units per ul of library volume; UDG, Enzymatics G5010L) to generate strand-specific libraries. Libraries were PCR-amplified for 12-15 cycles and size selected for fragments (200-500 for RNA-seq) by gel extraction (10% TBE gels, Life Technologies EC62752BOX). RNA-seq libraries were single-end sequenced for 76 cycles on an Illumina HiSeq 4000 (Illumina, San Diego, CA) according to manufacturer’s instruction.
Chromatin immunoprecipitation: Chromatin immunoprecipitation for histone modification H3K4me3, H3K27ac and Rpb2 and was performed as follows, using ~500,000 microglia per assay and n=2 independent biological replicates per conditions. First microglia were briefly thawed on ice and lysed by incubation in 1 ml of lysis buffer (0.5% IGEPAL CA-630, 10 mM HEPES pH 7.9, 85 mM KCl, 1 mM EDTA pH 8.0, in water) for 10 min on ice. Lysates were centrifuged at 800 RCF for 5 min at 4 °C, and pellets were resuspended in 200 ul of sonication/ immunoprecipitation buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EGTA, 0.1% Deoxcycholate, 0.5% Sarkosyl, in water). Sonication was performed with a Bioruptor Standard Sonicator (Diagenode) and consisted of two rounds of 15 min each, alternating stages of 30 sec “sonication-on” with 60 sec “sonication-off”. Twenty-two microliters of 10% Triton-X were then added to samples (1% final concentration) on ice, and lysates were cleared by centrifugation for 5 min at 18,000 RCF at 4 °C. Two microliters of supernatant were then set aside for input library sequencing controls. Supernatants were then immunoprecipitated on a rotator for two hours at 4 °C with antibody pre-bound to 17 ul Protein A Dynabeads (Life Technologies 10001D). The following antibodies were used: H3K4me3 (Millipore

value: WT

value: Ki67-Positive microglia

value: Male

tag: age at sacrifice (in days)


title: ATAC_Microglia_Ki67Positive_Ki67rfp_Male_PND75_PLX5622_17d_Recovery_3d_rep1

status: Public on Aug 18 2022

type: SRA

country: Canada

library_source: genomic

molecule: genomic DNA

organism: Mus musculus

tag: strain

tag: genotype

tag: cell type

tag: Sex

tag: treatment

value: Starting at 55 days of age, PLX5622 diet for 17 days; Recovery for 3 days


title: RGC105

city: Guangzhou

first: Sheng

last: Liu

address: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center

country: China

institute: Sun Yat-sen University

description: Retinal ganglion cells, scRNA-seq

library_source: transcriptomic single cell

data_processing:
Previously published pipeline STAR version 2.7.0
Assembly: mm10
Supplementary files format and content: Comma-Separated Values files and matrix files

molecule: total RNA

source_name: Retina

extract_protocol:
The retina is isolated and flat-mounted under dim red light (created with BioRender.com). A retinal ganglion cell is patched and its responses to blue and green spotlight stimuli are recorded. The cell content is aspirated. RNA is processed using Smart-seq2 protocol to get transcriptomic data.
the cDNA library was sequenced on the NovaSeq 6000 instrument and 150-bp paired-end reads were set up. Each sample was sequenced to approximately 10 million reads

value: Retinal ganglion cells

tag: tissue

value: Retina

tag: age

value: 7-9 weeks old


title: yHN21-ZA-HA_HAvsH3_20161220_Rep2

type: genomic

city: Montreal

first: Francois

last: Robert

state: QC

address: 110 av des Pins Ouest

institute: IRCM

description:
Biological replicate 2 of 2.  ZA-HA occupancy by ChIP-chip in WT cells. ZA-HA ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against HA and hybridized against H3 ChIP (Cy3-labeled).
SmthBED_yHN21-ZA-HA_HAvsH3_20161220_Rep1-2__S.cerevisiaeApr11_446228

hyb_protocol: ChIP and control samples were combined and dried by SpeedVac. Combined samples were resuspended in 110 µL of fresh hybridization mix (5 µL 10 mg/mL Salmon Sperm DNA (Thermo Fisher Scientific, 15632011), 5 µL 8 mg/mL Yeast tRNA (Thermo Fisher Scientific, 15401011) and 100 µL DIG Easy Hyb (Roche, 11603558001)) and incubated 5 min at 95˚C followed by 5 min at 45˚C. Samples were hybridized to Agilent microarrays in SureHyb enabled chambers (Agilent, G2534A) with rotation at 42˚C for at least 18 hr. Microarrays were washed on an orbital shaker for 5 min at room temperature with Wash I buffer (6X SSPE, 0.005% N-Lauroylsarcosine), followed by a second wash with pre-warmed (31˚C) Wash II buffer (0.06X SSPE) for 5 min at room temperature. The microarrays used were described before (Jeronimo and Robert, 2014) and were custom designed by Agilent Technologies and contain a total of about 180,000 Tm-adjusted 60-mer probes covering the entire yeast genome with virtually no gaps between probes.

scan_protocol: Microarrays were scanned using an InnoScan900 (Innopsys) at 2 µm resolution.

data_processing:
bed description: A genome browser-ready file for H2A.Z-HA occupancy by ChIP-chip (H2A.Z/H3) in WT cells.
bed method: The ChIP-chip data were normalized using the Limma Loess method, and replicates were combined as described previously (Ren et al. 2000, Science 290(5500):2306-9). The data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100 bp to generate data points in 10-bp intervals (Guillemette et al. 2005, PLoS Biol 3(12):e384).
Correction = Foreground-Background; Normalisation = limma loess (subgrid)

organism: Saccharomyces cerevisiae

label_protocol: The ChIP and Input samples were blunted as follow. 40 µL of ChIP DNA or about 400 ng of Input DNA were added to 70 µL of blunting mix (11 µL 10X NEBuffer 2 (NEB, B7002S), 0.5 µL 10 mg/mL BSA, 0.5 µL 20 mM dNTPs and 0.2 µL (0.6 units) of T4 DNA polymerase (NEB, M0203L)) and incubated at 12˚C for 20 min. Then, the blunted DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) in the presence of 280 mM of NaOAc pH 5.2 and 10 μg of glycogen, precipitated with ethanol and resuspended in 25 µL ddH2O. Blunted DNA was ligated to 100 pmol of annealed linkers (see Table S2) with 2.5 units of T4 DNA ligase (Thermo Fisher Scientific, 15224041) in the supplied buffer, in a final volume of 50 µL, overnight at 16˚C. Samples were precipitated with 6 µL of 3 M NaOAc pH 5.2 and 130 µL ethanol, and resuspended in 25 µL ddH2O. 15 µL of labeling mix (4 µL 10X ThermoPol Reaction buffer (NEB, B9004S), 2 µL of 5 mM aa-dUTP/dNTP mix (5 mM each dATP, dGTP and dCTP, 3 mM dTTP and 2 mM 5-(3-aminoallyl)-dUTP (Thermo Fisher Scientific, AM8439)) and 1.25 µL of 40 µM oligo 1) were added to each sample. Samples were placed in a thermocycler and when temperature reached 55˚C, 10 µL of enzyme mix (1 µL 10X ThermoPol Reaction buffer, 1 µL (5 units) Taq DNA polymerase (Thermo Fisher Scientific, 18038240) and 0.01 µL (0.025 units) Pfu DNA polymerase (Thermo Fisher Scientific, EP0502)) were added and the following program was run. 1) 72˚C, 5 min; 2) 95˚C, 2 min; 3) 32 cycles: 95˚C, 30sec; 55˚C, 30sec; 72˚C, 1 min; 4) 72˚C, 4 min; 5) 4˚C, forever. PCR products were purified using QIAquick PCR Purification kit (QIAGEN, 28106) with few modifications: PE buffer was replaced by Phosphate wash buffer (5 mM KPO4 pH 8.5 and 80% ethanol) and EB buffer was replace by Phosphate elution buffer (4 mM KPO4 pH 8.5). Eluates were dried by SpeedVac and pellets were resuspended in 4.5 µL of freshly prepared 0.1 M Na2CO3 pH 9.0 buffer. 4.5 µL of Cy5 Mono-Reactive NHS Ester Fluorescent Dye (ChIP samples) or Cy3 Mono-Reactive NHS Ester Fluorescent Dye (control samples Input DNA or no-tag ChIP) (GE Healthcare, PA25001 and PA23001) were added. Samples were incubated 1 hr in the dark at room temperature. 35 µL of 0.1 M NaOAc pH 5.2 were added and samples were purified using QIAquick PCR Purification kit according to the manufacturers’ instructions.

growth_protocol: 50 mL of yeast extract peptone (YEP) medium supplemented with 2% galactose was inoculated at OD600 0.1 from an overnight preculture in the same medium and allowed to grow at 30°C to an OD600 of 0.6 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 10 min and quenched with 125 mM glycine. Crosslinked cells were collected by centrifugation and washed twice with cold water. Cell pellets were snap-freezed in liquid nitrogen and stored at -80°C.

extract_protocol: Cell pellets were resuspended in 400 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. Cells were lysed using a vortex for 2 hrs at 4C. The lysate was then sonicated 30 cycles, 30 sec ON and 30 sec OFF at High settings (Diagenode Bioruptor). Soluble fragmented chromatin was recovered by centrifugation. 350 μL of the chromatin sample was taken per immunoprecipitation (IP) and, when necessary, 3.5 μL (1%) were saved as Input sample. 20 μL of Dynabeads coated with protein G pre-coupled with the indicated antibody were added to the chromatin sample and incubated overnight at 4°C. Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 200uL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 50°C for 1 hr and subsequently subjected to Proteinase K (5.5 μL 20% SDS, 7.5 μL 10 mg/mL Proteinase K) digestion at 50°C for 1 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 70 % EtOH. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-chip experiments.

value: W303a; htz1[delta]::CloNat; bar1[delta]::KANMx; ZA::HA-URA3

tag: antibody

value: anti-HA (12CA5 from Roche; 2 ug)

value: anti-H3 (ab1791 from Abcam; 2 ug)


title: yHN22-ZA-rII-HA_HAvsH3_20161220_Rep1

description:
Biological replicate 1 of 2.  ZA-rII'-HA occupancy by ChIP-chip in WT cells. ZA-rII'-HA ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against HA and hybridized against H3 ChIP (Cy3-labeled).
SmthBED_yHN22-ZA-rII-HA_HAvsH3_20161220_Rep1-2__S.cerevisiaeApr11_276022

value: W303a; htz1[delta]::CloNat; bar1[delta]::KANMx; ZA-rII'::HA-URA3


title: tpt_18HL3

city: Rostock

first: Andreas

middle: Sven

last: Richter

address: Albert-Einstein-Strasse 3

department: Institute for Biosciences

country: Germany

institute: University of Rostock

library_source: transcriptomic

data_processing:
base calling: CASAVA base recognition
removing reads containing adapter, reads containing ploy-N and low quality : Novogene pearl scripts
alignment: Hisat2 v2.0.5
feature count: featureCounts v1.5.0-p3
differential expression analysis: DESeq2 R package (1.20.0)
calculation of adjusted p-value: Benjamini and Hochberg’s
Genome_build: TAIR10 (ensemblplants_arabidopsis_thaliana_tair10_gca_000001735_1)
Supplementary_files_format_and_content: Table containing the FPKM for each transcript

organism: Arabidopsis thaliana

source_name: leaf tissue

growth_protocol: 21d on soil in short day conditions (10h light)

extract_protocol:
RNA extraction using citric acid (Onate-Sanchez & Vicente-Carbajosa, 2008) followed by Dnase I treatment and phenol:chloroform:isoamyl alcohol purification
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dUTP for directional library. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.

