title: RSG15

city: Delhi

first: Vivek

middle: T

last: Natarajan

state: Delhi

address: Mathura Road

country: India

institute: CSIR-IGIB

data_processing:
Quality check FastQC -0.11.5
Pre-processing to remove adaptor sequence Cutadapt - 0.11.5
Alignment HISAT2-2.0.4
Quantification Htseq-0.11.1
Normalised counts were generated using DESeq2
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include normalized count values for each Sample ...
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: B16 melanoma

growth_protocol: B16 cells are cultured at a density of 100 cells/sq.cm in DMEM-high glucose and cultured for 7 days in same media.

extract_protocol:
Macherey-Nagel RNA isolation kit protocol is used
RNA libraries were prepared for sequencing using standard Illumina protocols
TruSeq RNA Sample Prep Kit v2

treatment_protocol: No treatment is given. D3 -D7 cells are harvested to capture transition from depigmented to pigmented state

value: B16 melanocytes

tag: pigmentation state


title: Sample 16: P2.C.P.1

status: Public on Apr 19 2022

type: SRA

city: Cambridge

first: Irina

last: Mohorianu

address: Puddicombe Way

department: Wellcome-MRC Cambridge Stem Cell Institute

country: United Kingdom

institute: University of Cambridge

library_source: transcriptomic

data_processing:
The quality of the data was assessed using FastQC, MultiQC, PCAs, Jaccard Similarity Indices and MA plots
the reads were aligned using STAR v2.7.3a (parameters --outSAMtype BAM SortedByCoordinate --outReadsUnmapped Fastx --readFilesCommand gunzip -c)
Protein-coding gene quantification was performed using Subread featureCounts v2.0.0 (default parameters)
The raw expression levels were normalized with edgeR v3.26.8. After normalization, a noise filtering step was applied i.e. for each gene, if the maximum normalised expression level was <10 then the gene was excluded from the downstream analysis.
Statistical testing for differential expression of genes was performed using edgeR, with multiple-testing correction based on the Benjamini-Hochberg method. DE Genes were called on adj p-value < 0.05, and |log2(FC)| > 0.5.
The enrichment analysis was performed using g:profiler (Reimand et al., Nucleic Acids Research, 2016)
Genome_build: GRCh38.p13
Supplementary_files_format_and_content: raw.csv contains raw gene counts; normalized.csv contains edgeR-normalized expression levels after the application of the noise filter

molecule: total RNA

organism: Homo sapiens

source_name: Primary CLL cells

growth_protocol: Primary CLL cells were transfected with NSC or ZAP-70 siRNA and co-cultured with EL08 stromal cells in RPMI 1640 medium for totally 7 days.

extract_protocol:
Primary CLL cells were harvested after positivley selected by anti-CD19 beads and total RNA was extracted using the standard protocol from GenElute Mammalian Total RNA Miniprep Kit (Sigma/RTN70).
NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB #7420) was used with 100ng of total RNA for the construction of sequencing libraries. mRNA was isolated and purified by using NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB #7490) before cDNA synthesis.

treatment_protocol: For samples with anti-IgM treatment, primary CLL cells were co-cultured with EL08 stromal cells and treat with anti-IgM 4ug/1x10^6 CLL cells.

tag: patient

tag: disease state

value: Chronic Lymphocytic Leukemia

tag: treatment

value: NSC

tag: replicate

value: Rep 1


title: H9_day6_rep1

type: RNA

city: Ft Worth

first: Victor

last: Lin

address: 3500 Camp Bowie Blvd

country: USA

institute: UNTHSC

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: RSN normalization in R

source_name: cerebral organoids, WA09 derived_parent (unedited, did not undergo CRISPR editing)

label_protocol: Standard Illumina label protocol

extract_protocol: RNA was extracted with Ambion MirVana Isolation Kit

tag: cell type

tag: time point

value: replicate 1


title: Sample 12: P1.T.P.3

value: Rep 3


title: SW1353_RRBS_rep3_G20

city: Shanghai

first: Jianing

last: Wang

address: Dongchuan Road

country: China

institute: Shanghai JiaoTong University

library_source: genomic

data_processing:
RRBS reads were trimmed using Trim Galore, reads containing adapter were discarded.
Then used Bismark to align trimmed reads to hg38 genome
Next Bismark comes with a supplementarybismark_methylation_extractor script which operates on Bismark result files and extracts the methylation call for every single C analyzed.
Then we merged all the triplicate data to classify 100% ,50% and 0% methylation sites
Assembly: hg38 (GRCh38)
Supplementary files format and content: Tab-delimited text file includes chromosome position and methylation level for each 100%,50%,0% methylation fidelity CpG site .

