title: RNA-seq, human, iPSC-derived 3D cerebral organoids, day 105, FXS patient5

data_processing:
For day 28 samples, paired-end reads were first aligned to human genome (hg19) by Tophat v2.1.1 with default settings. FPKM values were calculated by Cufflinks v2.2.1. Pairwise differential expression was analyzed by Cuffdiff v2.2.1.
For day 105 samples, transcript abundances were quantified using salmon 0.11.1 and an hg38 (Gencode 28) annotation. Transcript abundances were then collapsed to gene abundances using tximport 1.6.0, and differentially expressed genes were identified using DESeq2 1.18.1.
Genome_build: hg19 (day 28 samples); hg38 (day 105 samples)
Supplementary_files_format_and_content: For day 28 samples, gene expression values are provided in Cufflinks-normalized fpkm_tracking format. For day 105 samples, gene expression values are provided as TPM values as calculated by salmon.

source_name: iPSC-derived 3D cerebral organoids

extract_protocol:
For day 28 samples, total RNA was extracted from fresh 3D cerebral organoids using TRIzol (Invitrogen). Samples were treated with DNase I and cleaned with RNA Clean & Concentrator-5 kit (Zymo Research). For day 105 samples, RNA was isolated using a Zymo Quick RNA Mini kit.
For day 28 samples, mRNA-seq libraries were generated from total RNA isolated from triplicated samples per condition. The library construction was performed by Novogene. An Agilent 2100 BioAnalyzer and DNA1000 kit were used to quantify amplified cDNA and to control the quality of the libraries. For day 105 samples, mRNA-seq libraries were created using a Kapa Hyperprep mRNA kit.

tag: individual

value: FXS patient5

tag: Sex

value: male

value: FXS

value: iPSC-derived 3D cerebral organoids

value: 105 days

tag: treatment of rna sample

value: Poly(A) selection

tag: biological replicate no.


title: wtAA_UV2hr_A3_1bp

status: Public on Apr 12 2021

type: SRA

city: Pullman

first: John

middle: J.

last: Wyrick

state: WA

address: Biotechnology Life Sciences 241

department: School of Molecular Biosciences

country: USA

institute: Washington State University

description: WT-anchor away (AA) control strain, treated with rapamycin

library_source: genomic

data_processing:
Library strategy: CPD-seq
Base calling by Torrent_Suite 5.0.4
Fastq files were separated into individual libraries using A1-A6 barcodes, and the barcode sequences were trimmed, as well as the 3' nucleotide of the sequencing read, using a custom perl script
Fastq files were aligned to the yeast genome (SacCer3 or SacCer3+pUC19) using bowtie2-2.2.6 software to generate SAM files
SAM files were converted to BAM files using samtools
BAM files were converted to BED files using BEDTOOLS2 bamtoBed
Damage coordinates were mapped using custom perl script
Only reads mapping to dipyrimidine sequences (i.e., TT, TC, CT, CC), corresponding to CPD lesions were retained in BED file using custom perl script.
BED files were converted to WIG file using COUNT function of IGVTools
Background values dipyrimidine sequences were generated in WIG file using custom perl script
Genome_build: SacCer3
Supplementary_files_format_and_content: WIG files containing counts of DNA CPD lesion reads filtered for only dipyrimidine mapping sequences and including background value of 0.0 at nucleotides associated with dipyrimidines with no mapped sequencing reads. A sequencing read count was  assigned to each nucleotide in the lesion-containing dipyrimidine sequence.

molecule: genomic DNA

organism: Saccharomyces cerevisiae

source_name: Yeast cells

growth_protocol: Yeast cultures were grown in YPD (yeast extract, peptone, dextrose) to OD600=~0.8 in a yeast shaker. Anchor away strains (indicated by -AA after name) were grown to OD600 ~0.8 for mutants and ~0.4 for WT and treated with 50mg/mL rapamycin for 3 hours to conditionally deplete the tagged nuclear proteins.

extract_protocol:
Yeast genomic DNA was extracted by mixing cell pellets with 250 μl of DNA lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA), 250 μl of PCI (Phenol:Chloroform:Isoamyl Alcohol=25:24:1), and 150 μl of acid-washed glass beads, and vortexed vigorously twice, 2 min each time. After adding 200 μl of TE (10mM Tris-HCl, pH 7.5, 1 mM EDTA) buffer, cell lysates were centrifuged and the supernatant was mixed with 1 ml of ethanol to precipitate DNA at room temperature for 5 min. DNA pellet was dissolved in 200 μl of TE and incubated with 2 μl of RNase A (ThermoFisher Scientific) at 37˚C for 1 hr to remove RNA. DNA was subsequently purified with PCI extraction, ethanol precipitated, and disolved in sterile deionized H2O. The purified genomic DNA was quantified and 5 μg of DNA was used for each sequencing library preparation.
CPD-seq procedure is adopted from the recently published emRiboSeq method (Nature Protocols 2015 10, 1433–1444). Briefly, DNA fragments were first ligated to a double-stranded adaptor DNA trP1, and free 3'-OHs were blocked by  terminal transferase and dideoxy-ATP.  CPD lesions were  cleaved with T4 endonuclease V and apurinic/apyrimidinic (AP) endonuclease, APE 1, to generage new 3'-OHs. DNA was then ligated to adaptor  A. Up to 6 different barcoded A adaptors (A1: AAGAGGAT; A2: TTCGTGAT; A3: CCTGAGAT; A4:ATCGCGAT; A5: TACTGGAT; A6: GAACTGAT) were used to generate libraries for different experiments. DNA was purified with Streptavidin beads and amplified for up to 6 cycles in a PCR reaction with primers complimentary to A and trP1, and the resulting DNA libraries were used for DNA sequencing.

treatment_protocol: Cells were collected and resuspended in sterile deionized H2O and irradiated with 125 J/m2 UVC. After UV irradiation, cells were spun down and resuspended in pre-warm YPD medium (yeast extract, peptone, dextrose)  for non-anchor away strains and YPD + rapamycin for anchor away strains to allow for repair. Aliquots (20 ml of yeast culture) were taken before UV irradiation (No UV), immediately after UV irradiation (0hr), and at 2 hours after UV irradiation (2hr). Cells were spun down and kept at -80˚C until genomic DNA isolation.

tag: strain

tag: genotype

value: WT-AA

tag: treatment

value: UV irradiated 125 J/m2

tag: repair time

value: 2 hr


title: hESC null 2

type: genomic

city: Irvine

first: Minji

last: Byun

state: California

address: 829 Health Science Rd, 311

department: Microbiology and Molecular Genetics

institute: University of California Irvine

hyb_protocol: Standard Illumina protocol

scan_protocol: Standard Illumina protocol

data_processing: Raw IDAT files were processed with minfi v1.32.0. Probes that have failed in one or more samples (detection pval >0.01) and cross reactive probes were removed.

label: Cy5 and Cy3

organism: Homo sapiens

source_name: human embryonic stem cell

label_protocol: Standard Illumina protocol

growth_protocol: Culture conditions for hESCs and their derivatives including macrophages can be found in the accompanying publication.

extract_protocol: Genomic DNA was extracted and purified from cells using Zymo Quick-DNA microprep kit according to standard instructions.

treatment_protocol: For the siRNA-treated samples, macrophages were transfected with indicated siRNA, washed after 24 hours, and incubated for additional 96 hours prior to genomic DNA extraction.

value: untreated

tag: cell type

value: human embryonic stem cell


title: RNA-seq, human, iPSC-derived 3D cerebral organoids, day 105, FXS patient4

city: Atlanta

first: Feiran

last: Zhang

state: GA

address: 615 Michael St NE

department: Human Genetics

institute: Emory University

library_source: transcriptomic

molecule: polyA RNA

growth_protocol: 3D cerebral organoids were generated from iPSC lines generated from male control and FXS patient dermal fibroblasts. Briefly, iPSCs colonies were detached from Matrigel coated plates with collagenase (1mg/ml; Invitrogen) treatment for 1hr and suspended in EB medium, comprising of FGF-2-free hESC medium supplemented with Dorsomorphin (2μM; Tocris) and A-83 (2μM; Tocris), in non-treated polystyrene plates for 4 days with a daily medium change. On days 5-6, half of the medium was replaced with induction medium consisting of DMEM/F-12, N2 Supplement (Thermofisher), 10μg/ml Heparin (Sigma), NEAA (1X), Glutamax (1X), WNT-3A (4 ng/ml; R&D Systems), CHIR99021 (1μM; Cellagentech), and SB-431542 (1μM; Cellagentech). On day 7, organoids were embedded in Matrigel (Corning) and continued to grow in induction medium for 6 more days. On day 14, embedded organoids were mechanically dissociated from Matrigel by pipetting up and down onto the plate with a 5ml pipette tip. Typically, 10 – 20 organoids were transferred to each well of a 12-well spinning bioreactor (SpinΩ) containing differentiation medium, consisting of DMEM:F12, N2 (1X), B27 (1X;  Invitrogen), 2-Mercaptoenthanol (100µM), NEAA (1X), Insulin (2.5μg/ml; Sigma). Media was changed every other day until harvested for RNA on day 28 or day 105.

value: FXS patient4

tag: tissue

tag: age


title: RNA-seq, human, iPSC-derived 3D cerebral organoids, day 105, FXS patient6

value: FXS patient6


title: Nonclassical monocytes WT2 [SAM24362393]

city: South San Francisco

first: Yasin

last: Senbabaoglu

address: 1 DNA Way

institute: Genentech

data_processing:
RNA-sequencing data were analyzed using HTSeqGenie (Wu et al., 2016)(Pau and Reeder, 2012) as follows:
first, reads with low nucleotide qualities (70% of bases with quality <23) or rRNA and adapter contamination were removed.
The reads that passed were then aligned to the mouse reference genome GRCm38.p5 using GSNAP (Wu and Nacu, 2010) version ‘2013-10-10-v2’ with alignment parameters: ‘-M 2 -n 10 -B 2 -i 1 -N 1 -w 200000 -E 1 --pairmax-rna=200000'.
Alignments of the reads that were reported by GSNAP as “uniquely mapping” were used for subsequent analysis.
Gene expression levels were quantified as Reads Per Kilobase of exon model per Million mapped reads normalized by size factor (nRPKM), defined as number of reads aligning to a gene in a sample / (total number of uniquely mapped reads for that sample x gene length x size factor).
Genome_build: GRCm38.p5
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

molecule: total RNA

organism: Mus musculus

source_name: Nonclassical monocytes WT

growth_protocol: Animals were maintained in a pathogen-free animal facility under 12hr light/12hr dark cycle with access to a standard rodent chow and water ad libitum

extract_protocol:
Non-classical monocytes were sorted (purity 98%) from the bone marrow of LAIR1−/− and LAIR1+/+. 5000-10000 cells were collected and RNA was extracted using Nucleospin RNA XS (Takara) and low input RNA sequencing was performed (Clontech).
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Bone marrow

value: Nonclassical monocytes

tag: library construction

value: HiSeq Rapid SBS Kit v2


title: D3_no_matrigel_2

city: Lausanne

first: Nicolas

last: Broguiere

address: AI 1208 Station 15

department: Sciences de la vie

country: Switzerland

institute: EPFL

data_processing:
The reads were trimmed for adapters with bcl2fastq v2.20.0
Alignment with star/2.7.0e with --quantMode GeneCounts
Genome_build: mm10
Supplementary_files_format_and_content: tab separated values containing transcript raw counts as lines for each condition as columns (i.e. XAV treated or not at each timepoint)

source_name: Mouse embryonic stem cells (CGR8)

growth_protocol: Mouse embryonic stem cells were aggregated in serum-free N2B27 medium, supplemented by Activin-A, Fgf2 and (KSR)

extract_protocol:
Trizol based RNA extraction
TruSeq stranded mRNA LT libraries were prepared according to Illumina protocol 15031047 Rev. E, starting from 300 ng of RNA, quantified by qubit DNA HS, profiled on TapeStation 4200, and sequenced on an Illumina HiSeq 4000 at a targeted depth of 36 Mreads/sample and paired-end read length of 81,8i,8i,81.
Illumina truseq stranded bulk RNAseq

treatment_protocol: EPI aggregates were treated with XAV939 from 0h to 72h.

value: Differentiated mES-cells


title: sRNA_seq_OE_WT_Dicer_rep2

city: Stanford

first: Mark

last: Kay

state: CA

address: 269 Campus Drive

institute: Stanford University

data_processing:
The sequencing data was processed by removing adapters with cutadapt (Version 1.16)
Trimmomatic (Version 0.33) was be used to filtering high-quality reads.
duplicated reads were removed with an in-house Perl script
5’ 8nt and 13nt degenerated barcode were removed for icSHAPE-MaP and RNA-seq respectively
Cleaned reads were mapped to hg38 reference by STAR(v2.7.1a).
For icSHAPE-MaP score calculation, Replicate samples were combined (with samtools merge),
Shapemapper (v2.1.4) was used to calculate final scores
Genome_build: sRNAs sequences less than ~200-nt in length were collected, such as miRNAs (from miRbase v22) (Kozomara et al., 2019), snoRNAs (from Gencode v26) (Harrow et al., 2012), snRNAs (from Gencode v26), tRNAs (from GtRNAdb v2.0) (Chan and Lowe, 2016), vault RNAs (from RefSeq v109) (O'Leary et al., 2016), Y RNAs (from RefSeq v109), and 5S rRNAs
Supplementary_files_format_and_content: Tab-seperated files. For reactivity score file, four columns represents RNA_name,position,nucleotide,score. For read count file, two columns represents RNA_name, read count. For files associated with miRNA-Seq, columns include "read ID", "Strand", "miRNA name", "start position", "end position", "sequence", "length", "mapped loci", and "raw reads per locus".