treatment_protocol: Shifted to 500µmol photons m-2 s-1 at the end of the night

value: leaf

tag: time of treatment

value: 500umol photons m-2 s-1


title: yHN21-ZA-HA_HAvsH3_20161220_Rep1

description:
Biological replicate 1 of 2.  ZA-HA occupancy by ChIP-chip in WT cells. ZA-HA ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against HA and hybridized against H3 ChIP (Cy3-labeled).
SmthBED_yHN21-ZA-HA_HAvsH3_20161220_Rep1-2__S.cerevisiaeApr11_446228


title: RGC106

description: Retinal ganglion cells, scRNA-seq


title: ATAC_Microglia_C57_Male_Adult_Basal_PND80_rep1

value: Ki67-Negative microglia


title: Merbarone.24h B3

city: Xiamen

first: Huifen

last: Cao

email: hfcao_bnu@163.com, hfcao@hqu.edu.cn

state: Fujian

address: 668 Jimei Road

department: School of Biomedical Sciences

institute: Huaqiao University

description: crosslinked MNase-treated nuclei

data_processing:
The SSB site mapping was performed the same way as in the SSiNGLe-ILM protocol (https://protocolexchange.researchsquare.com/article/pex-920/v2) with the exception that additional filter was used: a read-pair was used only if the first base of the read 2 aligned to the genome.
Genome_build: hg19(GRCh37) for human
Supplementary_files_format_and_content: BED files containing coordinates of single-strand breaks

organism: Homo sapiens

growth_protocol: Human CML leukemia cell line K562 was obtained from Cell Bank of Chinese Academy of Sciences. Cells were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, ExCell Bio) and 1% (v/v) pen-strep (Thermo Fisher Scientific) at 37 °C and 5% CO2.

extract_protocol:
Cells were either crosslinked with formaldehyde in vivo and fragmented with MNase for the standard SSiNGLe-ILM protocol, or directly used for genomic DNA extraction followed by the SSiNGLe on HMW DNA protocol. For SSiNGLe on HMW DNA, the DNA was extracted directly from K562 cells using the TIANamp Genomic DNA Kit (TIANGEN Biotech, DP304) according to the manufacturer?s protocol. One hundred nanograms of the DNA was used directly as input into the SSiNGLe-ILM protocol (https://protocolexchange.researchsquare.com/article/pex-920/v2) at the polyA-tailing step with only one modification at the Illumina library construction stage. The step 19 in the procedure corresponding to the 2nd round PCR was performed with all (instead of just the 4 µl used in the standard protocol) DNA from the 1st round PCR. The latter was purified with the 2x volume of the VAHTS DNA Clean Beads (Vazyme) before adding to the 2nd round PCR.
The libraries were prepared as described here: https://protocolexchange.researchsquare.com/article/pex-920/v2

treatment_protocol: For drug treatments, three million K562 cells were seeded at 1 million cells per ml of medium per well in 6-well plates. After 16 hr, the cells were separately treated with 0.1% DMSO, 20 ?M Z-DEVD-FMK (AbMole), 100 ?M ICRF-187 (Selleck) and 100 ?M merbarone (Merck) and incubated for 6, 12, 24, 36, or 48 hr. All drugs were dissolved in DMSO, concentration of which was kept at 0.1% in all treatments. Each treatment was done in two independent biological replicas. The cells were used as input in the standard SSiNGLe-ILM protocol on crosslinked nuclei (https://protocolexchange.researchsquare.com/article/pex-920/v2).

tag: cell line

value: chronic myelogenous leukemia cell line

value: treated with Merbarone for 24 hours


title: Merbarone.12h B4

description: crosslinked MNase-treated nuclei

value: treated with Merbarone for 12 hours


title: RGC107

description: Retinal ganglion cells, scRNA-seq


title: RGC115

description: Retinal ganglion cells, scRNA-seq


title: WT_18HL2


title: yHN20-H2A.Z-HA_HAvsH3_20161220_Rep2

description:
Biological replicate 2 of 2.  H2AZ-HA occupancy by ChIP-chip in WT cells. H2AZ-HA ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against HA and hybridized against H3 ChIP (Cy3-labeled).
SmthBED_yHN20-H2A.Z-HA_HAvsH3_20161220_Rep1-2__S.cerevisiaeApr11_549548

value: W303a; htz1[delta]::CloNat; bar1[delta]::KANMx; H2AZ::HA-URA3


title: RGC108

description: Retinal ganglion cells, scRNA-seq


title: Input_H3K4me3_Microglia_C57_Male_Adult_Basal_PND75_rep2

description: Input for ChIP_H3K4me3_Microglia_C57_Male_Adult_Basal_PND75_rep2

value: WT

tag: chip antibody


title: Merbarone.12h B3

description: crosslinked MNase-treated nuclei


title: WT_18HL3


title: tpt_18HL1


title: Merbarone.06h B3

description: crosslinked MNase-treated nuclei

value: treated with Merbarone for 6 hours


title: tpt_18HL2


title: Input_H3K4me3_Microglia_C57_Male_Adult_Basal_PND75_rep1

description: Input for ChIP_H3K4me3_Microglia_C57_Male_Adult_Basal_PND75_rep1

value: Male


title: yHN20-H2A.Z-HA_HAvsH3_20161220_Rep1

description:
Biological replicate 1 of 2.  H2AZ-HA occupancy by ChIP-chip in WT cells. H2AZ-HA ChIP sample (Cy5-labeled) was performed using a mouse monoclonal antibody against HA and hybridized against H3 ChIP (Cy3-labeled).
SmthBED_yHN20-H2A.Z-HA_HAvsH3_20161220_Rep1-2__S.cerevisiaeApr11_549548


title: RGC116

description: Retinal ganglion cells, scRNA-seq

value: Retinal ganglion cells


title: Merbarone.24h B4

description: crosslinked MNase-treated nuclei


title: WT_18HL1


title: Input_Rpb2_Microglia_Ki67Positive_C57_Male_PND8_rep1

description: Input for ChIP_Rpb2_Microglia_Ki67Positive_C57_Male_PND8_rep1

value: Ki67-Positive microglia


title: RGC109

description: Retinal ganglion cells, scRNA-seq

value: Retina

value: 7-9 weeks old


title: Merbarone.06h B4

description: crosslinked MNase-treated nuclei


title: ICRF_187.48h B4

description: crosslinked MNase-treated nuclei

value: treated with ICRF_187 for 48 hours


title: RGC117

description: Retinal ganglion cells, scRNA-seq


title: ChIP_Rpb2_Microglia_Ki67Positive_C57_Male_PND8_rep1

value: Rpb2 (GeneTex, GTX102535)


title: ICRF_187.48h B3

description: crosslinked MNase-treated nuclei


title: tpt_9HL3


title: tpt_9HL2


title: RGC118

description: Retinal ganglion cells, scRNA-seq


title: ICRF_187.36h B4

description: crosslinked MNase-treated nuclei

value: treated with ICRF_187 for 36 hours


title: ChIP_Rpb2_Microglia_C57_Male_Adult_Basal_PND60_rep2


title: ICRF_187.36h B3

description: crosslinked MNase-treated nuclei


title: RGC130

description: Retinal ganglion cells, scRNA-seq


title: WT_9HL3

value: leaf


title: RGC110

description: Retinal ganglion cells, scRNA-seq


title: tpt_9HL1

value: 500umol photons m-2 s-1


title: WT_9HL2


title: RGC121

description: Retinal ganglion cells, scRNA-seq


title: ICRF_187.24h B4

description: crosslinked MNase-treated nuclei

value: treated with ICRF_187 for 24 hours


title: ICRF_187.24h B3

description: crosslinked MNase-treated nuclei

value: chronic myelogenous leukemia cell line


title: WT_9HL1


title: ChIP_Rpb2_Microglia_C57_Male_Adult_Basal_PND60_rep1

value: Ki67-Negative microglia


title: Input_Rpb2_Microglia_C57_Male_Adult_Basal_PND65_rep2

description: Input for ChIP_Rpb2_Microglia_C57_Male_Adult_Basal_PND60_rep2


title: MKP_Vav1.Cre_3

city: Basel

first: DBM Bioinformatics

last: Core Facility

state: BS

address: Hebelstrasse 20

department: Departement of Biomedicine

country: Switzerland

institute: University of Basel

description: MKP, Vav1.Cre, replicate 3

data_processing:
Reads were aligned to the mm10 genome with STAR (version 2.7.0c) using the extra options '-outFilterMultimapNmax 10 --outSAMmultNmax 1' for handling multi-mapping reads.
Expression of RefSeq genes (annotation downloaded from UCSC 2015-12-18) was quantified using featureCounts (subread version 1.6.4) with extra options "-O --read2pos 5 –M –s 2 –p -B".
Genome_build: mm10
Supplementary_files_format_and_content: tab-separated values, text file with columns: 1. ENTREZ gene ID, 2. gene symbol, 3. gene name, 4. gene length (exon union model), 5. - 28. read counts per sample

source_name: Bone marrow

extract_protocol:
Bone marrow derived populations were FACS sorted and total RNA was extraced with PicoPure RNA preparation kit (invitrogen)
SmartSeq 2

treatment_protocol: Mice were treated with tamoxifen for 5 consequtive days. 12 weeks post-tamoxifen injections, indicated HSPC subsets were isolated by FACS sorting

tag: cell population

value: MKP

value: Vav1.Cre V617F (VF)

value: Bone marrow


title: RGC122

description: Retinal ganglion cells, scRNA-seq


title: K562.SSiNGLe-HMW_DNA.B1

description:
without fragmentation or crosslinked nuclei
SSiNGLe without fragmentation or crosslinking

data_processing:
The SSB site mapping was performed the same way as in the SSiNGLe-ILM protocol (https://protocolexchange.researchsquare.com/article/pex-920/v2) with the exception that additional filter was used: a read-pair was used only if the first base of the read 2 aligned to the genome.
Assembly: hg19(GRCh37) for human
Supplementary files format and content: BED files contain coordinates of the bases immediately upstream of single-strand breaks in the non-repeat portions of the genome. The 4th columns show the number of reads representing the SSB sites.

extract_protocol:
Cells were either crosslinked with formaldehyde in vivo and fragmented with MNase for the standard SSiNGLe-ILM protocol, or directly used for genomic DNA extraction followed by the SSiNGLe on HMW DNA protocol. For SSiNGLe on HMW DNA, the DNA was extracted directly from K562 cells using the TIANamp Genomic DNA Kit (TIANGEN Biotech, DP304) according to the manufacturer’s protocol. One hundred nanograms of the DNA was used directly as input into the SSiNGLe-ILM protocol (https://protocolexchange.researchsquare.com/article/pex-920/v2) at the polyA-tailing step with only one modification at the Illumina library construction stage. The step 19 in the procedure corresponding to the 2nd round PCR was performed with all (instead of just the 4 µl used in the standard protocol) DNA from the 1st round PCR. The latter was purified with the 2x volume of the VAHTS DNA Clean Beads (Vazyme) before adding to the 2nd round PCR. Total RNA was extracted using TRNzol Universal (Tiangen, DP424) and Total RNA kit I (Omega, R6834-02) according to the manufacturer’s protocols and quantified using Qubit 3.0 Fluorometer (Life technologies).
The libraries of SSBs were prepared as described here: https://protocolexchange.researchsquare.com/article/pex-920/v2. And the RNA-seq libraries were contructed based on rRNA depletion strategy by Novogene Corporation (Beijing).