molecule: genomic DNA

source_name: chondrosarcoma

growth_protocol: The cells were cultured with DMEM/F-12 medium (Biological Industries, Israel) containing 20% fetal bovine serum (Gibco BRL/Invitrogen, Carlsbad, CA, USA), 100μg/ml streptomycin, and 100IU/ml penicillin, and incubated with 37℃ and 5% CO2.

extract_protocol:
Genomic DNA from ARPE-19,Jurkat and SW1353 was extracted by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol.Two microgram of genomic DNA was used to construct the RRBS library by NEBNext Ultra II library kit and EZ DNA Methylation-Gold™ Kit (Zymo Research) following the manufacturer’s protocol. The quality of libraries was assessed with Agilent 2100 Bioanalyzer (Agilent, United States).
To evaluate the conversion error of bisulfite conversion, the methylated spike in and unmethylated spike in were used. We chose two different regions as the templates from the λDNA. One template was amplified with dNTP Mix to synthesize the unmethylated spike in, and the other one was amplified with 5-Methylcytosine dNTP Mix to synthesize the methylated spike in. Then the spike in were fragmented to about 300bp by Covaris (Covaris, USA). After constructing libraries using NEBNext Ultra II Library Prep Kit for Illumina® separately, the methylated spike in and unmethylated spike in were mixed together and spiked to the DNA at a ratio of 1% before bisulfite conversion. Two micrograms of genomic DNA were used for MspI (NEB, San Diego, California, USA) digestion. And the fragments were recovery by using AxyPrep PCR Clean kit. After constructing the sequencing library and mixing the spike in, bisulfite conversion was performed on the libraries by the EZ DNA Methylation-Gold Kit (Zymo Research, USA), which was also used to recovery the libraries. Epimark Hot Start Enzyme was used for PCR to amplify the libraries. To obtain the fragments exactly, the vertical TBE-PAGE electrophoresis was performed on the 6% TBE-PAGE Gel (Novex, Invitrogen, USA) to better separate the bands. After recovering the libraries from the gel, the Agilent 2100 Bioanalyzer system (Agilent, USA) was used for assessing the distribution of libraries.

treatment_protocol: Serum starvation for 24h before collecting the sample.

tag: tissue

value: chondrosarcoma

tag: cell line

value: Fibroblast Cells

value: Normal culture


title: RSG13

organism: Mus musculus

tag: strain


title: RSG14


title: Patient 15, Normal, Left Side colon cancer

city: INDIANAPOLIS

first: Ashiq

last: Masood

state: Indiana

address: 410 W. 10th Street

department: Heamatology and oncology

institute: Indiana University

description: 10x Genomics

data_processing:
Cell Ranger was utilized to process the raw gene expression matrices per sample and all samples from multiple patients were combined in R package (v3.6.3 2020-02-29] -- "Holding the Windsock"). Seurat package (v3.2.2) was used in this integrative multimodal analysis [44]. Genes detected in fewer than three cells and cells expressing less than 200 detected genes were filtered out and excluded from analysis. In addition, cells expressing > 25% mitochondria were removed. Cell cycle scoring was performed, (for the S phase and the G2M phase) and the predicted cell cycle phases were calculated. Doublet detection and any higher-order multiplets that were not dissociated during sample preparation were removed  via the DoubletFinder (v2.0.2) package using default settings [80]. Following quality control one normal colon sample (B-cac13) was discarded due to poor data quality. Finally, 49,859 cells remained and were utilized for downstream analysis.
Assembly: GRCh38/hg38
Supplementary files format and content: comma-separated values (CSV) file containing the raw gene expression.

source_name: Colorectal cancer

extract_protocol: Single-cell RNA sequencing (scRNA-seq) was performed using 10X Genomics Single Cell 5' Platform. Tumors and normal colon samples were enzymatically dissociated (Miltenyi), filtered through a 70-micron cell strainer, pelleted after centrifugation at 300 xg and resuspended in DAPI-FACS buffer (PBS, 0.04% BSA). Samples were sorted and viable singlets were gated on the basis of scatter properties and DAPI exclusion. Approximately 3000 cells were pelleted and resuspended in PBS, and cells underwent single cell droplet-based capture on 10X Chromium instruments according to the 10X Genomics Single Cell 5' Platform protocol. Transcriptome libraries were prepared post-fragmentation, end-repair, and A-tailing double-sided size selection, and subsequent adaptor ligation as per the manufacturer’s protocol. Illumina NextSeq 550 was used for library sequencing and data were mapped and counted using Cellranger-v3.1.0 (GRCh38/hg38).