source_name: 293T Dicer KO

growth_protocol: Cells were maintained in DMEM/HIGH glucose with L-glutamine, sodium pyruvate (Thermo Scientific HyClone) and 10% fetal bovine serum in a humidified incubator at 37°C with 5% CO2

extract_protocol:
To enrich sRNA <200nt, after RNA extraction by Trizol LS, the total RNA was subjected to gel size selection.
For icSHAPE-MaP, The 8 µL purified RNA was ligated with a 3′ linker by incubation with the 3′ ligation mix (6 µL PEG8000, 1 µL 3’ linker (10 µM), 1 µL DTT (100 mM), 2 µL 10×ligation buffer, 1 µL T4 RNA ligase KQ (NEB), 1 µL RiboLock) at 25 °C for 2 hrs, followed by inactivation of enzyme at 65°C for 20 min. The 1.2 µL RT primer (10 µM) was added to the 3’ ligation mix and annealed to excess 3’linker by 75 °C 5 min, 37 °C 15 min, 25 °C 15 min. The 5’ ligation mix ( 3 µL PEG8000, 3 µL 10 mM ATP, 1 µL 10×ligation buffer, 0.5 µL RiboLock, 0.5 µL 5′ linker (Key Resource Table, 20 µM), 1 µL T4 RNA ligase I (NEB)) was added directly and incubated at 25 °C for 2 hrs. The reaction was purified with a RNA concentration kit (Zymo). The 9 µL mutation buffer (50 mM Tris-HCl pH 8.0, 500 µM dNTPs, 75 mM KCl, 10 mM DTT, 6 mM MnCl2, 1 µL RiboLock) was added to the 10 µL purified RNA. The reaction mix was incubated at 42 °C for 2 min. Then 1 µL SuperScript II (Thermo Fisher) was added to the reaction mix. The reverse transcription mix was incubated at 42 °C for 3 hrs. cDNA products were purified with a DNA concentration kit (Zymo).PCR were set up with 20 µL eluted cDNA and PCR reaction mix (0.5 µL P5 primer (Key Resource Table, 20 µM), 0.5 µL P3 index primer (Key Resource Table, 20 µM), 0.4 µL 25×SYBR Green, 20 µL 2×Phusion HF Master Mix (NEB)). PCR was performed in a qPCR machine (Agilent, Mx3000P) to monitor the amplification procedure and was programmed as follows: stage I: 98 °C for 1 min; stage II: 98 °C for 15 s, 65°C for 30 s, 72 °C for 45 s, with limited cycles.PCR products were purified with a DNA concentration kit (Zymo) and a further size selection (150~330 nt) on a 6% Native PAGE gel.
For sRNA-seq(40-200nt), 1 µg sRNA was then incubated with the end-repairing mix (2 µL 5×PNK buffer, 2 µL T4 polynucleotide kinase (NEB), 1 µL Fast AP (Thermo Fisher), 1µL RiboLock, 4 µL nuclease-free water) at 37°C for 1 hr. The 3′ ligation mix (6 µL PEG8000, 1 µL 3′ linker (Key Resource Table, preadenylated and blocked at its 3′ end with SpC3, 10 µM), 1 µL DTT (100 mM), 1 µL 10×RNA ligation buffer, 1 µL T4 RNA ligase I (NEB)) was added directly to the end-repairing mix. The ligation condition was 25°C for 3 hrs. The ligation product was size-selected on a 6% Urea-PAGE gel (Thermo Fisher) for ~60-200 nt. Gel purification was performed by crushing the gel and incubating in the RNA elution buffer (20 mM Tris-HCl pH 7.0, 2 mM EDTA pH 8.0, 300 mM NaAc pH 5.2, RiboLock 40 U/mL) with vigorous shaking at 37°C overnight. The eluate was collected by centrifuging through 0.45 um Spin-X columns (Thermo Fisher), followed by ethanol precipitation.   For reverse transcription, 9 µL purified 3′-ligation product was mixed with 1 µL RT primer (Key Resource Table, 10 µM). Samples were heated to 70°C for 5 min in a PCR block, cooled to 25°C by stepping down 1°C every 3 sec and held at 25°C. Afterwards, 8 µL mutation buffer (50 mM Tris-HCl pH 8.0, 500 µM dNTPs, 75 mM KCl, 10 mM DTT, 6 mM MnCl2), 1 µL RiboLock, and 1 µL SuperScript II (Thermo Fisher) were added. The reaction mix was incubated at 25°C for 3 min, and then 42°C for 3 hr. cDNA products were purified by running on 6% Urea-PAGE gel. The following steps, including circularization, PCR amplification and PCR products purification, were done as previously described.
For miRNA-Seq (~20 nt), library construction, Illumina sequencing and data analysis were conducted as previously described (Gu et al., 2012), except for the following modification. Reverse transcription (RT) was set up with RNAs ligated with adaptors, 1 µL RT primer, 1×SSIV buffer, 500 µM dNTP, 5 mM DTT, 1 U/µL RiboLock, and 1 µL SuperScript IV (Thermo Fisher) in a total volume of 20 µL. The reaction was programmed as follows: 25°C for 3 min, 42°C for 10 min, 50°C for 30 min, and 80°C for 10 min. RT products were then ethanol precipitated and dissolved in 10 µL H2O. To set up PCR reactions, 5 µL of RT products were mixed with 1×Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB), 200 µM Solexa Forward and Reverse primers in a total volume of 50 µL. PCR was programmed as follows: stage I: 98°C for 30 s; stage II: 98°C for 10 s, 52°C for 30 s, 72°C for 15 s, repeating for total 22 cycles; Stage III: 72°C for 5 min. Multiplex sequencing was performed on an Illumina NextSeq 500 machine or on a MiSeq machine (1X36-bp) at the Stanford Functional Genomics Facility.

treatment_protocol: For Dicer RIP icSHAPE-MaP, Dicer KO 293T cells was transfected with plasmid encoded flag-tagged catalytic dead hDicer.Also, for sRNA-seq on WT/catalytic-dead Dicer overexpressed Dicer KO 293T cells, the Dicer KO 293T cells was transfected with plasmid encoded WT or calalytic-dead hDicer.

tag: cell line

value: 293T Dicer KO

value: Overexpression of WT hDicer


title: 2hr_UV_Sth1_A6

description: Repeat #2 of Sth1-anchor away (AA) strain, treated with rapamycin to deplete Sth1


title: Nonclassical monocytes WT1 [SAM24362392]


title: D7_XAV_1

value: XAV939 treatment between 0-72h


title: RNA-seq, human, iPSC-derived 3D cerebral organoids, day 105, control6

value: male

value: control

value: iPSC-derived 3D cerebral organoids


title: 0hr_UV_Sth1_A5

value: 0 hr


title: hESC null 1


title: 22RV1_shDouble_R1881_Rep3

city: Aurora

first: Lauren

last: Jillson

state: Colorado

address: 12801 E 17th Avenue, Room L18-6213, Mail stop 8303

department: Pharmacology

institute: University of Colorado Anschutz Medical Campus

data_processing:
Illumina NovaSeq 6000 Platform, PE150
Libraries were prepared using Novogene’s eukaryotic RNA-Seq method
Gene expression was quantified using default parameter of RSEM (v1.2.1) with Bowtie2 (v2.1.0) as the read aligning program and differential expression was calculated using EBSeq (v1.1.5). Reads were aligned to hg19 RefSeq annotated genes
Differential gene expression was calculated using the EdgeR package (v3.14.0).
Genome_build: hg19
Supplementary_files_format_and_content: The raw transcript counts (22RV1_compiled_raw) and RSEM-normalized values (22RV1_compiled_normalized)  below contain compiled data for each cell type deliniated by column headers.

source_name: 22RV1 prostate cancer cell line

growth_protocol: 22RV1 cells are maintained on uncoated culture dishes in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. 22RV1 cells were previously engineered with the following shRNAs using lentivirus transduction: shControl (puromycin and neomycin constructs), shMAP3K7 (puromycin), and shCHD1 (neomycin). We generated 2 groups to compare: 1. shControl(puro/neo)  4. shMAP3K7(puro)/shCHD1(neo).

extract_protocol:
After the above 4 hour treatments, total RNA was collected using the 5Prime Perfect RNA Cell Kit (Fisher Scientific) according to the manufacturer's protocol, including the optional DNAase treatment
After quality control, RNA samples were enriched for mRNA using oligo(dT) beads, fragmented for a 250-300 bp target range, then cDNA synthesis using random hexamer primers, second strand synthesis is performed using dNTPs, RNAas H and DNA polymerase I in Illumina synthesis buffer, Illumina sequencing adapters, then the double-stranded cDNA library is size selected and PCR enriched.

treatment_protocol: These 2 22RV1 cell types were plated on 6cm culture dishes in the above culture medium and left for 2 days. They were then hormone deprived by culturing in RPMI 1640 medium (-phenol red) with 10% charcoal-stripped serum and 1% penicillin/streptomycin for 2 additional days, reaching 80% confluency. Then they were treated with either vehicle (0.1% EtOH) or 1nM of the synthetic androgen R1881 for 4 hours beofre collection of RNA.

value: shMAP3K7_puro, shCHD1_neo

value: 1nM R1881, 4 hours

tag: passage


title: RNA-seq, human, iPSC-derived 3D cerebral organoids, day 105, control5

value: 105 days

value: Poly(A) selection


title: D6_XAV_1


title: sth1_2hrUV_A5_1bp

description: Sth1-anchor away (AA) strain, treated with rapamycin to deplete Sth1

value: UV irradiated 125 J/m2

value: 2 hr


title: sRNA_seq_OE_catalytic_dead_Dicer_rep1

value: Overexpression of catalytic-dead hDicer


title: 22RV1_shDouble_R1881_Rep2


title: RNA-seq, human, iPSC-derived 3D cerebral organoids, day 105, control4


title: D6_no_matrigel_1


title: sth1_0hrUV_A4_1bp


title: 22RV1_shDouble_R1881_Rep1


title: hESC haplo 2

value: untreated

value: human embryonic stem cell


title: rsc2_NoUV_A3_1bp


title: hESC null 3


title: D5_XAV_1

value: Differentiated mES-cells


title: rsc2_2hrUV_A5_1bp


title: 22RV1_shDouble_EtOH_Rep3

value: 0.1% Ethanol, 4hours


title: D5_no_matrigel_1


title: hESC haplo 1


title: 22RV1_shDouble_EtOH_Rep2


title: rsc2_0hrUV_A4_1bp


title: 22RV1_shDouble_EtOH_Rep1


title: D7_no_matrigel_1


title: D4_XAV_1


title: D4_no_matrigel_1


title: PAO1161 PA3458_3_RNA-seq

city: Warszawa

first: Adam

last: Kawałek

address: Pawinskiego 5A

department: Department of Microbial Biochemistry

country: Poland

institute: Institute of Biochemistry and Biophysics, PAS

description:
gene expression data from PA3458 overproducer, biological rep 3
PAO1161 PA3458_3

data_processing:
Sequencing data were quality-checked and filtered using FASTP version 0.20.0.
Reads were mapped to P. aeruginosa PAO11161 genome (CP032126) using Bowtie2 version 2.3.4.3 using default settings.
The number of reads mapping to individual genes was counted using FeatureCounts v 2.0.1 (with the -s2 option).
Differential expression analysis was conducted using edgeR ver 3.28.0.
Genome_build: CP032126
Supplementary_files_format_and_content: Raw counts of sequencing (FeatureCounts output summarized for all samples).
A table with DE data for all genes and normalized abundance measurements (EdgeR output).

organism: Pseudomonas aeruginosa

source_name: PA3458 overproducer (PA3458)

growth_protocol: The competent P. aeruginosa PAO1161 cells were prepared as described previously (Irani and Rowe, 1997) and transformed using pKKB2.11 carrying araC araBADp-PA3458 or empty vector (araC araBADp) to obtain PAO1161/ pKKB2.11 (araC araBADp-PA3458 ) and PAO1161/ pKGB8 (araC araBADp), respectively. Transformants were selected on L agar plates supplemented with 150 μg/ml chloramphenicol and were verified by isolation of plasmid DNA and its digestion. Bacteria from single colonies were used to inoculate L broth (DifcoTM L Broth, Lennox, BD) liquid cultures with supplementation (75 µg/ml chloramphenicol) and grown overnight with shaking at 37oC. Three independent overnight cultures for each strain were diluted 1:100 into fresh L broth with chloramphenicol (75 mg/ml) and 0.02% arabinose and grown with shaking at 37oC. Samples from the cultures were taken at regular intervals to measure the optical density at 600 nm (OD600). Aliquots of 2 ml from logarithmic phase cultures (OD600 = 0.5) were subjected to RNA extraction.

extract_protocol:
Total RNA was isolated from three independent replicates of each strain. 2 ml of cultures which reached optical density 0.4-0.6 at 600 nm were mixed with 4 ml of RNAprotect Bacteria Reagent (Qiagen). RNA was isolated with an RNeasy mini-kit (Qiagen) according to the manufacturer’s protocol. Total RNA was digested with DNase (TURBO DNA-free Kit, Ambion) to eliminate genomic DNA.
rRNA was depleted using Ribo-Zero rRNA Removal Kit (Bacteria) (MRZMB126,Illumina) according to manufacturer instructions. Libraries were prepared according to instructions accompanying the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, E7370S).