value: DMSO


title: ICRF_187.12h B4

description: crosslinked MNase-treated nuclei

value: treated with ICRF_187 for 12 hours


title: Input_H3K27ac_Microglia_Ki67Positive_C57_Male_PND90_PLX5622_17d_Recovery_3d_rep1

description: Input for ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_PND90_PLX5622_17d_Recovery_3d_rep1

value: WT

value: Male

value: Starting at 70 days of age, PLX5622 diet for 17 days; Recovery for 3 days


title: Input_H3K27ac_Microglia_Ki67Positive_C57_Male_PND90_PLX5622_17d_Recovery_3d_rep2

description: Input for ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_PND90_PLX5622_17d_Recovery_3d_rep2

value: Ki67-Positive microglia


title: ICRF_187.12h B3

description: crosslinked MNase-treated nuclei


title: MKP_Vav1.Cre_2

description: MKP, Vav1.Cre, replicate 2


title: ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_PND90_PLX5622_17d_Recovery_3d_rep1

value: H3K27ac (Active Motif, 39685)


title: Human5_SREBP_ChIPseq_Mock_24h

city: La Jolla

first: Aaron

middle: F

last: Carlin

state: California

address: 9500 Gilman Drive GPL Rm 118

department: Pathology and Medicine

country: USA

institute: University of California, San Diego

data_processing:
Basecalling, demultiplexing, and adapter trimming was performed using Illumina's bcl2fastq (v2.17.1.14)
RNA-seq and csRNA-seq reads aligned to the GRCh38/hg38 assembly using STAR were used to generate gene expression FPKM values using HOMER. Gene expression (RNA-seq) was measured using HOMER with the following parameters: analyzeRepeats.pl rna hg38 -count exons -raw -pc 3 -strand - -condenseGenes followed by DESeq normalized regularized logarithm (rlog) normalization values while accounting for individual donors in the design matrix. Gene FPKM values were defined for primary Gencode transcripts using HOMER (v4.10) using only strand-specific (RNA-seq = exonic reads) reads for each transcript. csRNA-seq reads aligned to the GRCh38/hg38 assembly from three replicates per condition (mock, ZIKV-, ZIKV+) were combined and TSRs identified using HOMER findcsRNATSS.pl with the corresponding combined input and RNA-seq tag directories.
ChIP-seq reads were aligned to the human genome (hg38) using bowtie2 (Langmead and Salzberg, 2012), keeping only reads that map to a single unique genomic location for further analysis (MAPQ > 10). ChIP-seq peaks were called using tags from three replicates per condition with input DNA as background using HOMER’s getDifferentialPeaksReplicates.pl using the “-style factor” (5-fold enrichment over background and FDR < 0.001).
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: RNA-seq tab-delimited text files include FPKM values for each sample. Tab-delimited BED files containing peak positions for each ChIP-seq experiment or combined replicates for csRNA-seq experiments.

source_name: Human blood monocytes derived

growth_protocol: Human peripheral blood was separated using Histopaque 1077 and centrifuged at 300xg for 30 min at 4˚C. The buffy coat was washed once, and red blood cells were lysed with molecular grade water. Monocytes were negatively selected from the buffy coat using the human pan monocyte isolation kit (Miltenyi Biotec) as described by the manufacturer. Cells were seeded at 1.5 x 10^6 cells/mL onto polystyrene plates and differentiated for 7 days in hDC media composed of RPMI 1640 Medium, GlutaMAX™ Supplement, 1% penicillin/streptomycin, 2.5% HEPES buffer, and freshly added cytokines including 100 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and 100 ng/ml recombinant human interleukin 4 (rhIL-4). Cells were incubated at 37˚C, 5% CO2 and media was changed every 2-3 days.

extract_protocol:
For RNA-seq and csRNA-seq, after ZIKV infection of hDCs for 24 hours, cells were collected and stained with Zombie Violet™ Fixable Viability stain (BioLegend) for 20 min at 4˚C. After washing, cells were fixed and permeabilized with 4% paraformadehyde (PFA), 0.1% saponin in the presence of RNasin Ribonuclease inhibitor (400 U/mL). After centrifugation, samples were resuspended in wash buffer (0.2% BSA, 0.1% saponin and 400 U/mL of RNasin Ribonuclease inhibitor). Sample were blocked with human Fc blocker diluted in staining buffer (1% Bovine serum albumin (BSA), 0.1% saponin, 1600 U/mL RNasin Ribonuclease inhibitor) for 5 min, 4˚C. Then, 4G2-AF647 antibody was added to samples and incubated for 30 min, 4˚C. Samples were centrifuged at 1000xg for 3 min, 4˚C. Cells were washed using wash buffer and centrifuged at 1000xg for 3 min, 4˚C. Samples were resuspended in sort buffer containing 0.5% BSA and 1600 U/mL of RNase inhibitor in 1XPBS and sorted into ZIKV+ or DENV+ and ZIKV− or DENV- cells on a FACSAria cell sorter (BD Biosciences). After sorting, cells were pelleted at 4oC, supernatant discarded and total RNA isolated using Ambion’s RecoverAll Total Nucleic Acid Isolation Kit [AM1975], starting at the protease digestion step. All steps were performed per manufacturer recommendations with the following modification. Cells were incubated in digestion buffer for 3 hours at 50oC supplemented with RNasin Plus RNase Inhibitor. RNA was treated with in column DNase per manufacturer instructions, eluted and RNA quality determined by BioAnalyzer using the Eukaryote Total RNA Pico Chip. Samples with RIN values greater than 8.0 were used for library preparation. For chromatin immunoprecipitation-sequencing (ChIP-seq) experiments, moDCs were cross-linked with 4 mM disuccinimidyl glutarate (DSG) in PBS for 30 minutes at RT followed by 1% formaldehyde for 15 minutes at RT and quenched with 0.125 M glycine. Preparation of moDCs for FACS was performed as described above for RNA-seq except that 1x cOmplete protease inhibitors (Roche) were included in all buffers instead of RNasin Plus RNase Inhibitor. After separation by FACS, cells were washed, separated into 500,000 cell aliquots, pelleted, snap-frozen and stored at -80°C.
For RNA-seq, RNA libraries were generated using the TruSeq stranded total RNA-seq [Illumina] kit per manufacturer instructions and single-end sequenced for 51 cycles on an Illumina Hi-Seq 2000 or NextSeq 500 per manufacturer instructions. For csRNA-seq, small RNAs of approximately 20–60 nt were size-selected from >1 µg of total RNA with generous spacing on a 15% acrylamide, 7M UREA and 1x TBE gel (Invitrogen EC6885BOX), eluted and precipitated over night at -80°C. Given the RNA fragmentation associated with de-crosslinking, input libraries were generated to eliminate potential false positives and ensure accurate TSS peak calling. csRNA-seq libraries were twice cap selected prior to decapping and libraries were generated as described above. Input libraries were treated with the RppH pyrophosphatase (NEB M0356) prior to adapter ligation to include the whole repertoire of small RNAs with 3’-OH. Samples were quantified by Qbit (Invitrogen). For ChIP-seq, Cells aliquots (500,000) were resuspended on ice in 130 µl RLNR1 lysis buffer (20 mM Tris/HCl pH7.5, 150mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.4% Na-Deoxycholate, 1% NP-40 alternative, 0.5 mM DTT, 1x protease inhibitor cocktail (DTT and protease inhibitors added immediately before use) and transferred to microtubes with an AFA Fiber (Covaris, MA). All subsequent steps were performed on ice or at 4°C. Chromatin was sheared by sonication in a 96 Place microTUBE Rack (Covaris cat#500282) using a Covaris E220 focused-ultrasonicator (Covaris, MA) for 20 cycles with the following settings: time, 60sec; duty, 5; PIP, 140; cycles, 200; amplitude, 0.0; velocity, 0.0; dwell, 0.0. Samples were recovered and spun at max speed for 10 mins and the pellet was discarded. 1 % of the sample was stored at -20°C for DNA input control and the remaining supernatant was transferred to PCR strips and brought up to a volume of 200 µl using RLNR1 lysis buffer. For ChIP, 20 μl Protein A/G Dynabeads per sample was washed 2x with 1 ml 0.5% BSA/TE/0.1%Tween20 (TET), resuspended in 0.5% BSA/TET, SREBP antibodies (Santa Cruz Biotechnology: sc-8984X, Thermo Fisher Scientific: PA1-337, R&D Systems: AF7119) added, and rotated for 1 hr at RT. The supernatant was removed, Dynabeads washed 1X with 0.1% BSA/TET using a magnet, resuspended in 10 µl RIPA buffer per IP reaction. Dynabeads in RIPA buffer were added to samples and rotated overnight at 4°C. Dynabeads were washed 3 times with Wash Buffer 1 (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), three times with Wash Buffer 3 (10 mM Tris-HCl pH, 250 mM LiCl, 1 mM EDTA, 1% Triton X- 100, 0.7% Na-Deoxycholate) and three times with TET (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.2% Tween20). Dynabeads were washed once with TE-NaCl (10 mM Tris-HCl pH 8, 1 mM EDTA, 50 mM NaCl) and resuspended in 25 μl TT (10 mM Tris-HCl pH 8, 0.05% Tween20). Input samples were resuspended in 25 μl TT and libraries were generated in parallel with ChIP samples. Library NEBNext End Prep and Adaptor Ligation were performed using NEBNext Ultra II DNA Library Prep kit (New England BioLabs) according to manufacturer instructions using barcoded adapters (NextFlex, Bioo Scientific). Libraries were incubated with RNase A and Proteinase K at 55°C for 1 hour and 65°C overnight. Libraries were PCR amplified for 14 cycles with NEBNext High Fidelity 2X PCR MasterMix (New England BioLabs, NEBM0541). Libraries were size selected for 225 – 350 bp fragments by gel extraction (10% TBE gels, Life Technologies).
Samples were sequenced using the Illumina NextSeq 500 platform using 51 cycles (ChIP-seq) or 75 cycles (csRNA-seq) single-end sequencing per manufacturer instructions. RNA-seq samples were single-end sequenced for 51 cycles on an Illumina HiSeq 4000 (Illumina, San Diego, CA)

treatment_protocol: After monocyte differentiation, on day 7 of culture, moDCs were infected with ZIKV clinical isolate SD001 at MOI 0.5, ZIKV PRVABC59, ZIKV-EGFP-BeH819015 and DENV UIS353 at MOI 1. Briefly, moDCs were incubated 2 hours with the virus, washed with PBS, and incubated in fresh medium for the for 24h at 37˚C, 5% CO2.