value: Colon,Left,Sigmoid

value: Colorectal cancer


title: SW1353_RRBS_rep2_G20


title: H9_day0_rep2

value: cerebral organoids, WA09 derived_parent (unedited, did not undergo CRISPR editing)

value: replicate 2


title: H9_day0_rep1


title: aorta_DIO_rep3

city: BeiJing

first: Yi

last: Zong

address: No. 38 Xue Yuan Road

institute: Peking University Health Science Center

hyb_protocol: 1. Add 500 μL of nuclease-free water to the vial containing lyophilized 10X Blocking Agent. Mix by gently vortexing.  2. Equilibrate water bath to 60°C.  3. For each microarray, add each of the components as indicated in the tables as below to a 1.5 mL nuclease-free microfuge tube:  For 1*244K array Amount  cyanine 3-labeled, linearly amplified cRNA 1.5ug  10X Blocking Agent 50ul  Nuclease-free water ×ul  25X Fragmentation Buffer 10ul  Total volume 250ul    For 2*105K array Amount  cyanine 3-labeled, linearly amplified cRNA 1. 5ug  10X Blocking Agent 25ul  Nuclease-free water ×ul  25X Fragmentation Buffer 5ul  Total volume 125ul    For 4*44K array Amount  cyanine 3-labeled, linearly amplified cRNA 1.65ug  10X Blocking Agent 11ul  Nuclease-free water ×ul  25X Fragmentation Buffer 2.2ul  Total volume 55ul    For 8*15K array Amount  cyanine 3-labeled, linearly amplified cRNA 0.6ug  10X Blocking Agent 5ul  Nuclease-free water ×ul  25X Fragmentation Buffer 1ul  Total volume 25ul    For 8*60K array Amount  cyanine 3-labeled, linearly amplified cRNA 0.6ug  10X Blocking Agent 5ul  Nuclease-free water ×ul  25X Fragmentation Buffer 1ul  Total volume 25ul      4. Mix well but gently on a vortex mixer.  5. Incubate at 60°C for exactly 30 minutes to fragment RNA.   6. Add 2x GEx Hybridization Buffer HI-RPM to the array to stop the fragmentation reaction.  Volumes per hybridization   Components 1*244K 2*105K 4*44K 8*15K 8*60k  cRNA from Fragmentation Mix 250ul 125ul 55ul 25ul 25ul  2* GEx Hybridization Buffer HI-RPM 250ul 125ul 55ul 25ul 25ul    7. Mix well by careful pipetting. Take care to avoid introducing bubbles. Do not mix on a vortex mixer; mixing on a vortex mixer introduces bubbles.  8. Spin for 1 minute at room temperature at 13,000 rpm in a microcentrifuge to drive the sample off the walls and lid and to aid in bubble reduction.  9. Place sample on ice and load onto the array as soon as possible.   10. Load a clean gasket slide into the Agilent SureHyb chamber base with the label facing up and aligned with the rectangular section of the chamber base. Ensure that the gasket slide is flush with the chamber base and is not ajar.  11. Slowly dispense the volume of hybridization sample onto the gasket well in a “drag and dispense” manner.  Volumes per hybridization   Components 1*244K 2*105K 4*44K 8*15K 8*60K  Volume Prepared 500ul 250ul 110ul 50ul 50ul  Hybridization Sample Volume 490ul 245ul 100ul 40ul 40ul    12. Slowly place an array “active side” down onto the SureHyb gasket slide, so that the “Agilent”-labeled barcode is facing down and the numeric barcode is facing up. Verify that the sandwich-pair is properly aligned.   13. Place the SureHyb chamber cover onto the sandwiched slides and slide the clamp assembly onto both pieces.  14. Hand-tighten the clamp onto the chamber.  15. Vertically rotate the assembled chamber to wet the gasket and assess the mobility of the bubbles.  16. Place assembled slide chamber in rotisserie in a hybridization oven set to 65°C. Set your hybridization rotator to rotate at 10 rpm.  17. Hybridize at 65°C for 17 hours.