treatment_protocol: All cultures were incubated at 37oC in L broth (DifcoTM L Broth, Lennox, BD) supplemented with 75 μg/mL chloramphenicol and 0.02% arabinose with shaking until reaching exponential phase of growth (OD600 = 0.5).

value: PAO1161/pKKB2.11 (araC araBADp-PA3458)

value: PA3458 overproducer (PA3458)

tag: growth phase

value: logarithmic phase of liquid culture


title: snf6_2hrUV_comb_1bp


title: hESC WT 3

value: wild type


title: hESC WT 1


title: D3_XAV_1

value: XAV939 treatment between 0-72h


title: hESC haplo 3


title: PAO1161 PA3458_2_RNA-seq

description:
gene expression data from PA3458 overproducer, biological rep 2
PAO1161 PA3458_2


title: PAO1161 PA3458_1_RNA-seq

description:
gene expression data from PA3458 overproducer, biological rep 1
PAO1161 PA3458_1


title: 22RV1_shControl_R1881_Rep2

value: shControl_puro, shControl_neo


title: hESC WT 2


title: snf6_0hrUV_comb_1bp

value: 0 hr


title: D6_XAV_2


title: snf6_NoUV_comb_1bp


title: Rep2_3percent_8days

data_processing:
The reads were trimmed for adapters with bcl2fastq v2.20.0
Alignment to mm10 with cellranger v3
Alignment, clustering and annotations with Seurat v3 + Harmony v1
Genome_build: mm10-3.0.0
Supplementary_files_format_and_content: matrix.mtx contains a matrix market sparse matrix with the raw counts of transcripts found for each cell barcode (columns) and gene (feature, lines). Features.tsv and barcodes.tsv contain the list of row and column names respectively. Additionally, metadata.tsv contains cell metadata, including dimensionality reductions (harmony aligned PCA coordinates, UMAP coordinates of overview and close-ups tissue by tissue), germ layers attributed to the cells, louvain clusters, condition of origin (days post aggregation/initiation of differentiation, epithelialization with pre-culture in 3% matrigel or not, replicate 1 or 2), as well as cell types from expert annotation and from automated transfer from the Pijuan-Sala et al. 2019 in vivo atlas.

source_name: Mouse embryonic stem cells (CGR8 line/129 strain)+ Mouse trophoblast stem cells (B5/EGFP strain)

growth_protocol: Mouse embryonic stem cells were aggregated in serum-free N2B27 medium, supplemented by Activin-A, Fgf2 and KSR. Mouse trophoblast stem cells were aggregated in 1:1 RPMI1640 /N2B27medium supplemented by FBS, Fgf4 and Heparin.

extract_protocol:
10X genomics chromium single cell controller
10X genomics single cell gene expression 3' chemistry v3

treatment_protocol: TS aggregates were treated with 2% matrigel in ETS medium from 0h to 72h. In indicated samples, EPI aggregates were treated with 3% matrigel in EPI medium from 0h to 72h.

tag: strain/cell type

tag: replicate

tag: days

tag: matrigel


title: 22RV1_shControl_EtOH_Rep3


title: 22RV1_shControl_EtOH_Rep2


title: scRNAseq barcoded PC9 rep3 rand

city: Jerusalem

first: Xue

last: Sun

address: Hebrew University, Givat Ram campus

department: Biochemistry department

country: Israel

institute: The Hebrew University of Jerusalem

description: library constructed with random hexamer primers

data_processing:
For all single-cell and Clone-Seq samples, the cell barcode and UMI are on Read 1, and the genomic barcode  is on Read 2 without a specific position. The structure of Read 1 is: [Barcode 1 7-10 bp] GAGTGATTGCTTGTGACGCCTT[Barcode 2 8bp][UMI 5bp]
The reads were first filtered based on the presence of two sample barcode components separated by the W1 adaptor sequence in Read 1. Barcodes for each read were matched against a list of the 768 pre-determined barcodes. Although the barcode was designed to be 3 SNPs away from each other, no mismatch was allowed in barcodes. The reads were UMI filtered, and single cell fastq files were produced.
Reads split into clone barcode-specific files were aligned using Bowtie2 (v2.3.4.3, defaut parameters) and RSEM (v1.2.31) to the mouse or human reference transcriptome. Alignments from Bowtie were filtered as follows: For each read, we retained at most one alignment per gene, across all isoforms, by choosing the alignment closest to the end of the transcript.
The Illumina output was analyzed using an in-house Perl script that produced a reads matrix. The resulting matrix was analyzed in R. For bulk data analysis the transcript per million (TPM) values were used to compare libraries.
Genome_build: hg38, mm9
Supplementary_files_format_and_content: single-cell and Clone-seq data: raw counts
Supplementary_files_format_and_content: UGI shifted primer processed data files raw counts of sequences with data in three columns: Single-cell barcode (BC1+BC2), UGI, and counts.

source_name: human lung adenocarcinoma cell line

growth_protocol: For standard tissue culture, PC9 cells were grown in DMEM supplemented with 10% fetal bovine serum, 50 μg/ml penicillin-streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate. ZHBTc4 ESCs cells were grown on 0.1% gelatin-coated standard tissue culture dishes and maintained in ESC medium (DMEM, 15% ESC-grade FBS, 50 μg/ml penicillin-streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol). To maintain pluripotency, 1000 U/ml LIF and 2i (1 μM PD0325901, 3 μM CHIR99021) were added to the culture medium.   For culture in hydrogel spheres, we used PEGDT (Mn ~10,000) and MALDEX hydrogel chemistry to encapsulate cells within spheres and grew them into clones. PC9 cells were encapsulated in PEGDT/MALDEX hydrogel with no additional cell adhesion peptides or remodeling supplements. ESCs cells were encapsulated in MALDEX and cell adhesion peptides containing cell recognition motifs of the extracellular matrix (RGD) and MMP-cleavable peptide sequence. For culturing encapsulated cells into clones, 500 μl aliquots of hydrogels were grown in a standard 48-well cell culture dish well with 500 μl standard cell growth medium as described above.

extract_protocol:
Single cells and single clone transcriptomes were extracted in microfluidic droplets. For scRNA-seq experiments, cells were loaded at 200,000 cells/ml in PBS containing 10% v/v OptiPrep and maintained in suspension using a magnetic micro-stirrer bar placed within the tube. For CloneSeq experiments, around 20,000 clones were resuspended in 900 μl PBS and 100 μl OptiPrep and maintained in suspension using a magnetic micro-stirrer bar placed within the tube. The device generates monodispersed droplets with volumes in the range of 2 nl for scRNA-seq and around 4-5 nl for CloneSeq. Single cell/clone lysates were subject to reverse transcription at 50°C without purification of RNA.
Cells were barcoded using the using inDrops platform (Klein AM et al., Cell. 2015), which makes use of the CEL-Seq protocol for library construction (Hashimshony et al., Cell Reports 2011).

treatment_protocol: To assess the impact of clonal cell origin on cellular states and variation, we produced PC9 cell lines that carry genomic barcodes. We designed and produced a plasmid pool containing a UGI region of 8 bp as a genomic barcode about 600 bp upstream of the BFP polyA region. For transfection, we grew 293T cells to 80% confluency and then incubated the cells for 30 min in conditioning medium (50 ml 293T medium supplemented with 500 μl L-glutamine and 500 μl Sodium-Pyruvate). The transfection solution contained 34.5 μl TransIT®-LT1 Transfection Reagent, 1 μg transfer plasmid (psPAX.2), 7 μg VSV-G (PMD2.G), and 3.5 μg of our BFP-UGI plasmid diluted to 1.5 ml with Opti-MEM I Reduced Serum Medium. Viruses were collected after 48 h and 72 h. For virus concentration, the PEG Virus Precipitation Kit was used. For infection, PC9 cells of 50% confluency were incubated for 30 min in 1 μl polybrene in 1 ml culture medium. PC9 cells were infected with the virus at an MOI of 1. Finally, BFP-positive cells were sorted by FACS to obtain the BFP PC9 cell line containing genetic barcodes.

tag: culture

value: hydrogel spheres

tag: protocol

value: scRNAseq (inDrop)

value: genomically barcoded


title: Rep2_0percent_8days

value: Mouse embryonic stem cells (CGR8 line/129 strain)+ Mouse trophoblast stem cells (B5/EGFP strain)


title: snf5_0hrUV_A2_1bp

tag: strain

value: UV irradiated 125 J/m2


title: scRNAseq barcoded PC9 rep2 BC

description: library constructed with UGI shifted primers


title: D3_no_matrigel_1


title: 22RV1_shControl_R1881_Rep1

tag: genotype

value: 1nM R1881, 4 hours


title: 22RV1_shControl_R1881_Rep3

tag: treatment


title: snf5_NoUV_A1_1bp

tag: repair time


title: scRNAseq barcoded PC9 rep3 BC


title: snf5_2hrUV_A3_1bp

value: 2 hr


title: scRNAseq barcoded PC9 rep2 rand


title: scRNAseq barcoded PC9 rep1 BC


title: Rep2_3percent_7days


title: Rep2_0percent_7days


title: 22RV1_shControl_EtOH_Rep1


title: WTNoUV_A1_1bp

value: Wild-type


title: scRNAseq barcoded PC9 rep1 rand


title: WT_2hrUV_A3_1bp


title: WT_0hrUV_A2_1bp


title: LAPC4_shDouble_R1881_Rep2

data_processing:
Illumina NovaSeq 6000 Platform, PE150
Libraries were prepared using Novogene’s eukaryotic RNA-Seq method
Gene expression was quantified using default parameter of RSEM (v1.2.1) with Bowtie2 (v2.1.0) as the read aligning program. Reads were aligned to hg19 RefSeq annotated genes
Differential gene expression was calculated using the EdgeR package (v3.14.0).
Genome_build: hg19
Supplementary_files_format_and_content: The raw transcript counts (LAPC4_compiled_raw) and RSEM-normalized values (LAPC4_compiled_normalized)  below contain compiled data for each cell type deliniated by column headers.

source_name: LAPC4 prostate cancer cell line

growth_protocol: LAPC4 cells are maintained on uncoated culture dishes in IMDM medium supplemented with 7.5% FBS and 1% penicillin/streptomycin. LAPC4 cells were previously engineered with the following shRNAs using lentivirus transduction: shControl (puromycin and neomycin constructs), shMAP3K7 (puromycin), and shCHD1 (neomycin). We generated 2 groups to compare: 1. shControl(puro/neo)  4. shMAP3K7(puro)/shCHD1(neo).

treatment_protocol: These 2 LAPC4 cell types were plated on 6cm culture dishes in the above culture medium and left for 2 days. They were then hormone deprived by culturing in IMDM medium (-phenol red) with 7.5% charcoal-stripped serum and 1% penicillin/streptomycin for 2 additional days, reaching 80% confluency. Then they were treated with either vehicle (0.1% EtOH) or 1nM of the synthetic androgen R1881 for 4 hours beofre collection of RNA.

value: shMAP3K7_puro, shCHD1_neo


title: LAPC4_shDouble_R1881_Rep3


title: scRNAseq PC9 3D

value: untreated


title: wtAAmag1_NoMMS_A1_1bp

data_processing:
Library strategy: NMP-seq
Base calling by Torrent_Suite 5.0.4
Fastq files were separated into individual libraries using A1-A6 barcodes, and the barcode sequences were trimmed, using a custom perl script
Fastq files were aligned to the yeast genome (SacCer3 or SacCer3+pUC19) using bowtie2-2.2.6 software to generate SAM files
SAM files were converted to BAM files using samtools
BAM files were converted to BED files using BEDTOOLS2 bamtoBed
Damage coordinates were mapped using custom perl script
Only reads mapping to G nucleotides, corresponding to 7meG lesions were retained in BED file using custom perl script.
BED files were converted to WIG file using COUNT function of IGVTools
Background values at G nucleotides were generated in WIG file using custom perl script
Genome_build: SacCer3
Supplementary_files_format_and_content: WIG files containing counts of DNA alkylation damage reads filtered for only G (7-methylguanine) mapping sequences and including background value of 0.0 for G nucleotides with no mapped sequencing reads.

growth_protocol: Yeast cultures (FH001, KB011, YAH049, KB012) were grown in YPD (yeast extract, peptone, dextrose) to OD600=~0.3 in a yeast shaker. 1x nocodozale and 1ug/mL rapamycin were added and cells were allowed to grow another 3 hours. G2/M arrest induced by nocodozale was confirmed via microscopy.