tag: donor id

value: monocyte-derived dendritic cell

tag: viral infection

value: mock infected

tag: viral strain

value: SREBP antibodies (Santa Cruz Biotechnology:sc-8984X, Thermo Fisher Scientific:PA1-337, R&D Systems:AF7119)

tag: time post infection viral infection

tag: experment type

value: SREBP ChIP-seq


title: ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_PND90_PLX5622_17d_Recovery_3d_rep2

tag: strain

tag: age at sacrifice (in days)


title: RGC124

description: Retinal ganglion cells, scRNA-seq

tag: cell type

value: Retinal ganglion cells

value: Retina

value: 7-9 weeks old


title: MKP_Vav1.Cre_1

description: MKP, Vav1.Cre, replicate 1

value: MKP

tag: genotype


title: Input_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND90_Cuprizone_4w_rep2

description: Input for ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND90_Cuprizone_4w_rep2

tag: Sex

tag: treatment

value: Cuprizone diet for 4 weeks prior sacrifice


title: RGC126

description: Retinal ganglion cells, scRNA-seq

tag: tissue

tag: age


title: MKP_Scl.N1_5

description: MKP, Scl.N1, replicate 5

value: Scl.Cre Exon12 (E12)

value: Bone marrow


title: RGC125

description: Retinal ganglion cells, scRNA-seq


title: ICRF_187.06h B4

description: crosslinked MNase-treated nuclei

value: chronic myelogenous leukemia cell line

value: treated with ICRF_187 for 6 hours


title: ICRF_187.06h B3

description: crosslinked MNase-treated nuclei


title: RGC127

description: Retinal ganglion cells, scRNA-seq


title: RGC123

description: Retinal ganglion cells, scRNA-seq


title: MKP_Scl.N1_2

description: MKP, Scl.N1, replicate 2


title: MKP_Scl.N1_4

description: MKP, Scl.N1, replicate 4


title: DMSO.48h B3

description: crosslinked MNase-treated nuclei

value: treated with DMSO for 48 hours


title: DMSO.48h B4

description: crosslinked MNase-treated nuclei


title: RGC129

description: Retinal ganglion cells, scRNA-seq


title: Input_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND8_rep2

description: Input for ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND8_rep2

value: WT

value: Male


title: RGC128

description: Retinal ganglion cells, scRNA-seq


title: ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND90_Cuprizone_4w_rep1

value: Ki67-Positive microglia

value: H3K27ac (Active Motif, 39685)


title: RGC136

description: Retinal ganglion cells, scRNA-seq


title: DMSO.36h B3

description: crosslinked MNase-treated nuclei

value: treated with DMSO for 36 hours


title: MKP_Scl.Cre.mJak2.KI_5

description: MKP, Scl.Cre.mJak2.KI, replicate 5

value: Scl.Cre mouse Jak2 knock-in


title: DMSO.24h B4

description: crosslinked MNase-treated nuclei

value: treated with DMSO for 24 hours


title: DMSO.36h B4

description: crosslinked MNase-treated nuclei


title: ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND8_rep2


title: MKP_Scl.Cre.mJak2.KI_4

description: MKP, Scl.Cre.mJak2.KI, replicate 4


title: Input_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND90_Cuprizone_4w_rep1

description: Input for ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND90_Cuprizone_4w_rep1


title: RGC137

description: Retinal ganglion cells, scRNA-seq

value: Retinal ganglion cells


title: ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND8_rep1


title: ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND90_Cuprizone_4w_rep2


title: DMSO.24h B3

description: crosslinked MNase-treated nuclei


title: DMSO.12h B4

description: crosslinked MNase-treated nuclei

value: treated with DMSO for 12 hours


title: PreMegE_Vav1.Cre_3

description: pre-MegE, Vav1.Cre, replicate 3

value: pre-MegE

value: Vav1.Cre V617F (VF)


title: PreMegE_Vav1.Cre_2

description: pre-MegE, Vav1.Cre, replicate 2


title: Input_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND90_Cuprizone_4w_rep2

description: Input for ChIP_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND90_Cuprizone_4w_rep2

value: Ki67-Negative microglia

value: Cuprizone diet for 4 weeks prior sacrifice


title: MKP_Scl.Cre.mJak2.KI_6

description: MKP, Scl.Cre.mJak2.KI, replicate 6

value: MKP

value: Bone marrow


title: RGC132

description: Retinal ganglion cells, scRNA-seq

value: Retina

value: 7-9 weeks old


title: ChIP_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND90_Cuprizone_4w_rep2

value: WT


title: PreMegE_Vav1.Cre_1

description: pre-MegE, Vav1.Cre, replicate 1


title: RGC133

description: Retinal ganglion cells, scRNA-seq


title: DMSO.12h B3

description: crosslinked MNase-treated nuclei

value: chronic myelogenous leukemia cell line


title: DMSO.06h B4

description: crosslinked MNase-treated nuclei

value: treated with DMSO for 6 hours


title: Input_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND90_Cuprizone_4w_rep1

description: Input for ChIP_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND90_Cuprizone_4w_rep1

value: Male


title: PreMegE_Scl.Cre.mJak2.KI_3

description: pre-MegE, Scl.Cre.mJak2.KI, replicate 3


title: RGC138

description: Retinal ganglion cells, scRNA-seq


title: Input_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND8_rep1

description: Input for ChIP_H3K27ac_Microglia_Ki67Positive_C57_Male_Adult_PND8_rep1

value: Ki67-Positive microglia


title: PreMegE_Scl.Cre.mJak2.KI_2

description: pre-MegE, Scl.Cre.mJak2.KI, replicate 2

value: Scl.Cre mouse Jak2 knock-in


title: DMSO.06h B3

description: crosslinked MNase-treated nuclei


title: RGC139

description: Retinal ganglion cells, scRNA-seq


title: ChIP_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND90_Cuprizone_4w_rep1

value: H3K27ac (Active Motif, 39685)


title: RGC134

description: Retinal ganglion cells, scRNA-seq

value: Retinal ganglion cells


title: RGC131

description: Retinal ganglion cells, scRNA-seq


title: Input_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND8_rep2

description: Input for ChIP_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND8_rep2


title: PreMegE_Scl.Cre.mJak2.KI_1

description: pre-MegE, Scl.Cre.mJak2.KI, replicate 1


title: LTHSC_Vav1.Cre_3

description: LTHSC, Vav1.Cre, replicate 3

value: LTHSC


title: RGC140

description: Retinal ganglion cells, scRNA-seq


title: ChIP_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND8_rep2


title: Input_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND8_rep1

description: Input for ChIP_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND8_rep1


title: ChIP_H3K27ac_Microglia_Ki67Negative_C57_Male_Adult_PND8_rep1


title: RGC135

description: Retinal ganglion cells, scRNA-seq


title: LTHSC_Vav1.Cre_1

description: LTHSC, Vav1.Cre, replicate 1


title: Input_H3K27ac_Microglia_C57_Male_Adult_Basal_PND65_rep2

description: Input for ChIP_H3K27ac_Microglia_C57_Male_Adult_Basal_PND60_rep2


title: RGC141

description: Retinal ganglion cells, scRNA-seq


title: LTHSC_Scl.N1_4

description: LTHSC, Scl.N1, replicate 4

value: Scl.Cre Exon12 (E12)


title: RGC142

description: Retinal ganglion cells, scRNA-seq


title: LTHSC_Scl.N1_2

description: LTHSC, Scl.N1, replicate 2


title: ChIP_H3K27ac_Microglia_C57_Male_Adult_Basal_PND65_rep2


title: Input_H3K27ac_and_Rpb2_Microglia_C57_Male_Adult_Basal_PND60_rep1

description: Input for ChIP_H3K27ac_Microglia_C57_Male_Adult_Basal_PND60_rep1 AND ChIP_Rpb2_Microglia_C57_Male_Adult_Basal_PND60_rep1


title: RGC143

description: Retinal ganglion cells, scRNA-seq


title: LTHSC_Scl.N1_11

description: LTHSC, Scl.N1, replicate 11


title: LTHSC_Scl.Cre.mJak2.KI_3

description: LTHSC, Scl.Cre.mJak2.KI, replicate 3


title: RGC148

description: Retinal ganglion cells, scRNA-seq


title: LTHSC_Vav1.Cre_2

description: LTHSC, Vav1.Cre, replicate 2

value: Vav1.Cre V617F (VF)


title: PFBA_4ppb_Rep4

type: RNA

city: West Lafayette

first: Jennifer

last: Freeman

state: IN

address: 550 Stadium Mall Drive

department: School of Health Sciences

institute: Purdue University

description: Gene expression 72 hpf following embryonic exposure (1-72 hpf)

hyb_protocol: 1.65 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55mL containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 55 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent custom zebrafish 4X180K Microarrays (060509) for 17 hours at 65°C in a rotating Agilent hybridization oven.  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).

scan_protocol: Arrays were scanned on an Agilent Technologies SureScan Microarray Scanner (Agilent Technologies, Santa Clara, CA).

data_processing: Array image data was extracted using Agilent Feature Extraction Software 12.0 (Agilent Technologies, Santa Clara, CA). Array image data was then uploaded to GeneSpring 14.9 (Agilent Technologies, Santa Clara, CA) for statistical analysis. Each gene list was imported into Ingenuity Pathway Analysis (IPA) for gene ontology and molecular pathway analysis.

organism: Danio rerio

source_name: whole fish

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared using the One-Color Low Input QuickAmp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAEasy column purification (QIAGEN, Valencia, CA).  Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

growth_protocol: Zebrafish (wild-type AB strain) were housed in a Z-Mod System (Aquatic Habitats, Apopka, FL) on a 14:10 hour light:dark cycle and maintained at 28ºC ± 1°C with a pH of 7.0-7.2 and conductivity range of 500-550 µS/cm.

extract_protocol: Total RNA was isolated using an RNeasy Mini Kit (Qiagen Sciences, Maryland, USA) following established protocols. RNA concentrations were measured by using a NanoDrop 1000 spectrophotometer and the quality was verified by absorbance ratio 260/280 nm.

treatment_protocol: Zebrafish embryos were exposed to 0, 4, 40, or 400 ppb PFBA from approximately 1 hpf through 72 hpf. Zebrafish were rinsed at 72 hpf, homogenized in Trizol (Life Technolgies, Carlsbad, CA) and flash frozen in liquid nitrogen and stored at -80°C until RNA extraction. Each replicate represents 45-50 pooled zebrafish aged 72 hpf and four biological replicates were completed (n=4).

tag: developmental stage

value: 72 hpf

value: whole fish

tag: dosage

value: 4 ppb


title: LTHSC_Scl.Cre.mJak2.KI_2

description: LTHSC, Scl.Cre.mJak2.KI, replicate 2

value: Bone marrow


title: ChIP_H3K27ac_Microglia_C57_Male_Adult_Basal_PND60_rep1

value: Ki67-Negative microglia

tag: chip antibody


title: RNA_dUTP_Microglia_Ki67Positive_Ki67rfp_Male_PND75_PLX5622_17d_Recovery_3d_rep2

description:
RNA-seq, dUTP, stranded
Microglia_RNA_dUTP_rawdata.txt
Microglia_RNA_dUTP_TPMnormalization.txt

molecule: polyA RNA

value: Starting at 55 days of age, PLX5622 diet for 17 days; Recovery for 3 days


title: LTHSC_Scl.Cre.mJak2.KI_1

description: LTHSC, Scl.Cre.mJak2.KI, replicate 1

tag: cell population


title: PFBA_400ppb_Rep4

value: 400 ppb


title: RGC111

description: Retinal ganglion cells, scRNA-seq

value: Retina

value: 7-9 weeks old


title: PFBA_4ppb_Rep3


title: PFBA_0ppb_Rep3

value: 0 ppb


title: PFBA_0ppb_Rep4


title: PFBA_40ppb_Rep3

value: 40 ppb


title: PFBA_40ppb_Rep4


title: RGC149

description: Retinal ganglion cells, scRNA-seq


title: RGC145

description: Retinal ganglion cells, scRNA-seq


title: RNA_dUTP_Microglia_Ki67Positive_Ki67rfp_Male_PND75_PLX5622_17d_Recovery_3d_rep1

value: Ki67-Positive microglia

value: Male


title: RGC147

description: Retinal ganglion cells, scRNA-seq

value: Retinal ganglion cells


title: RNA_dUTP_Microglia_Ki67Negative_Ki67rfp_Male_PND75_PLX5622_17d_Recovery_3d_rep2


title: R18057365-SAN-GFP-

city: Shanghai

first: yiwen

last: li

address: Yueyang road No.320

institute: Chinese Academy of Sciences

description: single-cell RNA-seq

data_processing:
Illumina software used for basecalling.
Cellranger R kit used for data normalization.
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: Matrix table with UMI for every gene and every sample. *-barcodes-*.tsv, *-genes-*.tsv, *-matrix-*.mtx files.