scan_protocol: 1. Assemble the slides into an slide holder.   2. Place assembled slide holders into scanner carousel.  3. Verify scan settings for one-color scans.   Parameters  Scan region Scan Area (61 x 21.6 mm)  Scan resolution (μm) 5  5μm scanning mode Single Pass  eXtended Dynamic range (selected)  Dye channel Green  Green PMT   XDR Hi 100%   XDR Lo 10%  4. Click Scan Slot m-n on the Scan Control main window where the letter m represents the Start slot where the first slide is located and the letter n represents the End slot where the last slide is located.

data_processing: 1. Open the Agilent Feature Extraction (FE) software.  2. Add the images (.tif) to be extracted to the FE Project.  3. Set FE Project Properties.  4. Check the Extraction Set Configuration.  5. Save the FE Project (.fep) by selecting File > Save As and browse for desired location.  6. Select Project > Start Extracting and export data to txt.

source_name: aorta

label_protocol: 1. Add 1ug of total RNA to a 1.5-mL microcentrifuge tube.   2. Add 1.2 μL of T7 Promoter Primer.   3. Use nuclease-free water to bring the total reaction volume to 11.5 μL.  4. Denature the primer and the template by incubating the reaction at 65°C in a circulating water bath for 10 minutes.  5. Place the reactions on ice and incubate for 5 minutes.  6. Immediately prior to use, gently mix the components listed in the following table for the cDNA Master Mix by adding in the order indicated, and put on ice.   Volume(ul) per reaction  5×First Strand Buffer 4  0.1M DTT 2  10mM dNTP mix 1  MMLV-RT 1  RNaseOut 0.5  Total volume 8.5  7. Briefly spin each sample tube in a microcentrifuge to drive down the contents from the tube walls and the lid. Return the tubes to ice.  8. Add 8.5 μL of cDNA Master Mix to each sample tube and mix by pipetting up and down.  9. Incubate samples at 40°C in a circulating water bath for 2 hours.  10. Move samples to a 65°C circulating water bath and incubate for 15 minutes.  11. Move samples to ice. Incubate for 5 minutes.  12. Spin samples briefly in a microcentrifuge to drive down tube contents from the tube walls and lid.  13. Immediately prior to use, gently mix the components listed in the following table in the order indicated for the Transcription Master Mix by pipetting at room temperature.   Volume(ul) per reaction  Nuclease-free water 15.3  4X Transcription Buffer 20  0.1 M DTT 6  NTP mix 8  50% PEG 6.4  RNaseOUT 0.5  Inorganic pyrophosphatase 0.6  T7 RNA Polymerase 0.8  Cyanine-3-CTP 2.4  Total volume 60  14. Add 60 μL of Transcription Master Mix to each sample tube. Gently mix by pipetting.  15. Incubate samples in a circulating water bath at 40°C for 2 hours.   16. Add 20 μL of nuclease-free water to  cRNA sample, for a total volume of 100 μL.  17. Add 350 μL of Buffer RLT and mix well by pipetting.  18. Add 250 μL of ethanol (100% purity) and mix thoroughly by pipetting. Do not centrifuge.  19. Transfer the 700 μL of the cRNA sample to an RNeasy mini column in a 2 mL collection tube. Centrifuge the sample at 4°C for 30 seconds at 13,000 rpm. Discard the flow-through and collection tube.  20. Transfer the RNeasy column to a new collection tube and add 500 μL of buffer RPE (containing ethanol) to the column. Centrifuge the sample at 4°C for 30 seconds at 13,000 rpm. Discard the flow-through. Re-use the collection tube.  21. Add another 500 μL of buffer RPE to the column. Centrifuge the sample at 4°C for 60 seconds at 13,000 rpm. Discard the flow-through and the collection tube.  22. Elute the cleaned cRNA sample by transferring the RNeasy column to a new 1.5 mL collection tube. Add appropriate RNase-free water (Passed-please see report ”Labeling Efficiency-QC”) directly onto the RNeasy filter membrane. Wait 60 seconds, then centrifuge at 4°C for 30 seconds at 13,000 rpm.  23. Maintain the cRNA sample-containing flow-through on ice. Discard the RNeasy column.  24. Take 1.5 μl of each sample to determine the yield and specific activity by using the NanoDrop ND-1000( Passed-please see report ”Labeling Efficiency-QC”).  From the main menu, choose MicroArray Measurement. Go to the Sample Type pull-down menu and select DNA-50. Blank the instrument with 1.5 μL of 1x labeling solution.   Use 1.5 μL of purified labeled genomic DNA for quantitation. Measure the absorbance at A260nm (DNA), A550nm (cyanine 3).   The specific activity (pmol dyes per μg genomic DNA) of the labeled genomic DNA can be obtained by the following calculation:  (Concentration of Cy3)  Specific Activity =——————————————— = pmol Cy3 per μg cRNA  (Concentration of cRNA) * 1000  * If the yield is <1.65 μg and the specific activity is <9.0 pmol Cy3 per μg cRNA do not proceed to the hybridization step. Repeat cRNA preparation.