extract_protocol:
Yeast genomic DNA was extracted by mixing cell pellets with 250 μl of DNA lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA), 250 μl of PCI,  (Phenol:Chloroform:Isoamyl Alcohol=25:24:1), and 150 μl of acid-washed glass beads, and vortexed vigorously twice, 2 min each time. After adding 200 μl of TE (10mM Tris-HCl, pH 7.5, 1 mM EDTA) buffer, cell lysates were centrifuged and the supernatant was mixed with 1 ml of ethanol to precipitate DNA at room temperature for 5 min. DNA pellet was dissolved in 200 μl of TE and incubated with 2 μl of RNase A (ThermoFisher Scientific) at 37˚C for ~1 hr to remove RNA. DNA was subsequently purified with PCI extraction, ethanol precipitated, and dissolved in sterile deionized H2O. The purified genomic DNA was quantified and ~5ug of DNA was used for each sequencing library preparation.
NMP-seq procedure is adopted from the recently published emRiboSeq method (Nature Protocols 2015 10, 1433Ð1444). Briefly, DNA fragments were first ligated to a double-stranded adaptor DNA trP1, and free 3'-OHs were blocked by  terminal transferase and dideoxy-ATP.  CPD lesions were  cleaved with Alkyladenine DNA glycosylase (AAG)  and apurinic/apyrimidinic (AP) endonuclease, APE 1, to generage new 3'-OHs. DNA was then ligated to adaptor  A. Up to 6 different barcoded A adaptors (A1: AAGAGGAT; A2: TTCGTGAT; A3: CCTGAGAT; A4: ATCGCGAT; A5: TACTGGAT) were used to generate libraries for different time points. DNA was purified with Streptavidin beads and amplified for 5-6 cycles in a PCR reaction with primers complimentary to A and trP1, and the resulting DNA libraries were used for DNA sequencing.

treatment_protocol: For repair time points experiments, MMS was added to the yeast cultures to the final concentrations of 0.4% and incubated for 10 minutes. Cells were collected and washed with sterile deionized H2O twice to remove MMS. Cells were resuspended in pre-warm YPD medium (yeast extract, peptone, dextrose) + rapamycin for repair. Aliquots (20 ml of yeast culture) were taken before MMS treatment (No MMS), immediately after MMS treatment (0hr), and at different repair time points as indicated. Cells were spun down and kept at -80˚C until genomic DNA isolation.

value: WT-AA + mag1<delta>


title: Rep2_0percent_6days


title: CloneSeq PC9 rep 2

value: CloneSeq


title: Rep2_3percent_5days


title: LAPC4_shDouble_EtOH_Rep3

value: 0.1% Ethanol, 4hours


title: LAPC4_shDouble_EtOH_Rep2


title: Rep2_0percent_5days


title: wtAAmag1_0hrMMS_A2_1bp

value: 0.4percent MMS for 10 min


title: CloneSeq PC9 rep 1


title: wtAA_2hrMMS_A3_1bp

value: WT-AA


title: Rep1_3percent_8days


title: sth1AAmag1_NoMMS_A3_1bp

value: Sth1-AA + mag1<delta>


title: LAPC4_shDouble_EtOH_Rep1


title: Rep1_0percent_8days


title: bulk-RNAseq PC9 3D rep 2

data_processing:
The cell barcode and UMI are on Read 1, and the genomic barcode for sample 4 to 7 (scRNAseq barcoded PC9 2D rand, scRNAseq barcoded PC9 2D BC,scRNAseq barcoded PC9 3D rand, scRNAseq barcoded PC9 3D BC) is on Read 2 without a specific position.
The structure of Read 1 is: [Barcode 1 7-10 bp]GAGTGATTGCTTGTGACGCCTT[Barcode 2 8bp][UMI 5bp]
The reads were first filtered based on the presence of two sample barcode components separated by the W1 adaptor sequence in Read 1. Barcodes for each read were matched against a list of the 768 pre-determined cell barcodes. 384 barcodes for each of Barcode 1 and Barcode 2. Although the barcode was designed to be 3 SNPs away from each other, no mismatch was allowed in barcodes. The reads were UMI filtered, and single cell fastq files were produced.
The genomic barcodes were inserted by a lentivirus BFP reporter plasmid that contains barcode at 3' end as described in "Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq". We used the Unique Genomic Index (UGI) in the plasmid as our genomic barcode. There is no pre-determined set for it. For the analysis, we looked for the constant region contains the Barcode region as the following structure: GTCGACGGATCC[UGI 8bp]GACTTAC
Reads split into clone barcode-specific files were aligned using Bowtie2 (v2.3.4.3, defaut parameters) and RSEM (v1.2.31) to the mouse or human reference transcriptome. Alignments from Bowtie were filtered as follows: For each read, we retained at most one alignment per gene, across all isoforms, by choosing the alignment closest to the end of the transcript.
The Illumina output was analyzed using an in-house Perl script that produced a reads matrix. The resulting matrix was analyzed in R. For bulk data analysis the transcript per million (TPM) values were used to compare libraries.
Genome_build: hg38, mm9

growth_protocol: For standard tissue culture, PC9 cells were grown in DMEM supplemented with 10% fetal bovine serum, 50 μg/ml penicillin-streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate. R1 ESCs cells were grown on 0.1% gelatin-coated standard tissue culture dishes and maintained in ESC medium (DMEM, 15% ESC-grade FBS, 50 μg/ml penicillin-streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol). To maintain pluripotency, 1000 U/ml LIF and 2i (1 μM PD0325901, 3 μM CHIR99021) were added to the culture medium.   For culture in hydrogel spheres, we used PEGDT (Mn ~10,000) and MALDEX hydrogel chemistry to encapsulate cells within spheres and grew them into clones. PC9 cells were encapsulated in PEGDT/MALDEX hydrogel with no additional cell adhesion peptides or remodeling supplements. ESCs cells were encapsulated in MALDEX and cell adhesion peptides containing cell recognition motifs of the extracellular matrix (RGD) and MMP-cleavable peptide sequence. For culturing encapsulated cells into clones, 500 μl aliquots of hydrogels were grown in a standard 48-well cell culture dish well with 500 μl standard cell growth medium as described above.

value: bulkRNAseq


title: LAPC4_shControl_R1881_Rep3

value: shControl_puro, shControl_neo


title: bulk-RNAseq PC9 3D rep 1

value: hydrogel spheres


title: Rep2_3percent_6days


title: LAPC4_shControl_R1881_Rep2


title: LAPC4_shDouble_R1881_Rep1


title: sth1AAmag1_0hrMMS_A4_1bp

value: 0 hr


title: bulk-RNAseq PC9 2D rep 2

value: standard tissue culture


title: sth1AA_2hrMMS_A5_1bp


title: bulk-RNAseq PC9 2D rep 1


title: Treatment D rep 4

city: Albemarle

first: Chandler

middle: J

last: Keck

address: 1510 Melchor rd

institute: NC State University

data_processing:
RNA-seq data were analyzed using CLC Genomics Workbench 20.0.3 (Qiagen Bioinformatics-Hilden, Germany) to identify differentially expressed genes
High quality reads were aligned to the Gallus gallus version-GRCg6a transcriptome.
RNA-seq data were mapped with a maximum number of allowed mismatches of 2, a minimum length and similarity fraction of 0.8, and a maximum number of hits per read of 10. Gene expression values were reported as total counts.
Genome_build: GRCg6a
Supplementary_files_format_and_content: xlsx file with differential expression comparisons of treatment D samples to treatment A samples using CLC genomics workbench

organism: Gallus gallus

source_name: Broiler liver tissue

extract_protocol:
On day 46, birds were euthanized and Liver tissue samples were taken, preserved in RNAlater, placed at 4°C for 24 hours then stored at -20°C. RNA was isolated with RNAeasy kit (QIAGEN-Hilden, Germany) according to manufactures protocol from liver samples of both the highest density (Treatment D) and the lowest density (Treatment A). Briefly, tissue samples were initially homogenized in kit lysis buffer and centrifuged. The supernatant was combined with equal volume 70% ethanol and loaded on the provided spin columns. Bound RNA was washed with provided buffers and eluted in nuclease free water. Final RNA sample yields were quantified spectrophotometrically using NanoDrop 2000 (Thermo Fisher Scientific-Waltham, MA). RNA samples were submitted to NC State Universities Genomic Sciences Laboratory for library preparation and sequencing using Illumina HiSeq (Illumina-San Diego, CA).
Final RNA sample yields were quantified spectrophotometrically using NanoDrop 2000 (Thermo Fisher Scientific-Waltham, MA). RNA samples were submitted to NC State Universities Genomic Sciences Laboratory for library preparation and sequencing using Illumina HiSeq (Illumina-San Diego, CA). Libraries were prepared according to Illumina's instructions and each library consisted of equal amounts pooled RNA from 4 samples of the same treatment and 4 libraries were prepared for each of the 2 treatments.

treatment_protocol: Stocking density served as treatment for this study with Treatment A having a final target stocking density of 24.4kg/m2 (5 lbs/ft2) and Treatment D having a final target stocking density of 39.1kg/m2 (8 lbs/ft2).

value: Liver

tag: Sex

value: Male

value: D


title: LAPC4_shControl_EtOH_Rep3


title: Rep1_3percent_7days

value: Mouse embryonic stem cells (CGR8 line/129 strain)+ Mouse trophoblast stem cells (B5/EGFP strain)


title: Rep1_3percent_6days


title: LAPC4_shControl_EtOH_Rep2


title: Rep1_0percent_6days


title: Rep1_0percent_7days


title: PAO1161 EV-F_1_control_for_PA3458_ChIP-seq

description: ChIP DNA data from empty vector control

data_processing:
Sequencing data were quality-checked and filtered using FASTP version 0.20.0.
Reads were mapped to P. aeruginosa PAO1161 genome (CP032126) using Bowtie2 version 2.3.4.3 using default settings.
Obtained *.sam files were sorted (samtools sort -n), run through samtools fixmate with the -m option, again sorted (samtools sort) and duplicates were marked with samtools markdup. Samtools ver. 1.9 was used.
The files were indexed and used to generate coverage *.bigwig files, normalized to 1× sequencing depth (RPGC), without binning and smoothing using the bamCoverage tool ver 3.3.0 included in deepTools.
PA3458 peaks were identified by comparing combined replicates of IP samples (F-PA3458) for each strain with negative control (empty vector EV-F) samples using the callpeak function of MACS2 ver 2.1.2 with default options for paired-end .BAM files, and 0.05 as false discovery rate (FDR) adjusted p-value cut off.
Genome_build: CP032126
Supplementary_files_format_and_content: Processed files in bigwig format represent normalized coverages of individual base pairs in the P. aeruginosa PAO1161 genome.
Reference genome is enclosed in fasta format (CP032126.fa) and the annotation used is enclosed as a .bed (CP032126.bed).
A modified .bed file with PAO1161 feature names changed to the names of corresponding PAO1 genes (PA… format) is included for convenience of data browsing e.g. using IGV (CP032126_PA_names.bed). These files are available on GSE163233.

source_name: empty vector control (EV-F)

growth_protocol: P. aeruginosa PAO1161 ΔPA3458 strain carrying pKKB2.12 (araC araBADp-PA3458-flag) as well as control strain carrying pABB28.3 (araC araBADp-flag) were obtained by conjugation of PAO1161 (leu-, r-, RifR) and E. coli S-17 donor strain carrying appropriate plasmid. Conjugants were selected on L agar plates supplemented with 300 μg/mL rifampicin and 150 μg/mL chloramphenicol. Two independent overnight cultures from each strain were inoculated from single colonies and grown overnight with shaking at 37oC. After that they were diluted 1:100 in L broth (DifcoTM L Broth, Lennox, BD) supplemented with 75 μg/mL chloramphenicol and 0.02% arabinose to induce the expression of PA3458-flag and grown with shaking at 37oC until reaching exponential phase with optical density about 0.5 (OD600).

extract_protocol:
Chromatin immunoprecipitation (ChIP) protocol was based on the method described earlier (Kawalek et al, 2018; Nucleic Acids Res. 46:9, 4592-4606). Two cultures of each strain with similar OD600 were combined, formaldehyde was added from a 37% stock to a final concentration of 1%, and the cultures were kept for 10 min at room temperature with gentle inverting. Glycine was added to a final concentration of 0.5 M and the cultures were incubated for 5 min at room temperature with gentle rotation. Cells were then harvested by centrifugation (20 min, 3000 x g, 25 mL of culture in 50 mL conical tubes, 4 tubes per strain), washed twice with ice-cold Tris-buffered saline (25 mM Tris-HCl, 150 mM NaCl, pH=7.6), resuspended in 1 mL of TBS, transferred to a fresh 1.5 mL tube and centrifuged (2 min, 6000 x g). The cell pellets were frozen at -70oC. The pellets were thawed on ice, suspended in 0.5 mL of lysis buffer [10 mM Tris-HCl, 20% (w/v) sucrose, 50 mM NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), pH=8.0] supplemented with 10 µl of Protease Inhibitor Cocktail (Sigma, PA8849) and 125 µL of lysozyme (20 mg/mL) and incubated for 30 min at 37oC. Four samples for each strain were combined in 15 mL. Subsequently, 1.5 mL of IP-buffer [50 mM Tris-HCl, pH=7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (m/v) sodium deoxycholate], 30 µL protease inhibitor cocktail, PMSF (phenylmethanesulfonyl fluoride) to a final concentration of 2 mM, SDS (sodium dodecyl sulphate) to a final concentration of 0.1% were added to each sample. The samples were sonicated 20 times for 20 s each with 1 min break on ice using a Microson ultrasonic cell disruptor Xl2000 (Misonix) with 5-mm tip and 90% of output power to obtain DNA fragments of an average length of 250-350 bp. DNA fragmentation was checked by agarose gel electrophoresis. After sonication, samples were aliquoted (1 mL in 1.5 mL tubes) and stored at -70oC. Sonicated lysate was thawed on ice and 150 μL of each strain variant was incubated with 20 μL of magnetic beads coupled with protein A (Dynabeads Protein A, Invitrogen, 10001D) previously separated from original suspension using Magnetic Separation Stand. Such pre-clearing step was performed for minimum 1 hour at 4oC with rotation of the mixtures. In the meantime 50 μL of magnetic beads were prepared for each reaction by separating from the suspension as above. It was then mixed with 6 μL anti-FLAG mouse antibodies  [DYKDDDDK Tag polyclonal  antibodies; 1 mg/mL; PA1-985B; Invitrogen] diluted in 200 μL of PBS with 0.05% Tween-20. Mixtures of magnetic beads and antibodies were incubated for 10 minutes at 4oC with gentle rotation. Beads with bound antibodies were then separated from the supernatant, washed once with 200 μL of the PBS with 0.05% Tween-20 solution and stored on ice. Pre-cleaned lysate was separated from the beads used for pre-incubation and added to the beads coated with antibodies. Mixture containing lysate and magnetic beads with antibodies was incubated at 4oC for 20 minutes with mixing on rotator. Beads were then collected and washed. Elution was performed twice for 15 minutes in 50 μL at 65oC in thermoblock with shaking (1400 rpm). Elutions from 6 parallel reactions were pooled and then 30 µL pipetted for SDS page analysis. Rest  of obtained eluates were incubated with 8 μL of RNase A (100 mg/mL, Qiagen, 19101) for 30 min at 65oC. Then, 40 μL of Proteinase K (20 mg/mL, Qiagen, 19133) was added and the samples were incubated for 1 h at 50oC followed by overnight incubation at 65oC. Next 40 μL of Proteinase K was added and the samples were again incubated for 1 h at 50oC. Such prepared probes were stored on ice and 24 μL of 3 M sodium acetate (pH=5) was added and water to end volume 700 μL. DNA purification was performed using Qiaquick Qiagen PCR purification Kit according to manufacturer’s instructions. The DNA was stored at -20oC.
NGS library was constructed using QiaSeq Ultralow Input Library kit. Sample was further quality checked on 1% agarose gel and concentration was measured using qPCR KAPA Library Quantification Kit.