source_name: Sinoatrial node region from P2 mice

extract_protocol:
10X Genomics kit was used for the construction of single-cell RNA-sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: ICR

value: Sinoatrial node region

value: HCN4-GFP- FACS sorted


title: R18057364-SAN-GFP+

description: single-cell RNA-seq

value: HCN4-GFP+ FACS sorted


title: PFBA_400ppb_Rep3

value: 72 hpf

value: whole fish


title: RGC144

description: Retinal ganglion cells, scRNA-seq


title: RGC146

description: Retinal ganglion cells, scRNA-seq


title: RNA_dUTP_Microglia_Ki67Positive_Ki67rfp_Male_PND8_rep2


title: RNA_dUTP_Microglia_Ki67Positive_Ki67rfp_Male_PND8_rep1


title: R18057363-RA

description: single-cell RNA-seq

value: Unsorted


title: RGC156

description: Retinal ganglion cells, scRNA-seq


title: RNA_dUTP_Microglia_Ki67Negative_Ki67rfp_Male_PND75_PLX5622_17d_Recovery_3d_rep1

value: Ki67-Negative microglia


title: RGC150

description: Retinal ganglion cells, scRNA-seq


title: RGC155

description: Retinal ganglion cells, scRNA-seq


title: PFBA_400ppb_Rep2


title: RNA_dUTP_Microglia_Ki67Negative_Ki67rfp_Male_PND8_rep2


title: APHSpleen_INVEN_Ter119_RNASEQ_2

city: Oxford

first: Mira

middle: Tony

last: Kassouf

state: England

address: John Radcliffe Hospital, Headly way

department: Weatherall Institute of Molecular Medicine

country: United Kingdom

institute: University of Oxford

data_processing:
Mapping: STAR
Duplicate removal: samtools rmdup
Assembly: mm9
Supplementary files format and content: text file, genes as feature counts for number of reads

source_name: primary erythroid cells

growth_protocol: Primary Ter119+ erythroid cells were obtained from the spleens of adult mice that were treated with phenylhydrazine (APH). Spleens were mechanically dissociated into single cell suspensions in cold phosphate-buffered saline (PBS; Gibco: 10010023)/10% fetal bovine serum (FBS; Gibco: 10270106) and passed through a 70 μm filter to remove clumps.

extract_protocol:
Cells were washed with cold PBS/10% FBS and resuspended in 10 μl of cold PBS/10% FBS per 106 cells and stained with a 1/100 dilution of anti-Ter119-PE antibody (BD Pharmingen 553673 2 μg/ml) at 4 °C for 20 minutes. Stained cells were washed with cold PBS/10% FBS and resuspended in 8 μl of cold PBS/0.5% BSA/2 mM EDTA and 2 μl of anti-PE MACS microbeads (Miltenyi Biotec: 130-048-801) per 106 cells and incubated at 4 °C for 15 minutes. Ter119+ cells were positively selected via MACS lineage selection (LS) columns (Miltenyi Biotec: 130-042-401) and processed for downstream applications. Purity of the isolated erythroid cells was routinely verified by Fluorescence-Activated Cell Sorting (FACS).
Total RNA was isolated from 105-2x106 Ter119+ erythroid cells lysed in TRI reagent (Sigma-Aldrich: T9424) using a Direct-zol RNA MiniPrep kit (Zymo Research: R2050). DNase I treatment was performed on the column as recommended in the manufacturer’s instructions but with an increased incubation of 30 min at room temperature (rather than 15 min).
For RNA-seq libraries, 1-2 μg of total RNA was depleted of rRNA and globin mRNA using the Globin-Zero Gold rRNA Removal Kit (Illumina: GZG1224) according to the manufacturer’s instructions. To enrich for mRNA, poly(A)+ RNA was isolated, strand-specific cDNA was synthesised, and the resulting libraries prepared for Illumina sequencing using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs: E7490) and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs: E7760) following the manufacturer’s instructions. Poly(A)+ and Poly(A)- RNA-seq libraries were sequenced on the Illumina Nextseq platform using a 75-cycle paired-end kit (NextSeq 500/550 High Output Kit v2.5: 20024906).

value: primary erythroid cells

tag: group

value: INVEN

tag: source

value: APH-treated ter119+ spleen cells


title: APHSpleen_INVEN_Ter119_RNASEQ_3


title: APHSpleen_WT_Ter119_RNASEQ_3

value: WT


title: PFBA_40ppb_Rep2


title: APHSpleen_INVEN_Ter119_RNASEQ_1


title: RNA_dUTP_Microglia_Ki67Negative_Ki67rfp_Male_PND8_rep1


title: RGC151

description: Retinal ganglion cells, scRNA-seq

value: Retina

value: 7-9 weeks old


title: PFBA_4ppb_Rep2

value: 4 ppb


title: RGC152

description: Retinal ganglion cells, scRNA-seq


title: RNA_dUTP_Microglia_C57_Male_Adult_Basal_PND90_rep2

value: Male


title: PFBA_400ppb_Rep1

value: 400 ppb


title: RNA_dUTP_Microglia_C57_Male_Adult_Basal_PND90_rep1


title: RGC157

description: Retinal ganglion cells, scRNA-seq

value: Retinal ganglion cells


title: PFBA_4ppb_Rep1


title: RGC153

description: Retinal ganglion cells, scRNA-seq


title: APHSpleen_WT_Ter119_RNASEQ_2


title: PFBA_40ppb_Rep1

value: 40 ppb


title: APHSpleen_WT_Ter119_RNASEQ_1


title: RGC154

description: Retinal ganglion cells, scRNA-seq


title: RGC163

description: Retinal ganglion cells, scRNA-seq


title: SUBJECT 10 Timepoint 2

city: Örebro

first: Robert-Jan

last: Brummer

address: Södra Grev Rosengatan 42 A

department: School of Medical Sciences

country: Sweden

institute: Örebro University

hyb_protocol: Affymetrix GeneChip 3000 7G

scan_protocol: Affymetrix GeneChip 3000 7G

data_processing: R Bioconductor “oligo” and “pd.hugene.1.1.st.v1” packages

label: biotin

source_name: biopsy

label_protocol: Ambion MessageAmp IIbiotin enhanced singleround amplification kit

growth_protocol: Healthy human study participants

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: Perfusion with Akkermansia muciniphila or placebo

tag: subject

tag: time

value: Placebo


title: RGC159

description: Retinal ganglion cells, scRNA-seq


title: SUBJECT 10 Timepoint 3


title: SUBJECT 09 Timepoint 3

value: Akkermansia


title: APHSpleen_INVEN_Ter119_H3K4me3_3

description: Genome-wide library of ChIPed chromatin

data_processing:
Trim_galore (to remove sequencing adaptors)
FLASH (to reconstruct paired end reads into single reads where possible)
Alignment to the genome (Bowtie) maintaining strict read order
Removal of PCR duplicates;
Combining data from multiple replicates (custom scripts)
Removal of ploidy regions
Assembly: mm9
Supplementary files format and content: BigWig

source_name: mouse derived Erythroid cells

extract_protocol:
To produce CD71+ erythroid cells in vitro from differentiated mESCs (WT or mutant): 24h prior to differentiation, mESCs were induced by passaging into IMDM base media, trypsinised and plated in differentiation media (IMDM suplemented serum and other reagents and growth factors) in either triple vent petri dishes (Thermo Fisher) or flat-bottom 96-well plates (Thermo Fisher) at 3x104 cells in 10 cm dishes or 250 cells per well of a 96-well plate for up to seven days without disruption. EBs were harvested and disaggregated in 0.25% trypsin for 3 minutes at 37°C. CD71-positive cells were isolated by magnetic column separation (LS Column, Miltenyi), according to the manufacturer’s instructions. Briefly, cells (from disaggregated EBs) were labelled with anti-mouse CD71-FITC (eBioscience 11-0711-85; 1:200) in staining buffer (PBS with 10% FCS; 500 ul per 10^7 cells) for 20 minutes at 4°C, washed, then incubated with MACS anti-FITC separation microbeads (Miltenyi; 10 ul  per 10^7 cells, following the manufacturer’s instructions) and bead-labelled cells were retained by LS columns. Cells were then processed for ChIP-Seq as in Hay D et al, Nature Genetics, 2016.
Cells were fixed with 1% Formaldehyde, lysed, sonicated and immunoprecipitated with the antibody of interest (CTCF: Merck Millipore 07-729 at 5ug/ml, H3K27me3: Cell Signaling 9733 at 2ug/ml, H3K4me3: Abcam 8580 at 2.5ug/ml). Libraries were prepared using the NEB Ultra II library preparation kit according to manufacturer’s instructions. Samples were sequenced using 40 bp paired end reads on the NextSeq platform.
The strategy was to characterise the chromatin state of primary mouse-derived or the CD71+ erythroid cells (WT and mutant) derived from mESC-EB in vitro differentitation system by isolating and analysing DNA regions bound by the antibodies of interest, genome-wide.

value: Primary erythroid cells derived from spleen from INVEN APH-treated mice are disaggregated and cells stained with ter119-PE antibody (BD Biosciences at a working concentration of 2ug/ml) then incubated with anti-PE MACS beads (Miltenyi) and purified using LS columns (Miltenyi), all following manufacturor's instructions.

value: H3K4me3 Abcam 8580 2.5ug/ml

value: Genome-wide

value: Ter119+  erythroid cells purified from the spleen of a INVEN Phenylhydrazine APH-treated mice, sample 2


title: RGC158

description: Retinal ganglion cells, scRNA-seq


title: APHSpleen_INVEN_Ter119_H3K4me3_2

description: Genome-wide library of ChIPed chromatin


title: APHSpleen_INVEN_Ter119_H3K27me3_3

description: Genome-wide library of ChIPed chromatin

value: H3K27me3 Cell Signaling 9733 2ug/ml


title: APHSpleen_INVEN_Ter119_H3K27me3_1

description: Genome-wide library of ChIPed chromatin

value: Ter119+  erythroid cells purified from the spleen of an INVEN Phenylhydrazine APH-treated mice, sample 1


title: SUBJECT 09 Timepoint 1


title: PFHXA_4ppb_Rep4

treatment_protocol: Zebrafish embryos were exposed to 0, 4, 40, or 400 ppb PFbA from approximately 1 hpf through 72 hpf. Zebrafish were rinsed at 72 hpf, homogenized in Trizol (Life Technolgies, Carlsbad, CA) and flash frozen in liquid nitrogen and stored at -80°C until RNA extraction. Each replicate represents 45-50 pooled zebrafish aged 72 hpf and four biological replicates were completed (n=4).