extract_protocol: 1. Homogenization  a) Cryopulverization of tissue  in a shallow container and cool thoroughly with either liquid nitrogen.  Place prefrozen tissue in the well of the mortar.  Remove the mortar from the shallow container, place it on the lab bench and pulverize the tissue with one or two blows to the pestle with a hammer.  Empty the powdered contents  homogenization，then add TRIzol  or extraction procedures.  2. Phase Separation  Incubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of TRIzol Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 2 to 3 minutes. Centrifuge the samples at 12,000 × g for 15 minutes at 4°C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIzol Reagent used for homogenization.  3. RNA Precipitation  Transfer the aqueous phase to a fresh tube. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIzol Reagent used for the initial homogenization. Incubate samples at 15 to 30°C for 10 minutes and centrifuge at 12,000 × g for 10 minutes at 4°C.   4. RNA Wash  Remove the supernatant. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIzol Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at 7,500 × g for 5 minutes at 4°C.  5. Redissolving the RNA  At the end of the procedure, air-dry the RNA pellet for 5-10 minutes. Dissolve RNA in 85ul or less RNase-free water by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55 to 60°C.

tag: gender

value: male

value: aorta

value: high-fat diet for four months without oral sitagliptin treatment


title: aorta_DIO_rep2


title: aorta_DIO+S_rep1

value: Oral SItagliptin treatment (3 mg/kg/d) of high-fat diet-fed


title: H9_day14_rep1


title: aorta_DIO+S_rep2


title: Patient 14, Normal, Left Side colon cancer

description: 10x Genomics

value: Colon,Left,Descending


title: RSG16

value: B16 melanocytes


title: aorta_DIO_rep1


title: aorta_DIO+S_rep3


title: Sample 11: P1.T.P.2

value: Chronic Lymphocytic Leukemia

value: Rep 2


title: SW1353_RRBS_rep2_G30


title: aorta_Veh_rep3

value: vehicle


title: Sample 5: P1.C.P.2

value: NSC


title: Patient 10, Normal, Left Side colon cancer

description: 10x Genomics


title: Sample 10: P1.T.P.1

value: Rep 1


title: Jurkat_RRBS_rep2_G30

source_name: peripheral blood

value: peripheral blood

value: Jurkat

value: lymphocyte cells


title: RSG12


title: Jurkat_RRBS_rep3_G30

value: Normal culture


title: Patient 11, Normal, Right Side colon cancer

description: 10x Genomics

value: Colon,Right,Ascending

value: Colorectal cancer


title: RSG09


title: RSG08


title: Patient 7, Normal, Right Side colon cancer

description: 10x Genomics

value: Colon,Right,Cecum/IC


title: Sample 4: P1.C.P.1


title: Sample 6: P1.C.P.3

value: Rep 3


title: Jurkat_RRBS_rep3_G20


title: RSG11


title: Patient 6, Normal, Left Side colon cancer

description: 10x Genomics

value: Colon,Left,Sigmoid


title: RSG10


title: Patient 4, Normal, Left Side colon cancer

description: 10x Genomics


title: Sample 20: P2.T.M.2


title: aorta_Veh_rep2

value: male

value: aorta


title: aorta_Veh_rep1


title: Jurkat_RRBS_rep2_G20


title: Sample 15: P2.C.M.3


title: A20_3Day_Nerve_Injury

city: San Francisco

first: Wulin

last: Tan

state: California

address: Room 345, Rock Hall, 1550 4th St

department: Anesthesia

institute: UCSF

description: Cx3cr1-yfp-creER/+; Tnfaip3 fl/fl 3 days after nerve injury

data_processing:
fastq files were processed by CellRanger 2.1.1.
Read alignment and count summarization were performed using CellRanger v2.1.1 CellRanger count.
Counts from different samples were aggregated using CellRanger aggr.
Genome_build: mm10
Supplementary_files_format_and_content: matrix.mtx files contain read counts for all cells in a matrix; genes.tsv files contain Ensembl IDs and gene symbols; barcode.tsv files contain the 10X barcodes to identify cells.