treatment_protocol: All cultures were incubated at 37°C in L broth (DifcoTM L Broth, Lennox, BD) supplemented with 75 μg/mL chloramphenicol and 0.02% arabinose with shaking until reaching exponential phase of growth (OD600 = 0.5).

value: PAO1161/pABB28.1 (araC BADp-flag)

value: empty vector control (EV-F)

value: logarithmic phase of liquid culture


title: PAO1161 PA3458-F_3_ChIP-seq

description: ChIP DNA data from PA3458-FLAG overproducer, replicate 3

source_name: PA3458 overproducer (PA3458-F)

value: PAO1161 delta_PA3458/pKKB2.12 (araC BADp-PA3458-flag)

value: PA3458 overproducer (PA3458-F)


title: LAPC4_shControl_EtOH_Rep1

value: 0.1% Ethanol, 4hours


title: Treatment D rep 3

tag: tissue


title: LAPC4_shControl_R1881_Rep1

value: 1nM R1881, 4 hours


title: Rep1_0percent_5days


title: Treatment D rep 1


title: WGBS-seq of untransfected control, replicate 2 [DYNC2LI1-GFP tagged line]

city: Dallas

first: Jin

last: Chen

state: TX

address: 5323 Harry Hines Blvd

institute: UT Southwestern Medical Center

data_processing:
For RNA-seq, linker sequences (AGATCGGAAGAGCACACGTCTGAACTC) were removed using FASTX-clipper (FASTX- Toolkit). The reads were then aligned to the human genome (GRCh37, hg19) using the STAR (Spliced Transcripts Alignment to a Reference, version 2.5) aligner against the Gencode Gene V24lift37 transcriptome annotation. Read quantification was carried out with featureCounts (Liao et al., 2014)
For the ChIP-seq, reads were aligned to the human genome (hg19) using bowtie v2.3.2 (Langmead and Salzberg, 2012) . Alignments were processed using deepTools2 bamCoverage (Ramírez et al., 2016) , normalizing reads to 1x average coverage. The resulting bigWig files were visualized on the Integrative Genomics Viewer (IGV).
For WGBS-seq, reads were first trimmed using fastp v0.21.0 (Chen et al., 2018) and then aligned to the human genome (hg38) using bwa mem v0.7.17 (Li, 2013) and methylCtools v1.0.0 (Hovestadt et al., 2014). Putative PCR duplicates were removed using sambamba v0.8.0 (Tarasov et al., 2015), and CpG methylation values were called using methylCtools. Further analyses were performed in R v4.0.2 using the bsseq v1.24.4 and DSS v2.36.0 packages.
Genome_build: hg19, hg38
Supplementary_files_format_and_content: BigWig files for ChIP-seq, counts file for RNA-seq, and methylation call files for WGBS-seq (final columns are Methylated and Unmethylated counts)

source_name: cell lysate

growth_protocol: HEK293T (female) cells were cultured in Dulbecco’s modified eagle medium (DMEM) in 10% FBS (HyClone or VWR), 100 units/mL streptomycin, 100 µg/ml penicillin, and 2 mM glutamine.

extract_protocol:
For RNA-seq, HEK293T cells that have maintained stable silencing of target genes were harvested 33 days (ITGB1 (CD29), CD81, and CD151) or 28 days (CLTA, HIST2H2BE, RAB11A) post CRISPRoff transfection. Cells were dislodged from plates with PBS, centrifuged at 500 × g for 5 min and washed again with PBS. Total RNA was extracted using Direct-zol RNA MiniPrep (Zymo R2051). For ChiP-seq, at 30 days post transfection, 10x106 cells were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched with 1.25 M glycine. Crosslinked cells were washed twice with cold PBS containing 1% HaltTM protease inhibitors (Thermo Fisher Scientific) and the cell pellets were flash frozen at –80 °C until sample preparation. Cells were lysed in lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 1% Igepal, 1% protease inhibitors) for 10 min on ice. Nuclei were isolated after spinning the suspension at 2000 rpm at 4 °C for 5 min. Nuclei were lysed at 4 °C for 10 min in nuclei lysis buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS, 1% protease inhibitors). Chromatin shearing was performed at 4°C using a Diagenode Bioruptor® Pico sonication device in 1.5 ml Bioruptor® Pico Microtubes. The shearing program was optimized to obtain 200-700 bp fragments (30 seconds on, 30 seconds off for 10 cycles). The sonicated samples were centrifuged at 13,000 rpm at 4 °C for 10 min and the supernatant was collected and diluted 5-fold in IP dilution buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Igepal, 0.25% deoxycholate, 1% protease inhibitors). A fraction of the input was saved and frozen (4% of total) prior to proceeding to immunoprecipitation. Immunoprecipitations were performed overnight at 4 °C using 5 µg of anti-H3K9me3 antibody (abcam ab8898). The washing steps were performed with PierceTM Protein A/G magnetic beads (Thermo Fisher Scientific) using the following protocol: once with IP wash buffer 1 (20 mM Tris pH 8, 2 mM EDTA, 50 mM KCl, 1% Triton X-100, 0.1% SDS), twice with high salt buffer (20 mM Tris pH 8, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP wash buffer 2 (10 mM Tris pH 8, 1 mM EDTA, 0.25 lithium chloride, 1% Igepal, 1% deoxycholate), and twice with TE buffer (10 mM Tris pH 8, 1 mM EDTA). Samples were eluted in elution buffer (50 mM sodium bicarbonate, 1% SDS) at 65 °C for 1 hour and reversed crosslinked initially in 300 mM NaCl and RNase A for 1 hour at 37 °C, followed by 63 °C overnight with Proteinase K (Thermo Fisher). The DNA samples were purified using a Zymo Clean & Concentrator kit.
For RNA-seq, library preparations were carried out using TruSeq Stranded mRNA Library Preparation Kit (Illumina RS-111-2101), starting with 1000 ng total RNA. The ChIP-seq library were prepared using the NEBNext UltraTM II DNA Library Prep kit (NEB). The WGBS-seq libraries were prepared using the EZ DNA Lightning kit (Zymo, Cat. No. D5030) and the Swift Accel-NGS Methyl-Seq DNA Library Kit (Cat. No. 30024), starting from 1 ug DNA.

tag: cell type

value: DYNC2LI1-GFP tagged line

tag: sgrna target

tag: crispr construct

value: CRISPRoff


title: PAO1161 PA3458-F_2_ChIP-seq

description: ChIP DNA data from PA3458-FLAG overproducer, replicate 2


title: Treatment D rep 2


title: Treatment A rep 4

value: A


title: PM_3

city: guangzhou

first: Xingyang

last: Li

state: Guangdong

address: 195, dongfengxi road

department: Department of Temporomandibular Joint Surgery

country: China

institute: Affiliated Stomatology Hospital of Guangzhou Medical University

data_processing:
RNA-seq reads were aligned to the mm10 genome assembly using STAR version 020201 with the "twopassMode"  configuration.
circRNA were identified using CIRI version 2.0 and find_circ version 2.0  with the default setting.
Genes expression estimate were calculated using Htseq and normalized by FPKM.
Genome_build: mm10
Supplementary_files_format_and_content: txt files were generated using RSEM.

source_name: Mouse bone marrow mesenchymal stem cells

growth_protocol: Mice BMSCs (P1) were purchased from Cyagen Co., Ltd. (Guangzhou, China). The BMSCs were expanded in proliferation medium (PM) containing Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA), 10% (v/v) fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin G and 100mg/ml streptomycin (Gibco) at 37°C in an incubator with 5% CO2 atmosphere and 100% relative humidity.

extract_protocol:
In this study, the total RNA of each sample was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, 1% agarose gels were used to monitor RNA degradation and contamination. RNA purity was checked using the NanoPhotometer spectrophotometer (Implen). RNA concentration was measured using the Qubit RNA Assay Kit in the Qubit 2.0 Fluorometer (Life Technologies). RNA integrity was evaluated by the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 System (Agilent Technologies).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: BMSCs were seeded into 48-well culture plates (2.5x104/well), and six-well culture plates (2x105). Osteogenic medium (OM: 100 nM dexamethasone + 0.2 mM ascorbic acid + 10 mM β-glycerophosphate from Sigma-Aldrich, St.Louis, MO, USA) was used to induce osteogenic differentiation in vitro. Cultures were treated with C2S (1, 10, 50 or 100 μg/ml) and osteogenic differentiation potential was further analyzed.

value: Bone marrow mesenchymal stem cells

value: proliferation medium culture for 7 days


title: WGBS-seq of untransfected control, replicate 1 [DYNC2LI1-GFP tagged line]

tag: cell line


title: PM_2


title: Treatment A rep 3


title: WGBS-seq of CRISPRoff with DYNC2LI1 targetingsgRNA, replicate 2 [DYNC2LI1-GFP tagged line]


title: WGBS-seq of CRISPRoff with DYNC2LI1 targetingsgRNA, replicate 1 [DYNC2LI1-GFP tagged line]


title: WGBS-seq of CRISPRoff with non-targeting sgRNA, replicate 2 [DYNC2LI1-GFP tagged line]

value: non-targeting


title: Treatment A rep 2

value: Liver


title: Rep1_3percent_5days


title: PM_1


title: cauda_P56_2

city: Nantong

first: Hao

state: Jiangsu Province

address: NO.16, Wenfeng xi Road

department: Medical school

institute: Nantong University

description:
mouse epididymis cauda
epididymis cauda segment scRNASeq

data_processing:
reads filtering and adapter trimming by Fastp 0.19.5
reads mapping done by STAR-2.7.3a
umi_tools was used for integrated scRNA-seq analyzing, including cell barcode extraction, cell id assignment, UMI counting
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: gene expression matrix file, containing UMI counts values for all the detected genes of each single cell in all the three epididymal regions

source_name: Cauda epididymis

extract_protocol:
The tissue pieces were digested in 2 ml GEXSCOPETM Tissue Dissociation Solution (Singleron Biotechnologies) at 37 ℃ for 15 min in a 15 ml centrifuge tube with continuous agitation. Following digestion, a 40-micron sterile strainer (Corning) was used to separate cells from cell debris and other impurities. To remove red blood cells, 2 mL GEXSCOPETM Red Blood Cell Lysis Buﬀer (Singleron) was added to the cell suspension and incubated at 25°C for 10 minutes. The mixture was then centrifuged at 1000 rpm for 5 min and cell pellet was re-suspended in PBS. Cells were counted with TC20 automated cell counter (Bio-Rad).
Single cell suspension was loaded onto a microfluidic chip (part of Singleron GEXSCOPETM Single Cell RNAseq Kit, Singleron Biotechnologies) and single cell RNA-seq libraries were constructed according to manufacturer’s instructions (Singleron Biotechnologies). The resulting single cell RNAseq libraries were sequenced on Illumina HiSeq X10 instrument with 150bp paired end reads.

value: epididymis

value: No treatment


title: WGBS-seq of CRISPRoff with non-targeting sgRNA, replicate 1 [DYNC2LI1-GFP tagged line]


title: corpus_P56_2

description:
mouse epididymis corpus
epididymis corpus segment scRNASeq

source_name: Corpus epididymis


title: PAO1161 PA3458-F_1_ChIP-seq

description: ChIP DNA data from PA3458-FLAG overproducer, replicate 1

tag: growth phase


title: WGBS-seq of untransfected control, replicate 2 [CLTA-GFP tagged line]

value: CLTA-GFP tagged line


title: C2S_3

value: C2S with osteogenic medium culture for 7 days


title: caput_P56_2

description:
mouse epididymis caput
epididymis caput segment scRNASeq

source_name: Caput epididymis


title: R+LDC C5a 100 #3

type: RNA

city: Jeonju-si, Jeollabuk-do,

first: Saehae

last: Kim

address: 567 Baekje-daero, Deokjin-gu,

department: Molecular biology

country: South Korea

institute: Jeonbuk National University

description: Gene expression after 3 hr in C5aR+LysoDC treated with rC5a (100 nM) #3

hyb_protocol: Labeled cRNA was applied microarray (Agilent technologies, 8x60K ) using Agilent’s Gene Expression Hybridization Kit.