value: 72 hpf

value: whole fish


title: PFBA_0ppb_Rep1

value: 0 ppb


title: RGC160

description: Retinal ganglion cells, scRNA-seq

value: Retina

value: 7-9 weeks old


title: SUBJECT 09 Timepoint 2


title: RGC161

description: Retinal ganglion cells, scRNA-seq


title: APHSpleen_INVEN_Ter119_H3K27me3_2

description: Genome-wide library of ChIPed chromatin


title: PFHXA_40ppb_Rep4


title: PFHXA_400ppb_Rep4


title: RGC162

description: Retinal ganglion cells, scRNA-seq


title: APHSpleen_INVEN_Ter119_CTCF_2

description: Genome-wide library of ChIPed chromatin

value: CTCF:Merck Millipore 07-729 at 5ug/ml


title: RGC292

description: Retinal ganglion cells, scRNA-seq


title: APHSpleen_INVEN_Ter119_CTCF_1

description: Genome-wide library of ChIPed chromatin


title: RGC001

description: Retinal ganglion cells, scRNA-seq


title: EB_CD71_INVEN_KO_CTCF_2

description: Genome-wide library of ChIPed chromatin

source_name: E14TG2a.IV mES derived Erythroid cells

value: CD71+ erythroid cells derived from differentiated INVEN mES cells

value: CD71+ eryrthoid cells, in vitro derived from INVEN KO mESCs, sample 2 (INVEN KO:a combination of INVEN and CTCF KO)


title: RGC002

description: Retinal ganglion cells, scRNA-seq

value: Retinal ganglion cells


title: RGC003

description: Retinal ganglion cells, scRNA-seq


title: SUBJECT 10 Timepoint 1

value: Placebo


title: PFHXA_400ppb_Rep3

value: 400 ppb


title: PFHXA_0ppb_Rep4


title: PFHXA_40ppb_Rep3

value: 40 ppb


title: SUBJECT 08 Timepoint 1


title: EB_CD71_CTCF_KO_CTCF_2

description: Genome-wide library of ChIPed chromatin

value: CD71+ eryrthoid cells, in vitro derived from CTCF KO mESCs, sample 2  (CTCF KO:homozygous for the deletion of the alpha globin 5' boundary elements, created by mutating two CTCF-binding sites HS38 and HS39.5)


title: EB_CD71_INVEN_KO_CTCF_1

description: Genome-wide library of ChIPed chromatin

value: Genome-wide

value: CD71+ eryrthoid cells, in vitro derived from INVEN KO mESCs, sample 1 (INVEN KO:a combination of INVEN and CTCF KO)


title: SUBJECT 07 Timepoint 2

value: Akkermansia


title: PFHXA_4ppb_Rep3

value: 4 ppb


title: SUBJECT 08 Timepoint 3


title: PFHXA_0ppb_Rep3

value: 72 hpf


title: EB_CD71_CTCF_KO_CTCF_1

description: Genome-wide library of ChIPed chromatin

value: CD71+ eryrthoid cells, in vitro derived from CTCF KO mESCs, sample 1 (CTCF KO:homozygous for the deletion of the alpha globin 5' boundary elements, created by mutating two CTCF-binding sites HS38 and HS39.5)


title: PFHXA_40ppb_Rep2

value: whole fish


title: SUBJECT 08 Timepoint 2


title: PFHXA_400ppb_Rep2


title: aCD4_tumor_5

city: Nedlands

first: Caitlin

middle: M

last: Tilsed

state: WA

address: 6 Verdun St

country: Australia

institute: NCARD,UWA

data_processing:
Image analysis was performed in real time by the NovaSeq Control Software (NCS) v1.7.0 and Real Time Analysis (RTA) v3.4.4, running on the instrument computer. RTA performs real-time base calling on the NovaSeq instrument computer. Then the Illumina bcl2fastq 2.20.0.422 pipeline was used to generate the sequence data.
read alignment with Kallisto
count quantification with tximport
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: count matrix with counts generated from txi import on kallisto aligned samples

source_name: AB1-HA tumor

growth_protocol: subcutaneous tumor growth

extract_protocol:
Sample homogenization using QIAGEN tissue rupture and RNA extraction using QIAGEN RNeasy Plus Mini Kit
Human, Mouse, Rat, Bacteria, Globin RNA depletion + NovaSeq 100bp PE

treatment_protocol: 100ug anti-CD4 or PBS day -3, day 0, day 7 with respect to tumor innoculation.

value: tumor

value: BALB/c

value: 8-10 weeks

tag: gender

value: m


title: EB_CD71_INVEN_CTCF_2

description: Genome-wide library of ChIPed chromatin

value: CD71+ erythroid cells derived from differentiated INVEN mES cells

tag: strain

value: CTCF:Merck Millipore 07-729 at 5ug/ml

value: CD71+ eryrthoid cells, in vitro derived from INVEN mESCs, sample 2 (INVEN:homozygous for the inversion of the alpha globin super-enhancer and encompassing both Nprl3 and Mpg genes, ~48kb)


title: SUBJECT 07 Timepoint 1


title: PFHXA_0ppb_Rep2

value: 0 ppb


title: aCD4_tumor_3


title: SUBJECT 06 Timepoint 3

value: Placebo


title: PFHXA_4ppb_Rep2


title: aCD4_tumor_4


title: PFHXA_400ppb_Rep1

value: 400 ppb


title: PFHXA_40ppb_Rep1

value: 40 ppb


title: SUBJECT 07 Timepoint 3


title: EB_CD71_INVEN_CTCF_1

description: Genome-wide library of ChIPed chromatin

value: Genome-wide

value: CD71+ eryrthoid cells, in vitro derived from INVEN mESCs, sample 1 (INVEN:homozygous for the inversion of the alpha globin super-enhancer and encompassing both Nprl3 and Mpg genes, ~48kb)


title: aCD4_tumor_2


title: INVEN_APHSpleen_Promoter_Capture_Sample 3

description: sequences enriched from a 3C library for their interaction with the Capture points in promoters of all 42 genes within 5.6Mb around the Alpha Globin locus

data_processing:
Trim_galore (to remove sequencing adaptors)
FLASH (to reconstruct paired end reads into single reads where possible)
In silico restriction enzyme digestion of FASTQ file (dpnIIPE.pl)
Alignment to the genome (Bowtie) maintaining strict read order
Removal of PCR duplicates; Parsing of informative reads and mapping to restriction enzyme fragments (CCanalyser2.pl)
Combining data from multiple replicates (custom scripts)
Removal of ploidy regions and off target capture (custom scripts)
Analysis in R to normalise between tracks using the total number of informative interactions
Assembly: mm9
Supplementary files format and content: Custom combined data format (restriction enzyme fragment \t sample 1 \t sample 2 \t etc.)
Library strategy: Capture-C

extract_protocol:
To produce CD71+ erythroid cells in vitro from differentiated mESCs (WT or mutant mouse ESC line): 24h prior to differentiation, mESCs were induced by passaging into IMDM base media, trypsinised and plated in differentiation media (IMDM suplemented serum and other reagents and growth factors) in either triple vent petri dishes (Thermo Fisher) or flat-bottom 96-well plates (Thermo Fisher) at 3x104 cells in 10 cm dishes or 250 cells per well of a 96-well plate for up to seven days without disruption. EBs were harvested and disaggregated in 0.25% trypsin for 3 minutes at 37°C. CD71-positive cells were isolated by magnetic column separation (LS Column, Miltenyi), according to the manufacturer’s instructions. Briefly, cells (from disaggregated EBs) were labelled with anti-mouse CD71-FITC (eBioscience 11-0711-85; 1:200) in staining buffer (PBS with 10% FCS; 500 ul per 10^7 cells) for 20 minutes at 4°C, washed, then incubated with MACS anti-FITC separation microbeads (Miltenyi; 10 ul  per 10^7 cells, following the manufacturer’s instructions) and bead-labelled cells were retained by LS columns. Cells were then processed for Capture-C as in Davies J et al, Nature Methods.
The protocol used for generation of the 3C libraries was similar to previous methods with minor modifications. Briefly, cells were crosslinked with 2% formaldehyde (10 minutes, room temperature); quenched with cold glycine; washed in phosphate buffered saline; resuspended in cold lysis buffer (tris 10mM, NaCl 10mM, NP40 0.2%, complete proteinase inhibitor (Roche)) and snap frozen to -80.  Cells were thawed on ice, washed in milliq dH2O and Dounce homogenised on ice (x 40 strokes). Cells were then resuspended with 0.25% SDS and restriction enzyme buffer and incubated at 37C for 1h at 1400rpm on a Comfort Thermomixer (Eppendorf) followed by a further incubation of 1h following the addition of triton X100 (final concentration 1.67%). An overnight digestion was performed using Dpn II (500U /ml (NEB) at 37C / 1400 rpm). The digested chromatin was ligated overnight (Fermentas HC Ligase final concentration 10U/ml) at 16 degrees at 1400 rpm on the Thermomixer.  The samples were then decrosslinked overnight at 65C with Proteinase K (Roche) followed by a 30 min incubation at 37C with RNAse (Roche). Phenol/Chloroform extraction was then performed followed by an Ethanol precipitation and a wash with 70% Ethanol. Digestion efficiencys were assessed by gel electrophoresis (1% agarose) and RT-PCR (Taqman), which showed digestion efficiencies in excess of 80%.  DNA content of the Dpn II 3C libraries were quantitated using a Qubit fluorometer (Life technologies) and 5-10ug of each library was sheared using a Covaris S2 in milliq dH2O. Covaris settings used were: duty cycle 10%, Intensity 5, Cycles/burst 200, Time 6 cycles of 60seconds, Set Mode Frequency sweeping Temperature 4 to 7 degrees. Following shearing DNA was purified using AMPureXP beads (Agencourt) and DNA quality assessed on a Bioanalyser 2100 using a DNA High Sensitivity Chip (Agilent). DNA end repair and adapter ligation was performed using the NEB Next or NEB Ultra II DNA sample preparation reagent kits, depending on the amount of DNA available, using the standard protocol.  Biotinylated capture oligonucleotides were designed to the ends of the viewpoint fragments.  Where possible 1-2ug of each adapter ligated library were hybridized with the biotinylated capture oligonucleotides, using  the Nimblegen SeqCap reagents and an adapted protocol.  The quality of the resultant captured library was assessed by Agilent tapestation or bioanalyser (D1000). The resulting libraries were sequenced using Illumina Next-Seq.
The experimental strategy was to generate very high complexity randomly sonicated 3C libraries with ligated Illumina sequencing adaptors (ideally with several hundred thousand fold coverage).  Oligonucleotide capture technology was then used to pull down the restricion fragments containing the viewpoints of interest and their interacting partners, which were subsequently idendified by high throughput sequencing.  The power of the technique is that multiple samples and genes can be analysed simultaneously by using differently indexed samples as we did with all the samples and most prominently with the erythroid SE (183 viewpoints in 6 samples) and promoter (42 viewpoints in 6 samples) captures

value: Ter119+  erythroid cells purified from the spleen of a WT Phenylhydrazine APH-treated mice, sample 3

tag: protocol

value: As detailed in Protocols below

tag: analysis


title: INVEN_APHSpleen_Promoter_Capture_Sample 2

value: Ter119+  erythroid cells purified from the spleen of a WT Phenylhydrazine APH-treated mice, sample 2


title: aCD4_tumor_1


title: SUBJECT 06 Timepoint 1


title: SUBJECT 05 Timepoint 2

value: Akkermansia


title: SUBJECT 06 Timepoint 2


title: PFHXA_0ppb_Rep1

value: 72 hpf

value: whole fish


title: INVEN_APHSpleen_Promoter_Capture_Sample 1

value: Ter119+  erythroid cells purified from the spleen of a WT Phenylhydrazine APH-treated mice, sample 1


title: PBS_tumor_6

value: tumor

value: BALB/c

value: 8-10 weeks

value: m

value: PBS


title: PFHXA_4ppb_Rep1

value: 4 ppb


title: SUBJECT 05 Timepoint 3


title: PBS_tumor_5


title: WT_APHSpleen_Promoter_Capture_Sample 2


title: WT_APHSpleen_Promoter_Capture_Sample 3


title: PBS_tumor_4


title: PFOA_40ppb_Rep4

treatment_protocol: Zebrafish embryos were exposed to 0, 4, 40, or 400 ppb PFOA from approximately 1 hpf through 72 hpf. Zebrafish were rinsed at 72 hpf, homogenized in Trizol (Life Technolgies, Carlsbad, CA) and flash frozen in liquid nitrogen and stored at -80°C until RNA extraction. Each replicate represents 45-50 pooled zebrafish aged 72 hpf and four biological replicates were completed (n=4).