source_name: lumbar microglia

extract_protocol:
lumbar cords were dissected. microglia were FACS sorted by cx3cr1-YFP(+).
Chromium Next GEM Automated Single Cell 3′ Library and Gel Bead Kit v3.1 (10X genomics)

treatment_protocol: Bilateral sciatic nerve transection

value: lumbar cord

value: microglia

value: 3 days after nerve injury


title: Patient 16, Tumor, Left Side colon cancer

description: 10x Genomics

value: Colon,Left,Rectosigmoid


title: RSG07

value: B16 melanocytes


title: ARPE-19_RRBS_rep2_G30

source_name: retina

value: retina

value: Epithelial Cells


title: Sample 14: P2.C.M.2

value: Chronic Lymphocytic Leukemia

value: NSC

value: Rep 2


title: Sample 19: P2.T.M.1

value: Rep 1


title: ARPE-19_RRBS_rep3_G20

value: Normal culture


title: ARPE-19_RRBS_rep2_G20


title: Patient 14, Tumor, Left Side colon cancer

description: 10x Genomics

value: Colon,Left,Descending

value: Colorectal cancer


title: ARPE-19_RRBS_rep3_G30


title: Patient 13, Tumor, Right Side colon cancer

description: 10x Genomics

value: Colon,Right,Cecum/Appendix


title: A20_2Day_Nerve_Injury

description: Cx3cr1-yfp-creER/+; Tnfaip3 fl/fl 2 days after nerve injury

value: 2 days after nerve injury


title: Sample 13: P2.C.M.1


title: A20_1Day_Nerve_Injury

description: Cx3cr1-yfp-creER/+; Tnfaip3 fl/fl 1 day after nerve injury

value: 1 day after nerve injury


title: PANC1 cercosporamide poly rep 3

city: Stockholm

first: Ola

last: Larsson

department: Department of oncology-pathology

country: Sweden

institute: Karolinska Institutet

data_processing:
The Bcl to FastQ conversion was performed using bcl2fastq_v2.20.0.422 from the CASAVA software suite. The quality scale used is Sanger / phred33 / Illumina 1.8+.
RNA sequencing read quality was assessed using FastQC v.0.11.8 and MultiQC.
Sequencing adapters and reads mapping to ribosomal RNA were removed using BBDuk from the BBTools suite (http://jgi.doe.gov/data-and-tools/bb-tools/) prior to alignment to hg38 using HISAT2 with default settings.
Reads were summarized using the featureCounts function of the Rsubread R package with default settings and RefSeq gene definitions.
Assembly: GRCh38
Supplementary files format and content: Matrix of raw gene expression counts based on RefSeq gene definitions.

source_name: pancreatic ductal adenocarcinoma

growth_protocol: PANC1 cells were cultured in RPMI 1640 media with GlutaMAX, supplemented to a final concentration of 10% Fetal Bovine Serum (FBS, Gibco), and penicillin (100 units/mL)/streptomycin (100 ug/mL). Cells were kept in a humidified incubator at 37°C in 5% CO2.

extract_protocol:
Polysome profiling was performed on four replicates of PANC1 cells treated with MNK2 inhibitors or DMSO as previously described (Ristau et al. Methods Mol Biol, 2022). Briefly, total cytosolic RNA was isolated from aliquots of cell lysates using TRIzol reagent. The remaining lysates were layered onto optimized sucrose density gradients (5:34:55% [w/v]) allowing polysomes to be fractionated. Fractions corresponding to mRNAs associated with more than three ribosomes were collected in TRIzol reagent and pooled, allowing isolation of efficiently translated polysome-associated mRNA.
Smart-seq2 RNA sequencing libraries were prepared as previous described (Picelli et al. Nature Protocols, 2014) using 10 ng of RNA input per sample. Pre-amplification PCR was performed with 12 cycles, and 100 pg of cDNA was used for tagmentation. After tagmentation, adapter-ligated fragments were amplified with 14 PCR cycles.