scan_protocol: :The microarray chip was scanned using Agilent’s DNA microarray scanner and the raw signal density was acquired from feature Extraction software.

data_processing: The differential gene expression was classified by Benjamini-Hochberg's false discovery ratio method. GeneSpringGX12software. Gene ontology

source_name: C5aR+ LysoDC_C5a 100 nM #3

label_protocol: labeled cRNA (complementary RNA) was generated from Agilent’s Low RNA Input Linear Amplification kit with 500ng total RNA. Labeled cRNA was also measured using Nanodrop spectrophotometer.

growth_protocol: Sorted C5aR+ LysoDCs cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 1% penicillin/streptomycin, 1 mM sodium pyruvate, CSF-1 (0.1 μg/mL), and CFS-2 (0.5 μg/mL).

extract_protocol: Total RNA was prepared using an RNeasy PLUS Micro kit according to the manufacturer’s protocols.

treatment_protocol: Sorted C5aR+ LysoDCs were stimulated with recombinant C5a protein (100 nM) for 3 hours.

value: Peyer's patch


title: Dicer_RIP_input_icSHAPE_MaP_rep3

data_processing:
Library strategy: icSHAPE-MaP
The sequencing data was processed by removing adapters with cutadapt (Version 1.16)
Trimmomatic (Version 0.33) was be used to filtering high-quality reads.
duplicated reads were removed with an in-house Perl script
5’ 8nt and 13nt degenerated barcode were removed for icSHAPE-MaP and RNA-seq respectively
Cleaned reads were mapped to hg38 reference by STAR(v2.7.1a).
For icSHAPE-MaP score calculation, Replicate samples were combined (with samtools merge),
Shapemapper (v2.1.4) was used to calculate final scores
Genome_build: sRNAs sequences less than ~200-nt in length were collected, such as miRNAs (from miRbase v22) (Kozomara et al., 2019), snoRNAs (from Gencode v26) (Harrow et al., 2012), snRNAs (from Gencode v26), tRNAs (from GtRNAdb v2.0) (Chan and Lowe, 2016), vault RNAs (from RefSeq v109) (O'Leary et al., 2016), Y RNAs (from RefSeq v109), and 5S rRNAs
Supplementary_files_format_and_content: Tab-seperated files. For reactivity score file, four columns represents RNA_name,position,nucleotide,score. For read count file, two columns represents RNA_name, read count. For files associated with miRNA-Seq, columns include "read ID", "Strand", "miRNA name", "start position", "end position", "sequence", "length", "mapped loci", and "raw reads per locus".

value: 293T Dicer KO

value: Input of hDicer RIP for icSHAPE-MaP


title: WGBS-seq of CRISPRoff with CLTA targetingsgRNA, replicate 2 [CLTA-GFP tagged line]

value: CLTA


title: cauda_P56_1


title: C2S_2


title: Treatment A rep 1

value: Male


title: R+LDC C5a 0 #3

description: Gene expression in C5aR+LysoDC #3

source_name: C5aR+ LysoDC_C5a 0 nM #3


title: corpus_P56_1


title: R+LDC C5a 100 #2

description: Gene expression after 3 hr in C5aR+LysoDC treated with rC5a (100 nM) #2

source_name: C5aR+ LysoDC_C5a 100 nM #2


title: R+LDC C5a 100 #1

description: Gene expression after 3 hr in C5aR+LysoDC treated with rC5a (100 nM) #1

source_name: C5aR+ LysoDC_C5a 100 nM #1


title: C2S_1


title: Dicer_RIP_input_icSHAPE_MaP_rep2

source_name: 293T Dicer KO


title: R+LDC C5a 0 #1

description: Gene expression in C5aR+LysoDC #1

source_name: C5aR+ LysoDC_C5a 0 nM #1


title: OM_3

value: osteogenic medium culture for 7 days


title: Dicer_RIP_input_icSHAPE_MaP_rep1


title: WGBS-seq of CRISPRoff with non-targeting sgRNA, replicate 2 [CLTA-GFP tagged line]


title: miRNA amiR-SEAP1 rep2

city: Lincoln

first: Chi

state: NE

address: 1901 Vine Street

institute: University of Nebraska - Lincoln

data_processing:
Illumina software used for basecalling.
Sequenced reads were trimmed for adaptor sequence then mapped to TAIR10 using HISAT2
Reads counts for each gene or miRNA with trimmed mean of M values (TMM) normalization method. Robinson and Oshlack Genome Biology 2010.
Genome_build: TAIR10
Supplementary_files_format_and_content: Tab-delimited plain text files: the first column is Gene ID or MicroRNA gene IDs and the second column is raw reads counts
Supplementary_files_format_and_content: RNA-Seq: abundance measurements FPKM
Supplementary_files_format_and_content: miRNA-Seq: raw counts

organism: Arabidopsis thaliana

source_name: inflorescence

growth_protocol: 16 hour light; 8 hour dark;  at 22 oC

extract_protocol:
Flower inflorescences were collected and total RNA was extracted by Trizol.  For small RNA-seq, Illumina TruSeq Small RNA Sample Prep Kits was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina mRNA library protocol and Illumina small RNA library protocol

tag: ecotype

value: Columbia (Col)

value: inflorescence


title: WGBS-seq of CRISPRoff with CLTA targetingsgRNA, replicate 1 [CLTA-GFP tagged line]

value: CRISPRoff


title: OM_2

value: Bone marrow mesenchymal stem cells


title: caput_P56_1


title: R+LDC C5a 0 #2

description: Gene expression in C5aR+LysoDC #2

source_name: C5aR+ LysoDC_C5a 0 nM #2


title: OM_1


title: WGBS-seq of CRISPRoff with non-targeting sgRNA, replicate 1 [CLTA-GFP tagged line]


title: Dicer_RIP_icSHAPE_MaP_NAIN3_rep2

description:
Dicer bound sRNA(<200nt)
RIP_icSHAPE-MaP_shape.txt.gz

value: hDicer RIP treated with NAIN3 for icSHAPE-MaP


title: Dicer_RIP_icSHAPE_MaP_NAIN3_rep3


title: WGBS-seq of untransfected control, replicate 1 [CLTA-GFP tagged line]


title: miRNA WT rep 2

value: WT Col


title: Dicer_RIP_icSHAPE_MaP_NAIN3_rep1


title: caput_P42


title: K27me3_rGBM_(20200323_JS_Hs_CNT7RK27)

city: Seattle

first: Jorja

last: Henikoff

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

description: scCUT&Tag (10x GEM) for native nuclei from a recurrent glioblastoma (GBM) a sample extracted an autopsy.

data_processing:
Genome_build: UCSC HG38
A. iCell8 system: 1. We used a custom python script to demultiplex and annotate fastq reads in the following format: Antibody_CellType_Rep_Cell#, e.g K27me3_K562_r1_54. 2. we used Bowtie2 2.3.4.3 with options --end-to-end --very-sensitive --no-mixed --no-discordant -q --phred33 -I 10 -X 700 to map the paired-end 25bp reads to HG38 of Homo sapiens genomic sequence obtained from UCSC. 3. We extracted properly paired reads from Homo sapiens alignments to generate a fragment bed files <chr> <start> <stop> <barcode> <duplicateCount>. Custom software to analyse these files is available from https://github.com/Henikoff/scCUT-Tag. B. 10X system: Cell Ranger ATAC v1.1 from 10X company with default parameters was used to do the demultiplexing, alignment to HG38 and read quantification.

source_name: CUT&TAG

tag: antibody

value: H3K27me3, Cell Signaling Technology, Cat# 9733; Lot# 8

value: rGBM


title: miRNA WT rep 1


title: lymphnode_CD45pos_day14_antigen

first: Jay

middle: R.

last: Hesselberth

state: CO

department: Biochemistry and Molecular Gentetics

institute: University of Colorado School of Medicine

description:
ADT_CD45pos_d14
Single-cell antigen tracking library from the 10X Genomics 3' expression kit (v3 chemistry).
processed data file:
CD45pos_d14_barcodes.tsv.gz
CD45pos_d14_features.tsv.gz
CD45pos_d14_matrix.mtx.gz
d14_DC_count_matrix.tsv.gz
d14_DC_metadata.tsv.gz
d14_FRC_count_matrix.tsv.gz
d14_FRC_metadata.tsv.gz
d14_LEC_count_matrix.tsv.gz
d14_LEC_metadata.tsv.gz

data_processing: Single-cell RNA-seq and antigen tag libraries were processed using the 10X Genomics cellranger software (3.1.0). Analysis was performed using the R packages Seurat (3.1.4) and scrunchy (0.0.0.900). Cell type identification was performed with clustifyr (0.0.0.900) using references built from published data.

source_name: Lymph node

extract_protocol:
Two weeks following vaccination, popliteal lymph nodes were removed from 15 mice, digested, and filtered. One-third of cells were stained with CD11c (N418), CD11b, B220, and a live/dead dye (Tonbo). Live cells were sorted into CD11c+, CD11b+, B220+, and ungated live cells which were recombined at a 4:4:1:1 ratio, respectively. The cell mixture was washed with 0.1% BSA in PBS as described in the 10X Genomics Cell Prep Guide.
10X Genomics single-cell 3' expression kit combined with CITE-seq protocol.

treatment_protocol: 6-8 week old C57BL/6 mice were vaccinated with 1E3 colony forming units of Vaccinia Western Reserve combined with 10 ug of OVA-psDNA in 50 uL per footpad.

value: WT C57BL/6

value: Lymph node

tag: time point


title: Dicer_RIP_icSHAPE_MaP_DMSO_rep1

description: Dicer bound sRNA(<200nt)

value: hDicer RIP treated with DMSO for icSHAPE-MaP


title: Dicer_RIP_icSHAPE_MaP_DMSO_rep2

description: Dicer bound sRNA(<200nt)


title: K27me3_GBM_(20190910_CNT7_S1)

description: scCUT&Tag (10x GEM) for native nuclei from a glioblastoma (GBM) a sample extracted from a patient.

value: GBM


title: HCT116-CYGB rep3

city: Ningbo

first: YANG

last: XI

address: Fenghua Road

department: School of medicine

institute: Ningbo University

data_processing:
The sequencing quality were assessed with Fast QC (Version 0.11.5) and then low quality data were filtered using NGSQC (v0.4), quality value of over 50% bases of the read was less than 5, and trimmed the adapter sequence, and removed the reads containing poly-N using in-house perl scripts.
The clean reads were then aligned to the reference genome using HISAT2 (Johns Hopkins University, USA) with default parameters (Liu et al., 2015). The processed reads from each sample were aligned using HISAT (Johns Hopkins University, USA) against the corresponding hg19 reference genome. The gene expression analyses were performed withStringTie. DESeq2 was used to analyze the DEGs between samples. Thousands of independent statistical hypothesis testing were conducted on DEGs, separately. Then a p-value was obtained, which was corrected by FDR method. And Corrected P-value (q-value) was calculated by correcting using BH method. p-value or q-value were used to conduct significance analysis. Parameters for classifying significantly DEGs are ≥2- fold differences (|log2FC|≥1, FC: the fold change of expressions) in the transcript abundance and q < 0.05 (Parreira et al., 2016). By searching the ENSEMBL, NCBI, Uniprot, GO, and KEGG databases, the BLAST (Basic Local Alignment Search Tool) alignment was performed to determine the functional annotation of DEGs. The best matches were selected to annotate the DEGs. Finally, A GO functional enrichment analysis and KEGG pathway enrichment analysis were performed for the DGEs using the Goseq R package and KOBAS 3.0 software (Available online: http://kobas.cbi.pku.edu.cn) . GO terms and pathway terms with P-value(or Q-value ) less than 0.05 were considered significantly enriched by target genes.
Genome_build: hg19
Supplementary_files_format_and_content: count file

source_name: HCT116 cell line

growth_protocol: The human HCT116 cells were cultured in RPMI 1640 medium (Corning, USA) supplemented with 10 % fetal bovine serum and 100 U/ml penicillin-streptomycin (Sigma-Aldrich, USA) at 37 ℃ in an atmosphere of 5 % CO2.

extract_protocol:
Total RNA were extracted with Trizol (Tiangen, Beijing) and assessed with Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit Fluorometer (Invitrogen). Total RNA samples that meet the following requirements were used in subsequent experiments: RNA integrity number (RIN) > 7.0 and a 28S:18S ratio > 1.8.
The cluster formation and sequencing were performed on the NovaSeq 6000 platform following the manufacturer’s standard CapitalBio Technology (Beijing, China) and sequencing protocols.

treatment_protocol: We constructed CYGB gene expressing cells by lentivirus in HCT116 cells.