title: PFOA_400ppb_Rep4

value: 400 ppb


title: SUBJECT 04 Timepoint 3


title: WT_APHSpleen_Promoter_Capture_Sample 1


title: PFOA_0ppb_Rep4

value: 0 ppb


title: PFOA_4ppb_Rep4


title: PFBA_0ppb_Rep2


title: PBS_tumor_3


title: INVEN EBd7 CD71+ R1 Capture sample 2 (INVEN_EB19_Enhancers)

description: sequences enriched from a 3C library for their interaction with the Capture point in the Alpha Globin locus as detailed in the sample name

value: Chromatin from purified CD71+ erythroid cells derived from differentiated WT mES cells, Rhbdf1 and Mpg promoters as Capture point, sample 2

value: Alpha Globin Locus


title: PBS_tumor_2


title: INVEN EBd7 CD71+ R1 Capture sample 3 (INVEN_EB22_Enhancers)

value: Chromatin from purified CD71+ erythroid cells derived from differentiated WT mES cells, Rhbdf1 and Mpg promoters as Capture point, sample 3


title: INVEN EBd7 CD71+ R1 Capture sample 1(INVEN_EB10_Enhancers)

value: Chromatin from purified CD71+ erythroid cells derived from differentiated WT mES cells, Rhbdf1 and Mpg promoters as Capture point, sample 1

value: As detailed in Protocols below


title: PBS_tumor_1


title: S6_RNAseq_KO_rep3

city: Houston

first: Yejing

last: Ge

state: Texas

department: Cancer Biology

institute: The University of Texas, MD Anderson Cancer Center

description: Sample 6

data_processing:
For Sample 1-6, raw sequencing 150-bp paired-end reads were aligned to the Mus musculus reference genome (version mm10 from Genome Reference Consortium GRCm38) using STAR 2.6.0b aligner. Aligned reads were quantified against the reference annotation (GRCm38) to obtain RPKM (transcripts per million reads) and raw counts using  HTSeq (python/3.6/anaconda3/bin/htseq-count, with options -m union -a 10 -s  no), respectively.
For Sample 7-10, libraries were sequenced using PE150 on a HiSeq4000 lane with about 25-30M reads output per sample and trimmed to obtain SE50 reads and demultiplexed. Raw sequencing 50-bp single-end reads were aligned to the Mus musculus reference genome (version mm10 from Genome Reference Consortium GRCm38) or the Homo sapiens reference genome (version hg19 from Genome Reference Consortium GRCh37) for mouse and human samples, respectively, using the Bowtie (https://sourceforge.net/projects/bowtie-bio/files/bowtie/1.0.1/) pipeline with ( -n 1 -m 1 --best --strata ). Unique reads mapped to a single genomic location (allowing default mismatchesr) were kept for peak identification using the Model based Analysis of Chip-seq 2 (MACS2) (Zhang et al., 2008) pipeline with  parameters ( --nomodel).
For Sample 11, libraries were sequenced using pair end PE150 on a HiSeq4000 lane with about 50M reads output per sample and trimmed to obtain single end SE50 reads and demultiplexed. Raw sequencing 50-bp single end reads were aligned to the genomic builder for Mus musculus (version mm10 from Genome Reference Consortium GRCm38) as previously described (Buenrostro et al., 2013). Briefly, mapping was performed using Bowtie package (https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.3/) with the default parameters. Unique reads mapped to a single genomic location (allowing default mismatches) were kept for peak identification using the Model based Analysis of Chip-seq 2 (MACS2) (Zhang et al., 2008) pipeline with parameters (--nomodel).
Genome_build: hg19 (GRCh37), mm10 (GRCm38)
Supplementary_files_format_and_content: Sample 1-6: *.star.ensembl.htseqcount.gene.txt: tab-delimited text files, gene counts.
Supplementary_files_format_and_content: Sample 7-10: *.macs2_peaks.narrowPeak: bed files, ChIP peaks.
Supplementary_files_format_and_content: Sample 11: *.macs2_peaks.narrowPeak: bed files, ATAC peaks.

source_name: skin

growth_protocol: E17.5 mice skin epithelium or HaCaT cells.

extract_protocol:
For Sample 1-8 and Sample 11, skin was removed and digested with dispase. Then epidermis was peeled from dermis, treated with trypsin and purified based on sort markers indicated.
For Sample 9 and Sample 10, HaCaT cells were treated with trypsin and collected to do ChIP-Seq.
For RNAseq, total RNAs were submitted to Novogene for quality control and polyA enriched eukaryotic mRNA-seq library preparation. For ChIP-Seq and ATAC-Seq, after tagmentation reactions with TnD transposase, libraries that passed QC were then pooled and sequenced.

tag: cell type

value: embryonic skin epithelia

tag: genotype

tag: sort markers

value: DAPI-, CD45- and Integrin A6+


title: SUBJECT 04 Timepoint 2

tag: treatment

value: Akkermansia


title: SUBJECT 03 Timepoint 3


title: SUBJECT 05 Timepoint 1


title: SUBJECT 03 Timepoint 2


title: S5_RNAseq_Het_rep3

description: Sample 5


title: PFOA_400ppb_Rep3

value: 72 hpf

tag: tissue

value: whole fish


title: SUBJECT 04 Timepoint 1


title: WT EBd7 CD71+ R1 Capture sample2 (WT_EB9_Enhancers)

value: Chromatin from purified CD71+ erythroid cells derived from differentiated WT mES cells, alpha globin enhancer sequence R1 as Capture point, sample 2


title: PFOA_4ppb_Rep3

tag: developmental stage


title: PFOA_400ppb_Rep2

tag: dosage


title: SUBJECT 03 Timepoint 1


title: S4_RNAseq_KO_rep2

description: Sample 4


title: WT EBd7 CD71+ R1 Capture sample1 (WT_EB8_Enhancers)

value: Chromatin from purified CD71+ erythroid cells derived from differentiated WT mES cells, alpha globin enhancer sequence R1 as Capture point, sample 1


title: APHSpleen_INVEN_Ter119_ATAC_3

description: Genome-wide library of transposed elements

data_processing:
Trim_galore (to remove sequencing adaptors)
Alignment to the genome Bowtie 1 standard parameters with -m set to 2
Removal of PCR duplicates;
plot density of alignments in a moving 300bp window with an moving increment of 30 bp
Assembly: mm9
Supplementary files format and content: BigWig

extract_protocol:
Spleen from WT APH-treated mice are disaggregated and cells stained with ter119-PE antibody then incubated with anti-PE MACS beads (Miltenyi) and purified using LS columns (Miltenyi), all following manufacturor's instructions.
To produce CD71+ erythroid cells in vitro from differentiated mESCs (WT or mutant): 24h prior to differentiation, mESCs were induced by passaging into IMDM base media, trypsinised and plated in differentiation media (IMDM suplemented serum and other reagents and growth factors) in either triple vent petri dishes (Thermo Fisher) or flat-bottom 96-well plates (Thermo Fisher) at 3x104 cells in 10 cm dishes or 250 cells per well of a 96-well plate for up to seven days without disruption. EBs were harvested and disaggregated in 0.25% trypsin for 3 minutes at 37°C. CD71-positive cells were isolated by magnetic column separation (LS Column, Miltenyi), according to the manufacturer’s instructions. Briefly, cells (from disaggregated EBs) were labelled with anti-mouse CD71-FITC (eBioscience 11-0711-85; 1:200) in staining buffer (PBS with 10% FCS; 500 ul per 10^7 cells) for 20 minutes at 4°C, washed, then incubated with MACS anti-FITC separation microbeads (Miltenyi; 10 ul  per 10^7 cells, following the manufacturer’s instructions) and bead-labelled cells were retained by LS columns. Cells were then processed for ATAC-Seq as in (Buenrostro et al, 2013).
Assay for transposition of active chromatin sequencing (ATAC-Seq) was performed as previously published (Buenrostro, 2013). 60000-80000 cells were used per biological replicate. Cells were lysed and nuclei were isolated prior to transposition with Tn5 transposase (Nextera, Illumina) for 30 minutes at 37°C. DNA was purified using a MinElute kit (Qiagen). Libraries were amplified and barcoded using the NEBNext 2xMastermix (NEB) and the custom primers as published in Buenrostro et al., 2013. ATAC-Seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent). Due to the broad range of DNA fragment sizes found in these libraries, quantitation with the Qubit for DNA concentration was found to be highly variable and was omitted. The libraries were quantified using the universal library quantification kit (KAPA Biosystems). Samples were sequenced using 40 bp paired end reads (NextSeq platform).
The strategy was to survey regions of open chromatin in our cell population of interest (primary mouse derived erythroid cells or CD71+ EB-derived cells) as they mark regulatory genomic regions. The modified Tn5 can in one step identify open chromatin fragments, cut, and ligate adaptors. The fragments can then be multiplexed and sequenced

value: Ter119+  erythroid cells purified from the spleen of a WT Phenylhydrazine APH-treated mice, sample 3


title: PFOA_0ppb_Rep3


title: S3_RNAseq_KO_rep1

description: Sample 3


title: WT EBd7 CD71+ R1 Capture sample3 (WT_EB11_Enhancers)

value: Chromatin from purified CD71+ erythroid cells derived from differentiated WT mES cells, alpha globin enhancer sequence R1 as Capture point, sample 3


title: SUBJECT 02 Timepoint 3


title: SUBJECT 02 Timepoint 2


title: PFOA_40ppb_Rep3

value: 40 ppb


title: PFOA_40ppb_Rep2


title: S2_RNAseq_Het_rep2

description: Sample 2


title: APHSpleen_INVEN_Ter119_ATAC_1

value: Ter119+  erythroid cells purified from the spleen of a WT Phenylhydrazine APH-treated mice, sample 2


title: SUBJECT 01 Timepoint 2


title: APHSpleen_INVEN_Ter119_H3K4me3_1

description: Genome-wide library of ChIPed chromatin

value: Primary erythroid cells derived from spleen from INVEN APH-treated mice are disaggregated and cells stained with ter119-PE antibody (BD Biosciences at a working concentration of 2ug/ml) then incubated with anti-PE MACS beads (Miltenyi) and purified using LS columns (Miltenyi), all following manufacturor's instructions.

tag: chip antibody

value: H3K4me3 Abcam 8580 2.5ug/ml

value: Genome-wide

value: Ter119+  erythroid cells purified from the spleen of an INVEN Phenylhydrazine APH-treated mice, sample 1


title: SUBJECT 01 Timepoint 3


title: PFOA_0ppb_Rep2

value: 0 ppb


title: PFOA_4ppb_Rep2

value: 4 ppb


title: S1_RNAseq_Het_rep1

description: Sample 1

value: embryonic skin epithelia


title: RNA_2019_03_22_AdNP_LungRes_69plus_1_month

status: Public on Aug 17 2022

city: Atlanta

first: Chris

last: Scharer

state: GA

address: 1510 Clifton Rd, Suite 3086A

department: Microbiology and Immunology

institute: Emory University

data_processing:
Raw sequencing reads were mapped to the mm10 genome using STAR and the UCSC KnownGene reference transcriptome.
Reads mapping to exons were summarized for each unique ENTREZ gene using the GenomicRanges package in R/Bioconductor and normalized to reads per kilobase per million (RPKM). Genes were filtered for detection based on being expressed at greater than or equal to 3 reads per million in all samples for at least one group.
Assembly: mm10 (GRCm38)
Supplementary files format and content: CD8.TEM.TRM.AdNP.X31.detected.RPKM.csv: Annotation file containing FPKM normalized read counts for each sample.

source_name: CD8 T cell

extract_protocol:
Cells were FACS sorted into RLT+1%Bme using the indicated markers. Total RNA was purified using the Quick-RNA MicroPrep Kit (Zymo Research).
All purified RNA was used as input for the SMART-seq v4 cDNA synthesis kit (Takara). Final libraries were constructed using 200 pg of cDNA as input for the NexteraXT kit with the NexteraXT indexing primers (Illumina).