treatment_protocol: Four million PANC1 cells were seeded in 15 cm plates 24 hours prior to treatment prior to treatment with CID661578 (40 uM), cercosporamide (5 uM), or DMSO. Treatments were administered in complete growth media, and were applied to cells for 6 hours.

value: Human pancreatic ductal adenocarcinoma cell line

value: cercosporamide (5 uM), 6hrs

tag: sample identifier

tag: molecule subtype

value: polysome-associated mRNA


title: PANC1 cercosporamide poly rep 4


title: Sample 9: P1.T.M.3

value: Rep 3


title: Patient 12, Tumor, Right Side colon cancer

description: 10x Genomics

value: Colon,Right,Cecum


title: Sample 8: P1.T.M.2


title: PANC1 cercosporamide total rep 4

value: total cytosolic RNA


title: YFP_3Day_Nerve_Injury

description: Cx3cr1-yfp-creER/+ 3 days after nerve injury

value: lumbar cord

value: microglia

value: 3 days after nerve injury


title: YFP_2Day_Nerve_Injury

description: Cx3cr1-yfp-creER/+ 2 days after nerve injury


title: A20_Naive

description: Cx3cr1-yfp-creER/+; Tnfaip3 fl/fl naïve

value: naïve


title: PANC1 cercosporamide poly rep 2


title: Sample 7: P1.T.M.1

value: Chronic Lymphocytic Leukemia

value: Rep 1


title: Sample 3: P1.C.M.3

value: NSC


title: Sample 22: P2.T.P.2

value: Rep 2


title: Patient 15, Tumor, Left Side colon cancer

description: 10x Genomics

value: Colon,Left,Sigmoid

value: Colorectal cancer


title: YFP_1Day_Nerve_Injury

description: Cx3cr1-yfp-creER/+ 1 day after nerve injury

value: 1 day after nerve injury


title: E20/p53.6 : Pik3ca/p53   double gain of function mutant tongue sample 6

city: New York

first: Richard

middle: A

last: Friedman

state: NY

address: 1130 St. Nicholas Av

department: Medicine

institute: Columbia University Irving  Medical Center

data_processing:
Bases were called with RTA (Illumina).
Adaptors were trimmed and reads were converted from BCL to Fasta format with bcl2fastq (version 1.8.4).
Fastq files were trimmed of low quality bases and adapters with Trimmomatic
Reads were mapped to the genome with Rubread.
Reads were assigned to genes and counted with featurecounts
Genome_build: mm10
Supplementary_files_format_and_content: text file with raw counts for each gene

source_name: LSL- PIK3CAH1047R;LSL-p53+/R172H;K14-Cre squamous epithelium from mouse tongue

extract_protocol: TruSeq Stranded mRNA LT Sample Prep Kit

value: squamous epithelium

tag: genotype/variation


title: PANC1 cercosporamide total rep 2

value: Human pancreatic ductal adenocarcinoma cell line

value: cercosporamide (5 uM), 6hrs


title: Patient 11, Tumor, Right Side colon cancer

description: 10x Genomics

value: Colon,Right,Ascending


title: Patient 8, Tumor, Right Side colon cancer

description: 10x Genomics

value: Colon,Right,Cecum


title: Sample 2: P1.C.M.2


title: YFP_Naive

description: Cx3cr1-yfp-creER/+ naïve


title: Patient 9, Tumor, Left Side colon cancer

description: 10x Genomics


title: E20/p53.5: Pik3ca/p53   double gain of function mutant tongue sample 5


title: Sample 1: P1.C.M.1


title: PANC1 cercosporamide total rep 1

value: total cytosolic RNA


title: PANC1 cercosporamide total rep 3


title: Bleo+HB 90 2

city: Jerusalem

first: Jacob

last: Rachmilewitz

department: Goldyne Savad Institute of Gene Therapy

country: Israel

institute: Hadassah-Hebrew University Medical Center

data_processing:
Library quality control: FASTQC version 0.11.5
Quality and adapter trimming: trim_galore (uses cutadapt version 1.10)
Mapping: Tophat2 version 2.1.0 (uses Bowtie2 version 2.2.6)
Gene counting: HTseq-count version 0.11.2
Normalization and differential expression analysis: DESeq2 R package version 1.24.0
Genome_build: GRCh38

source_name: primary human fibroblast

extract_protocol: Illumina TruSeq RNA Library Preparation Kit v2

value: Treated for 30 min with Bleomycin collected 90 min after new medium with HB-EGF was added