value: CYGB overexpression

value: human colorectal cancer cells

tag: passage


title: mRNA amiR-SEAP1 rep4


title: sRNA_icSHAPE-MaP_in_vitro_NAIN3_rep2

description:
sRNA(<200nt)
sRNA_icSHAPE-MaP_invitro_shape.txt.gz

value: WT

value: In vitro sRNAs treated with NAI-N3 for icSHAPE-MaP


title: lymphnode_CD45neg_day14_antigen

description:
ADT_CD45neg_d14
Single-cell antigen tracking library from the 10X Genomics 3' expression kit (v3 chemistry).
processed data file:
CD45neg_d14_barcodes.tsv.gz
CD45neg_d14_features.tsv.gz
CD45neg_d14_matrix.mtx.gz
d14_DC_count_matrix.tsv.gz
d14_DC_metadata.tsv.gz
d14_FRC_count_matrix.tsv.gz
d14_FRC_metadata.tsv.gz
d14_LEC_count_matrix.tsv.gz
d14_LEC_metadata.tsv.gz

extract_protocol:
Two weeks following vaccination, popliteal lymph nodes were removed from 15 mice, digested, and filtered. For two-thirds of cells, cells were stained with CD45 PE followed by magnetic bead isolation using the Miltenyi bead isolation kit. CD45 negative cells that passed through the column were washed with 0.1% BSA in PBS as described in the 10X Genomics Cell Prep Guide.
10X Genomics single-cell 3' expression kit combined with CITE-seq protocol.


title: mRNA amiR-SEAP1 rep3


title: HCT116-CYGB rep2


title: sRNA_icSHAPE-MaP_in_vitro_NAIN3_rep1


title: lymphnode_CD45pos_day14_mrna

description:
GEX_CD45pos_d14
Single-cell RNAseq library from the 10X Genomics 3' expression kit (v3 chemistry).
processed data file:
CD45pos_d14_barcodes.tsv.gz
CD45pos_d14_features.tsv.gz
CD45pos_d14_matrix.mtx.gz
d14_DC_count_matrix.tsv.gz
d14_DC_metadata.tsv.gz
d14_FRC_count_matrix.tsv.gz
d14_FRC_metadata.tsv.gz
d14_LEC_count_matrix.tsv.gz
d14_LEC_metadata.tsv.gz


title: miRNA amiR-SEAP1 rep1


title: cauda_P42

value: epididymis

value: No treatment


title: mRNA amiR-SEAP1 rep1

value: Columbia (Col)

value: inflorescence


title: HCT116-CYGB rep1


title: lymphnode_CD45neg_day14_mrna

description:
GEX_CD45neg_d14
Single-cell RNAseq library from the 10X Genomics 3' expression kit (v3 chemistry).
processed data file:
CD45neg_d14_barcodes.tsv.gz
CD45neg_d14_features.tsv.gz
CD45neg_d14_matrix.mtx.gz
d14_DC_count_matrix.tsv.gz
d14_DC_metadata.tsv.gz
d14_FRC_count_matrix.tsv.gz
d14_FRC_metadata.tsv.gz
d14_LEC_count_matrix.tsv.gz
d14_LEC_metadata.tsv.gz


title: lymphnode_CD45pos_day2_antigen

description:
ADT_CD45pos_d2
Single-cell antigen tracking library from the 10X Genomics 3' expression kit (v3 chemistry).
processed data file:
CD45pos_d2_barcodes.tsv.gz
CD45pos_d2_features.tsv.gz
CD45pos_d2_matrix.mtx.gz
d2_DC_count_matrix.tsv.gz
d2_DC_metadata.tsv.gz
d2_FRC_count_matrix.tsv.gz
d2_FRC_metadata.tsv.gz
d2_LEC_count_matrix.tsv.gz
d2_LEC_metadata.tsv.gz

extract_protocol:
Two days following vaccination, popliteal lymph nodes were removed from 15 mice, digested, and filtered. One-third of cells were stained with CD11c (N418), CD11b, B220, and a live/dead dye (Tonbo). Live cells were sorted into CD11c+, CD11b+, B220+, and ungated live cells which were recombined at a 4:4:1:1 ratio, respectively. The cell mixture was washed with 0.1% BSA in PBS as described in the 10X Genomics Cell Prep Guide.
10X Genomics single-cell 3' expression kit combined with CITE-seq protocol.


title: mRNA amiR-SEAP1 rep2


title: K27ac_PBMC_(20200624_PBMCCNTK27Ac_S3)

description: scCUT&Tag (10x GEM) for native nuclei from an anonymous peripheral blood mononuclear cells (PBMCs) donor.

value: H3K27ac, Millipore Sigma, Cat# MABE647

value: PBMCs


title: corpus_P42


title: HCT116-MOCK rep3

value: wild type


title: sRNA_icSHAPE-MaP_in_vivo_NAIN3_rep2

description:
sRNA(<200nt)
sRNA_icSHAPE-MaP_invivo_shape.txt.gz

extract_protocol:
To enrich sRNA <200nt, after RNA extraction by Trizol LS, the total RNA was subjected to gel size selection.
For icSHAPE-MaP, The 8 µL purified RNA was ligated with a 3′ linker by incubation with the 3′ ligation mix (6 µL PEG8000, 1 µL 3’ linker (10 µM), 1 µL DTT (100 mM), 2 µL 10×ligation buffer, 1 µL T4 RNA ligase KQ (NEB), 1 µL RiboLock) at 25 °C for 2 hrs, followed by inactivation of enzyme at 65°C for 20 min. The 1.2 µL RT primer (10 µM) was added to the 3’ ligation mix and annealed to excess 3’linker by 75 °C 5 min, 37 °C 15 min, 25 °C 15 min. The 5’ ligation mix ( 3 µL PEG8000, 3 µL 10 mM ATP, 1 µL 10×ligation buffer, 0.5 µL RiboLock, 0.5 µL 5′ linker (Key Resource Table, 20 µM), 1 µL T4 RNA ligase I (NEB)) was added directly and incubated at 25 °C for 2 hrs. The reaction was purified with a RNA concentration kit (Zymo). The 9 µL mutation buffer (50 mM Tris-HCl pH 8.0, 500 µM dNTPs, 75 mM KCl, 10 mM DTT, 6 mM MnCl2, 1 µL RiboLock) was added to the 10 µL purified RNA. The reaction mix was incubated at 42 °C for 2 min. Then 1 µL SuperScript II (Thermo Fisher) was added to the reaction mix. The reverse transcription mix was incubated at 42 °C for 3 hrs. cDNA products were purified with a DNA concentration kit (Zymo).PCR were set up with 20 µL eluted cDNA and PCR reaction mix (0.5 µL P5 primer (Key Resource Table, 20 µM), 0.5 µL P3 index primer (Key Resource Table, 20 µM), 0.4 µL 25×SYBR Green, 20 µL 2×Phusion HF Master Mix (NEB)). PCR was performed in a qPCR machine (Agilent, Mx3000P) to monitor the amplification procedure and was programmed as follows: stage I: 98 °C for 1 min; stage II: 98 °C for 15 s, 65°C for 30 s, 72 °C for 45 s, with limited cycles.PCR products were purified with a DNA concentration kit (Zymo) and a further size selection (150~330 nt) on a 6% Native PAGE gel.
For sRNA-seq(40-200nt), 1 µg sRNA was then incubated with the end-repairing mix (2 µL 5×PNK buffer, 2 µL T4 polynucleotide kinase (NEB), 1 µL Fast AP (Thermo Fisher), 1µL RiboLock, 4 µL nuclease-free water) at 37°C for 1 hr. The 3′ ligation mix (6 µL PEG8000, 1 µL 3′ linker (Key Resource Table, preadenylated and blocked at its 3′ end with SpC3, 10 µM), 1 µL DTT (100 mM), 1 µL 10×RNA ligation buffer, 1 µL T4 RNA ligase I (NEB)) was added directly to the end-repairing mix. The ligation condition was 25°C for 3 hrs. The ligation product was size-selected on a 6% Urea-PAGE gel (Thermo Fisher) for ~60-200 nt. Gel purification was performed by crushing the gel and incubating in the RNA elution buffer (20 mM Tris-HCl pH 7.0, 2 mM EDTA pH 8.0, 300 mM NaAc pH 5.2, RiboLock 40 U/mL) with vigorous shaking at 37°C overnight. The eluate was collected by centrifuging through 0.45 um Spin-X columns (Thermo Fisher), followed by ethanol precipitation.   For reverse transcription, 9 µL purified 3′-ligation product was mixed with 1 µL RT primer (Key Resource Table, 10 µM). Samples were heated to 70°C for 5 min in a PCR block, cooled to 25°C by stepping down 1°C every 3 sec and held at 25°C. Afterwards, 8 µL mutation buffer (50 mM Tris-HCl pH 8.0, 500 µM dNTPs, 75 mM KCl, 10 mM DTT, 6 mM MnCl2), 1 µL RiboLock, and 1 µL SuperScript II (Thermo Fisher) were added. The reaction mix was incubated at 25°C for 3 min, and then 42°C for 3 hr. cDNA products were purified by running on 6% Urea-PAGE gel. The following steps, including circularization, PCR amplification and PCR products purification, were done as previously described.
For miRNA-Seq (~20 nt), library construction, Illumina sequencing and data analysis were conducted as previously described (Gu et al., 2012), except for the following modification. Reverse transcription (RT) was set up with RNAs ligated with adaptors, 1 µL RT primer, 1×SSIV buffer, 500 µM dNTP, 5 mM DTT, 1 U/µL RiboLock, and 1 µL SuperScript IV (Thermo Fisher) in a total volume of 20 µL. The reaction was programmed as follows: 25°C for 3 min, 42°C for 10 min, 50°C for 30 min, and 80°C for 10 min. RT products were then ethanol precipitated and dissolved in 10 µL H2O. To set up PCR reactions, 5 µL of RT products were mixed with 1×Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB), 200 µM Solexa Forward and Reverse primers in a total volume of 50 µL. PCR was programmed as follows: stage I: 98°C for 30 s; stage II: 98°C for 10 s, 52°C for 30 s, 72°C for 15 s, repeating for total 22 cycles; Stage III: 72°C for 5 min. Multiplex sequencing was performed on an Illumina NextSeq 500 machine or on a MiSeq machine (1X36-bp) at the Stanford Functional Genomics Facility.

value: In vivo sRNAs treated with NAI-N3 for icSHAPE-MaP


title: mRNA WT rep 4

value: WT Col


title: lymphnode_CD45pos_day2_mrna

description:
GEX_CD45pos_d2
Single-cell RNAseq library from the 10X Genomics 3' expression kit (v3 chemistry).
processed data file:
CD45pos_d2_barcodes.tsv.gz
CD45pos_d2_features.tsv.gz
CD45pos_d2_matrix.mtx.gz
d2_DC_count_matrix.tsv.gz
d2_DC_metadata.tsv.gz
d2_FRC_count_matrix.tsv.gz
d2_FRC_metadata.tsv.gz
d2_LEC_count_matrix.tsv.gz
d2_LEC_metadata.tsv.gz

value: WT C57BL/6


title: mRNA WT rep 3


title: HCT116-MOCK rep2


title: sRNA_icSHAPE-MaP_in_vivo_NAIN3_rep1


title: sRNA_icSHAPE_MaP_DMSO_rep2

value: DMSO treated sRNAs for icSHAPE-MaP as control


title: sRNA_icSHAPE_MaP_DMSO_rep1


title: HCT116-MOCK rep1


title: mRNA WT rep 2


title: mRNA WT rep 1


title: lymphnode_CD45neg_day2_antigen

description:
ADT_CD45neg_d2
Single-cell antigen tracking library from the 10X Genomics 3' expression kit (v3 chemistry).
processed data file:
CD45neg_d2_barcodes.tsv.gz
CD45neg_d2_features.tsv.gz
CD45neg_d2_matrix.mtx.gz
d2_DC_count_matrix.tsv.gz
d2_DC_metadata.tsv.gz
d2_FRC_count_matrix.tsv.gz
d2_FRC_metadata.tsv.gz
d2_LEC_count_matrix.tsv.gz
d2_LEC_metadata.tsv.gz

extract_protocol:
Two days following vaccination, popliteal lymph nodes were removed from 15 mice, digested, and filtered. For two-thirds of cells, cells were stained with CD45 PE followed by magnetic bead isolation using the Miltenyi bead isolation kit. CD45 negative cells that passed through the column were washed with 0.1% BSA in PBS as described in the 10X Genomics Cell Prep Guide.
10X Genomics single-cell 3' expression kit combined with CITE-seq protocol.

value: Lymph node


title: lymphnode_CD45neg_day2_mrna

description:
GEX_CD45neg_d2
Single-cell RNAseq library from the 10X Genomics 3' expression kit (v3 chemistry).
processed data file:
CD45neg_d2_barcodes.tsv.gz
CD45neg_d2_features.tsv.gz
CD45neg_d2_matrix.mtx.gz
d2_DC_count_matrix.tsv.gz
d2_DC_metadata.tsv.gz
d2_FRC_count_matrix.tsv.gz
d2_FRC_metadata.tsv.gz
d2_LEC_count_matrix.tsv.gz
d2_LEC_metadata.tsv.gz


title: D17_Parenchyma_ROI15_Geometric

status: Public on Apr 11 2021

city: Boston

first: Yered

middle: Hammurabi

last: Pita-Juarez

state: MA

address: 330 Brookline Ave

department: Pathology

institute: Beth Israel Deaconess Medical Center

description: Autopsies at Beth Israel Deaconess Medical Center were performed on a cohort of 5 patients with positive SARS-CoV-2 nasopharyngeal swab on admission. Consent for autopsy and research was obtained from the healthcare proxy or the next of kin. Ultrasound-guided biopsies were performed within 3 hours of death on a gurney in the hospital morgue. The body was not removed from the bag until staff was wearing standard precautions and an N95 mask. Biopsies were obtained through 13G coaxial guide with 14 core biopsy and 20 mm sample length. Multiple core biopsies were acquired from each organ by tilting the coaxial needle a few degrees in different directions. Cores for snRNA-Seq were flash-frozen and stored at -80˚C until assayed.