value: TRM CD69+

tag: sorting markers

value: CD8+ i.v. antibody- NP+ CD69+ CD103-

tag: infection

value: AdNP

tag: days post infection

tag: host organ

value: Lung


title: PFOA_400ppb_Rep1

value: 72 hpf

value: whole fish

value: 400 ppb


title: RNA_2019_03_22_x31_LungRes_DP_1_month

value: TRM CD69+CD103+

value: CD8+ i.v. antibody- NP+ CD69+ CD103+


title: RNA_2019_03_22_AdNP_spleen_TEM_1_month

value: TEM

value: CD8+ CD62L- NP+

value: Spleen


title: RNA_2019_03_22_AdNP_LungRes_DP_1_month


title: RNA_2019_02_06_AdNP_LungRes_DP_1_year


title: RNA_2019_03_22_x31_spleen_TEM_1_month


title: RNA_2019_02_06_AdNP_spleen_TEM_1_year


title: RNA_2019_02_06_AdNP_LungRes_69plus_1_year


title: SUBJECT 02 Timepoint 1

tag: subject

tag: time

value: Akkermansia


title: RNA_2019_02_01_AdNP_LungRes_DP_1_year


title: RNA_2019_02_01_AdNP_spleen_TEM_1_year


title: RNA_2019_01_30_AdNP_LungRes_DP_1_month


title: PFOA_40ppb_Rep1

value: 40 ppb


title: PFOA_4ppb_Rep1


title: RNA_2019_01_30_AdNP_LungRes_69plus_1_month


title: aCD4_tumor_6

value: tumor

tag: strain

value: BALB/c

tag: age

value: 8-10 weeks

tag: gender

value: m


title: RNA_2019_01_30_x31_LungRes_DP_1_month

tag: cell type


title: RNA_2019_03_22_x31_LungRes_69plus_1_month

value: TRM CD69+

value: CD8+ i.v. antibody- NP+ CD69+ CD103-


title: PFOA_0ppb_Rep1

value: 0 ppb


title: RNA_2019_01_30_AdNP_spleen_TEM_1_month

value: AdNP


title: RNA_2019_01_30_x31_LungRes_69plus_1_month

value: Lung


title: NTC_L25

city: ROCHESTER

first: Douglas

middle: Glenn

last: Brownfield

state: Minnesota

address: 200 1ST ST SW

department: PCCM

institute: Mayo Clinic

description: Sample used for quality control. No quantitative processed data produced for this sample.

data_processing:
CASAVA 1.8.2 was used to separate out the data for each single cell using unique barcode combinations from the Nextera XT preparation and to generate *.fastq files.Â
adapter sequences were removed using cutadapt-1.2.1
trimming by base quality was performed and low complexity reads were removed using prinseq-lite-0.20.3
paired reads were aligned against mm10 using bowtie2-2.1.0 and tophat-2.0.8
Fragments Per Kilobase of transcript Per Million mapped reads (FPKM) values were computed using cufflinks-2.0.2
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample. Column headers are: tracking_id, gene_ID, gene short name, tss_id, locus, q0_FPKM, q0_FPKM_low, q0_FPKM_hi

organism: synthetic construct

source_name: negative control

extract_protocol:
To obtain cells from the embryonic  lung, the pulmonary vasculature was cleared and lungs were gently incubated with digestion buffer prior to trituration. All tissue was digested at 37C for 15 min using dispase and DNase I, triturated and subsequently filtered using 100µm and 40µm strainers to obtain a single cell suspension. Mesenchymal and endothelial cells were enriched by magnetic-assisted cell sorting using CD45, Pecam, and EpCAM depletion. Cells were loaded into the Fluidigm C1 microfluidic platform, where single cells were captured. Lysis of single cells, reverse-transcription of mRNA into cDNA as well as preamplification of cDNA occured within the microfluidic device using reagents provided by Fluidigm as well as the SMARTer Ultra Low RNA kit for Illumina (Clontech). External RNA spike-in transcripts (ERCC spike-in Mix, Ambion) were added to all single cell lysis reactions. As a bulk control, cDNA was prepared from approximately 200 cells in a PCR tube using the same protocol and reagents as used for the single cells.
Libraries were prepared using Illumina Nextera XT kit per illumina's protocols.

value: No cells, ERCC spike-ins only


title: B_Pro

first: Dihan

last: Lu

state: Guangdong

address: No.58, Zhong Shan Er Lu, Guangzhou, P. R. China.

department: Department of Anesthesiology

institute: First Affiliated Hospital, Sun Yat-sen University

description: One sample from hippocampus of C57/BL6 in Propofol group

data_processing:
Cellranger standard processing
**Cell filter**  Seurat allows to easily explore QC metrics and filter cells, we can visualize gene and molecule counts, plot their relationship, and exclude cells with a clear outlier number of genes detected as potential multiplets. Followed criteria were used to filter cells:  1.gene counts 500-Inf per cell.  2.UMI counts  3. the percentage of mitochondrial genes < 25% %(customized).  4.DoubletFinder for detecting doublets.
**Cell clustering**  Cell clustering contains the following steps:  1)Normalizing the data  After removing unwanted cells from the dataset, the next step is to normalize the data. By default, we employ a global-scaling normalization method “LogNormalize” that normalizes the gene expression measurements for each cell by the total expression.
2)PCA(Principal component analysis)  To overcome the extensive technical noise in any single gene for snRNA-seq data, Seurat clusters cells based on their PCA scores, with each PC essentially representing a ‘metagene’ that combines information across a correlated gene set.
4)UMAP (Uniform Manifold Approximation and Projection) visualization  Seurat continues to use UMAP as a powerful tool to visualize and explore these datasets. The UMAP aims to place cells with similar local neighborhoods in high-dimensional space together in low-dimensional space.
Genome_build: Mus_musculus.GRCm38.96
Supplementary_files_format_and_content: gene-barcode counts
Supplementary_files_format_and_content: gene files
Supplementary_files_format_and_content: cell barcode files

source_name: hippocampus

extract_protocol:
Mice hippocampus were isolated rapidly with cuting into pieces <0.5cm and were homogenized using a glass dounce tissue grinder (Sigma, #D8938). First, nuclei were isolated with EZ PREP buffer(Sigma, #NUC-101). The number of isolated nuclei was adjusted to 300000 nuclei/ml with suspension buffer and Ficoll PM-400 was added to the nuclei suspension buffer. Second, nuclei were loaded at a concentration of 200 nuclei per microliter. Third, the emulsion was kept on ice for 30 minutes to allow release of nuclear RNA.
Single-cell suspensions were loaded to 10x Chromium to capture  single nuclei according to the manufacturer’s instructions of 10X Genomics Chromium Single-Cell 3’ kit (V3) .The following cDNA amplification and library construction steps were performed according to the standard protocol. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run,150bp) by LC-Bio Technology co.ltd., (HangZhou,China) at a minimum depth of 20,000 reads per cell.

treatment_protocol: PND7 mice were injected with low-dose Propofol and sacrificed at PND10

tag: agent

value: low-dose Propofol

value: brain


title: RNA_2019_01_30_x31_spleen_TEM_1_month

value: TEM


title: A_control

description: One sample from hippocampus of C57/BL6 in Control group

value: Control


title: SUBJECT 01 Timepoint 1


title: Hip.HNTBI3

city: Los Angeles

first: Xia

last: Yang

state: CA

address: 2000A, 610 Charles E Young Dr S

institute: UCLA

description:
Drop-Seq; whole cell
DropSeq.Hip.24hrs.TBI.HN.digital_expression.mtx
DropSeq.Hip.24hrs.TBI.HN.features.tsv
DropSeq.Hip.24hrs.TBI.HN.barcodes.tsv
DropSeq.Hip.24hrs.TBI.HN.metaData.tsv

data_processing:
Base calls performed with bcl2fastq2.15.
Read 2 was aligned with Star v2.5.0c.
Each aligned read was tagged with its mate-pair UMI and cell barcode.
Each aligned exonic read was tagged with a gene name.
A digital gene expression matrix was built by counting the number of unique UMIs per gene within each cell; columns are cells, and rows are genes.
Drop-seq samples were processed using Drop-seq Tools  were processed using cell ranger.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Digital gene expression matrices (genes x cells) in sparse matrix format. Compressed, flat text files with tab delimiters. Requires the corresponding features (genes) and barcodes (cell names) files. Meta Data files include the cell type labels.

source_name: hippocampus HumaninTBI, 24 hours

extract_protocol:
The protocol by Brewer et al. was used to suspend cells at a final concentration of 100 cells/μl in 0.01% BSA-PBS by digesting freshly dissected hippocampus and frontal cortex tissue with papain (Worthington, Lakewood, NJ, USA). Briefly, hippocampi from the ipsilateral side of the brain and frontal corticies were rapidly dissected on ice. The hippocampi and cortices were transferred into 4 ml HABG (Fisher Scientific, Hampton, NH, USA) and incubated in water bath at 30 °C for 8 min. The supernatant was discarded and the remaining tissue was incubated with papain (12 mg in 6 ml HA-Ca) at 30 °C for 30 min. After incubation, the papain solution was removed from the tissue and washed with HABG three times. Using a siliconized 9-in Pasteur pipette with a fire-polished tip, the solution was triturated approximately ten times in 45 s. Next, the cell suspension was carefully applied to the top of the prepared OptiPrep density gradient (Sigma Aldrich, St. Louis, MO, USA) and floated on top of the gradient. The gradient was then centrifuged at 800 g for 15 min at 22 °C. We aspirated the top 6 ml containing cellular debris. To dilute the gradient material, we mixed the desired cell fractions with 5 ml HABG. The cell suspension containing the desired cell fractions was centrifuged for 3 min at 22 °C at 200 g, and the supernatant containing the debris was discarded. Finally, the cell pellet was loosened by flicking the tube and the cells were re-suspended in 1 ml 0.01% BSA (in PBS). This final cell suspension solution was passed through a 40-micron strainer (Fisher Scientific, Hampton, NH, USA) to discard debris, followed by cell counting.
Barcoded single cells, or STAMPs (single-cell transcriptomes attached to microparticles), and cDNA libraries were generated following the drop seq protocol from Macosko et al11. and version 3.1 of the online Drop-seq protocol [http://mccarrolllab.com/download/905/]. Briefly, single cell suspensions at 100 cells/μl, EvaGreen droplet generation oil (BIO-RAD, Hercules, CA, USA), and ChemGenes barcoded microparticles (ChemGenes, Wilmington, MA, USA) were co-flowed through a FlowJEM aquapel-treated Drop-seq microfluidic device (FlowJEM, Toronto, Canada) at recommended flow speeds (oil: 15,000 μl/hr, cells: 4000 μl/hr, and beads 4000 μl/hr) to generate STAMPs. The following modifications were made to the online published protocol to obtain enough cDNA as quantified by a high sensitivity BioAnalyzer (Agilent, Santa Clara, CA, USA) to continue the protocol: (1) The number of beads in a single PCR tube was 4000. (2) The number of PCR cycles was 4 + 11 cycles. (3) Multiple PCR tubes were pooled. The libraries were then checked on a BioAnalyzer high sensitivity chip (Agilent, Santa Clara, CA, USA) for library quality, average size, and concentration estimation. The samples were then tagmented using the Nextera DNA Library Preparation kit (Illumina, San Diego, CA, USA) and multiplex indices were added. After another round of PCR, the samples were checked on a BioAnalyzer high sensitivity chip for library quality check before sequencing. A cell doublet rate of 5.6% was obtained by running the microfluidic device without the lysis buffer and counting the percentage of cell doublets through three separate runs.

tag: strain/backgorund

value: Humanin/TBI

value: 24 hours

value: hippocampus

value: 10 weeks


title: Hip.HNTBI4

tag: treatment


title: RNA_2019_02_01_AdNP_LungRes_69plus_1_year


title: Hip.HNTBI2

tag: tissue


title: Hip.HNTBI1