data_processing:
Library strategy: NanoString GeoMx DSP RNA
FastQ files were compiled and demultiplexed with bcl2fastq
Adapter trimming with trim galore, trmGaloreOpts = " --hardtrim5 26 --dont_gzip"
Merge overlapping R1 and R2 with flash2, flash2Opts = "-m 26 -e 26 -f 26 -s 1 -r 27"
Extract UMIs in bowtie2, umiExtractOpts = " --bc-pattern=NNNNNNNNNNNNNN"
Align RTS_IDs (probe barcodes) using bowtie2, bowtie2Opts = " --end-to-end -L 4 --trim5 0 --trim3 0 --norc"
Deduplication using UMI-tools, umiDedupOpts = " --edit-distance-threshold=1“
The resulting DCC files were converted to an expression count matrix. Target counts were generated by taking the geometric mean of all probe counts against the same gene target in each well.
Normalization: The 75th percentile of the target counts of each probe pool in each AOI were calculated and normalized to the geometric mean of the 75th percentiles across all AOIs to give normalization factors. Target counts were divided by these normalization factors to give normalized expression counts.
Genome_build: N/A, sequencing of synthetic tags and alignment to whitelist of RTS_IDs (probe barcodes) found in the config.ini output from the GeoMx DSP platform, see attached NanoString GeoMx .pkc files
Supplementary_files_format_and_content: Digital Count Conversion (DCC) file format outputted from GeoMx NGS Pipeline, contains software versions, scan attributes, GeoMx NGS pipeline parameters and output metrics, Q30 scores, and list of deduplicated counts

source_name: Parenchyma

extract_protocol:
Autopsy FFPE tissues from COVID-19 infected patents were processed following the GeoMx DSP slide prep user manual (MAN-10087-04). Autopsy slides were baked in oven at 60°C for at least 1 hour, and then deparaffinized and hydrated by Leica Biosystems BOND RX. Proteinase K was added prior to the incubation of incubated with RNA probe mix (WTA and COVID-19 spike-in panel). After overnight incubation, slides were washed with buffer and stained with CD68-594 (Novus Bio, NBP2-34736AF647), CD45-647 (Novus Bio, NBP2-34527AF647), and PanCK-488 (eBioscience, 53-9003-82) and Syto83 (ThermoFisher, S11364) for 1 hour, and loaded to the GeoMx DSP machine to scan 20X fluorescent images. Regions of interest (ROIs) were placed by referencing COVID viral staining on serial section, in particular V-nCoV2019-S RNAscope (Cat: 848568-C3) staining and SARS-CoV-2 spike glycoprotein (AbCam ab272504) IHC staining. Oligos were then UV-cleaved and collected into 96-well plates. Oligos from each AOI was uniquely indexed using Illumina’s i5 x i7 dual-indexing system. 4 uL of a GeoMx DSP sample was used in the PCR reaction. PCR reactions were purified with two rounds of AMPure XP beads (Beckman Coulter) at 1.2x bead-to-sample ratio. Libraries were paired-end sequenced (2x75) on a NovaSeq up to 3 billion total aligned reads. Fastq files were processed by the NanoString DND pipeline to generate count files for each target probe, and saved as DCC files.
DSP collection sample plates were dried down by sealing the plates with aeroseal and placed at room temperature overnight. Following resuspension of the wells with 10 μL of DEPC-treated water the plates were incubated for 10 min at room temperature before briefly spun down. 96-well PCR plates were prepared by mixing 2 μL PCR mix (NanoString), 4 μL of index primer mix (NanoString) and 4 μL of DSP sample. The following PCR program was used to amplify the Illumina sequencing compatible libraries; 37°C for 30 min, 50°C for 10 min, 95°C for 3 min, followed by 18 cycles of (95°C for 15 sec, 65°C for 1 min, 68°C for 30 sec), 68°C for 5 min and a final hold at 4°C. The indexed libraries were pooled with a 8-channel pipette by combining 4 μL per well from the 12 columns into 8-well strip tubes. The combined 48 μl pools in the 8-well strip were incubated with 58 μL AMPure XP beads (Beckman Coulter) (1.2x bead to sample ratio) for 5 min. The 8-well strip was placed on a magnetic stand for 5 min before removal of the supernatant. The beads were then washed twice with 200 μl of 80% ethanol and dried for 1 min before being eluted with 10 μL elution buffer (10mM Tris-HCl pH 8, 0.05% Tween-20). The 8 samples were combined into two pools of 40 μL each and incubated with 48 μL AMPure buffer (2.5M NaCl, 20% PEG 8000) for 5 min. Following magnetic incubation for 5 min and removal of supernatants the beads were washed twice with 200 μL of 80% ethanol and dried for 1 min before eluted with 17 μL elution buffer. The two pools were combined to a final elute of 34 μL. Sequencing library size was assessed with a DNA high sensitivity bioanalyzer assay (Agilent Technologies) and the expected library size of 161 bp was observed. Library concentration was assessed with Qubit dsDNA high sensitivity assay (Thermo Fisher Scientific).

tag: segment

value: Geometric Segment

tag: morphology

value: Inflamed Alveoli

tag: assay

value: WTA

tag: race

value: White

tag: ethnicity

value: Not Hispanic

value: M

tag: age

tag: organ failure

value: Lung, heart, kidney

tag: s/s to death (days)

tag: donor


title: TNF t48h rep2

city: Belvaux

first: Luc

last: Grandbarbe

address: 7, Avenue des Hauts Fourneaux

department: Life Sciences Research Unit

country: Luxembourg

institute: University of Luxembourg

description: TNF t48h rep2

hyb_protocol: The cDNA was hybridized onto Affymetrix GeneChip Mouse Gene 1.0 ST Arrays according to the GeneChip WT Terminal Labeling and Hybridization User Manual for use with the Ambion Whole Transcript Expression kit (P/N 702808 Rev.6)

scan_protocol: Arrays were scanned with the Affymetrix® GeneChip® Scanner 3000

data_processing: data were imported into R (v3.3.1) and processed by the oligo package. The normalization was performed with the RMA function and the quantile method and the summarization at transcript cluster

label: biotin

source_name: TNF

label_protocol: The cDNA was labeled according to the GeneChip WT Terminal Labeling and Hybridization User Manual for use with the Ambion Whole Transcript Expression kit (P/N 702808 Rev.6)

growth_protocol: Primary cultures of neurospheres (NSPs) were obtained from embryonic  murine neural stem cells derived from the ventricular zone of embryonic day 14 (E14) C57BL/6J mouse embryos (Harlan, The Netherlands)

extract_protocol: total RNA was extracted, using RNA NOWTM reagent (Ozyme, France) according to the manufacturers instructions. Total RNA integrity and purity were assessed using the Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip kits (Agilent Technologies).

treatment_protocol: NSPs were differentiated into astrocytes on poly-L-ornithine-pre-coated 6 wells plates with DMEM containing 10% FBS (Fetal Bovine Serum; Gibco), penicillin (100 U/ml; Lonza), and streptomycin (100 g/ml; Lonza). Cells were differentiated with and without TNF (50 ng/ml; R&D Systems) and then incubated for 6, 24, 48, or 72h at 37°C in 5% CO2.

tag: cell type

value: neural progenitor cells-derived astrocytes

tag: treatment

value: TNF

tag: time


title: TNF t24h rep3

description: TNF t24h rep3


title: control t48h rep3

description: control t48h rep3

source_name: control (FCS)

value: control


title: D17_Parenchyma_ROI14_Geometric

value: Normal Alveoli

tag: Sex


title: TNF t48h rep3

description: TNF t48h rep3


title: TNF t6h rep3

description: TNF t6h rep3


title: control t24h rep3

description: control t24h rep3


title: control t6h rep3

description: control t6h rep3

source_name: FCS


title: control t72h rep3

description: control t72h rep3


title: TNF t24h rep2

description: TNF t24h rep2


title: TNF t72h rep1

description: TNF t72h rep1


title: control t72h rep1

description: control t72h rep1


title: control t48h rep2

description: control t48h rep2


title: TNF t6h rep2

description: TNF t6h rep2


title: control t0h rep1

description: control t0h rep1


title: control t0h rep3

description: control t0h rep3


title: TNF t48h rep1

description: TNF t48h rep1


title: control t48h rep1

description: control t48h rep1


title: control t72h rep2

description: control t72h rep2


title: TNF t6h rep1

description: TNF t6h rep1


title: control t6h rep1

description: control t6h rep1


title: control t24h rep2

description: control t24h rep2


title: control t6h rep2

description: control t6h rep2


title: control t0h rep2

description: control t0h rep2


title: TNF t24h rep1

description: TNF t24h rep1


title: D17_Parenchyma_ROI6_Geometric


title: D17_Parenchyma_ROI9_Geometric


title: D17_Parenchyma_ROI11_Geometric


title: D17_Parenchyma_ROI8_Geometric


title: D17_Parenchyma_ROI5_Geometric


title: D17_Parenchyma_ROI10_Geometric


title: D17_Parenchyma_ROI4_Geometric

value: Bronchial Epithelium


title: D17_Parenchyma_ROI2_Geometric


title: D17_Parenchyma_ROI3_Geometric


title: D17_Parenchyma_ROI7_Geometric


title: D16_Parenchyma_ROI25_Geometric

value: Black/ AA

value: F

value: Lung, heart


title: D16_Parenchyma_ROI24_Geometric


title: D17_Parenchyma_ROI1_Geometric


title: D16_Parenchyma_ROI19_Geometric


title: D16_Parenchyma_ROI20_Geometric


title: control t24h rep1

description: control t24h rep1


title: D16_Parenchyma_ROI15_Geometric


title: D15_Parenchyma_ROI12_Geometric

value: Artery

value: Lung, kidney


title: D16_Parenchyma_ROI14_Geometric

value: Geometric Segment


title: D16_Parenchyma_ROI16_Geometric


title: D16_Parenchyma_ROI18_Geometric

value: Inflamed Alveoli


title: D15_Parenchyma_ROI10_Geometric

value: WTA


title: D15_Parenchyma_ROI9_Geometric


title: D16_Parenchyma_ROI23_Geometric


title: D15_Parenchyma_ROI13_Geometric


title: D17_Parenchyma_ROI13_Geometric

value: White


title: D15_Parenchyma_ROI11_Geometric


title: D16_Parenchyma_ROI17_Geometric

value: Not Hispanic


title: D15_Parenchyma_ROI7_Geometric


title: D15_Parenchyma_ROI5_Geometric


title: D15_Parenchyma_ROI6_Geometric


title: D15_Parenchyma_ROI3_Geometric


title: D15_Parenchyma_ROI4_Geometric


title: D15_Parenchyma_ROI2_Geometric


title: D14_Parenchyma_ROI30_Geometric

value: Lung


title: D15_Parenchyma_ROI1_Geometric


title: D14_Parenchyma_ROI27_Geometric


title: D17_Parenchyma_ROI12_Geometric

value: M

value: Lung, heart, kidney


title: D14_Parenchyma_ROI28_Geometric


title: D14_Parenchyma_ROI26_Geometric


title: D14_Parenchyma_ROI25_Geometric


title: D14_Parenchyma_ROI23_Geometric


title: D14_Parenchyma_ROI24_Geometric


title: D14_Parenchyma_ROI21_Geometric


title: D14_Parenchyma_ROI19_Geometric


title: D14_Parenchyma_ROI11_Geometric


title: D14_Parenchyma_ROI17_Geometric


title: D14_Parenchyma_ROI14_Geometric


title: D14_Parenchyma_ROI10_Geometric


title: D14_Parenchyma_ROI13_Geometric


title: D14_Parenchyma_ROI9_Geometric


title: D15_Parenchyma_ROI8_Geometric


title: D14_Parenchyma_ROI12_Geometric


title: D14_Parenchyma_ROI7_Geometric


title: D14_Parenchyma_ROI6_Geometric


title: TNF t72h rep2

description: TNF t72h rep2

value: neural progenitor cells-derived astrocytes

value: TNF


title: D14_Parenchyma_ROI29_Geometric


title: D14_Parenchyma_ROI4_Geometric


title: D14_Parenchyma_ROI3_Geometric


title: D14_Parenchyma_ROI5_Geometric


title: D14_Parenchyma_ROI20_Geometric

value: Normal Alveoli


title: D14_Parenchyma_ROI2_Geometric


title: D14_Parenchyma_ROI1_Geometric


title: D13_Parenchyma_ROI16_Geometric


title: D13_Parenchyma_ROI15_Geometric


title: D14_Parenchyma_ROI18_Geometric


title: D13_Parenchyma_ROI13_Geometric


title: D13_Parenchyma_ROI9_Geometric


title: D13_Parenchyma_ROI11_Geometric


title: D13_Parenchyma_ROI12_Geometric


title: D13_Parenchyma_ROI8_Geometric


title: D13_Parenchyma_ROI7_Geometric


title: D13_Parenchyma_ROI5_Geometric


title: D13_Parenchyma_ROI10_Geometric


title: D13_Parenchyma_ROI6_Geometric


title: D13_Parenchyma_ROI2_Geometric


title: D13_Parenchyma_ROI4_Geometric