title: PANC1 CID total rep 4


title: Patient 10, Tumor, Left Side colon cancer

description: 10x Genomics

value: Colon,Left,Descending


title: E20/p53.2: Pik3ca/p53   double gain of function mutant tongue sample 2


title: Patient 7, Tumor, Right Side colon cancer

description: 10x Genomics

value: Colon,Right,Cecum/IC


title: Bleo 90 2

value: Treated for 30 min with Bleomycin collected 90 min after new medium added


title: Patient 6, Tumor, Left Side colon cancer

description: 10x Genomics


title: E20/p53.3: Pik3ca/p53   double gain of function mutant tongue sample 3


title: Bleo+HB 90 1


title: E20/p53.4: Pik3ca/p53   double gain of function mutant tongue sample 4


title: Patient 2, Tumor, Left Side colon cancer

description: 10x Genomics


title: E20.6: Pik3ca gain of function mutation tongue sample 6

source_name: Loxp-STOP-Loxp (LSL)-PIK3CAH1047R  squamous epithelium from mouse tongue

value: Loxp-STOP-Loxp (LSL)-PIK3CAH1047R


title: PANC1 CID total rep 3


title: E20.5: Pik3ca gain of function mutation tongue sample 5


title: PANC1 CID total rep 2


title: PANC1 CID poly rep 4

value: polysome-associated mRNA


title: Bleo+HB 90 3


title: mock+HB 90 2

value: collected 90 min after new medium with HB-EGF  was added


title: PANC1 CID poly rep 1


title: PANC1 cercosporamide poly rep 1


title: Patient 5, Tumor, Right Side colon cancer

description: 10x Genomics


title: Patient 3, Tumor, Right Side colon cancer

description: 10x Genomics

value: Colon,Right,Hepatic Flexure


title: PANC1 CID poly rep 3


title: E20/p53.1: Pik3ca/p53   double gain of function mutant tongue sample 1


title: mock+HB 90 1


title: mock+HB 90 3


title: Bleo 90 1


title: E20.4: Pik3ca gain of function mutation tongue sample 4


title: mock 90 3

value: collected 90 min after new medium added


title: E20.2: Pik3ca gain of function mutation tongue sample 2


title: Patient 4, Tumor, Left Side colon cancer

description: 10x Genomics

value: Colorectal cancer


title: Specimen_38

city: Philadelphia

first: Yijing

last: Su

state: PA

address: Levy 326

department: Department of Oral Medicine

institute: University of Pennsylvania

data_processing:
Library strategy: split-seq
Basecalls performed using BCL2fastq
RNAseq reads were aligned to the hg38 genome assembly using STAR 2.7.6a
Data processing was preprocessed using Drop-seq-1.13
Individual samples should be demutiplex using provided barcode sequence file.
Assembly: hg38
Supplementary files format and content: abundance measurements
Supplementary files format and content: cell barcode sequence

source_name: human hippocampus

extract_protocol:
Tissue were minced with a razor blade and homogenized using a tissue grinder in a 1 ml of HB buffer (1 mM DTT), 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor, 0.3% IGEPAL-630, 0.25 M sucrose, 25 mM MgCl2, 20 mM Tricine-KOH) for 5 to 10 strokes, then filtered through a 40 mm strainer, underlayer with a cushion buffer (0.5 mM MgCl2, 0.5 mM DTT, EDTA-free protease inhibitor, 0.88 M sucrose) and centrifuged at 2800g for 10 minutes in a swinging bucket centrifuge at 4 ºC. Nuclei were collected as pellets. Nuclei were then centrifuged for 3 mins at 500g at 4 ˚C. The pellet was resuspended in 1 ml of cold PBS-RI (1x PBS + 0.05U/μl RNase Inhibitor). The nuclei were passed through a 40 μm strainer. 3 ml of cold 1.33% formaldehyde solution was then added to 1 ml of cells. Nuclei were fixed for 10 mins before adding 160 μl of 5% Triton X-100. Nuclei were then permeabilized for 3 mins and centrifuged at 500g for 3 mins at 4 ˚C. Nuclei were resuspended in 500 l of PBS-RI before adding 500 μl of cold 100 mM Tris-HCl pH 8. Then, nuclei were spun down at 500g for 3 mins at 4 ˚C and resuspended in 300 μl of cold 0.5 X PBS-RI. Finally, nuclei were again passed through a 40 μm strainer and then counted on a hemocytometer, diluted to 1,000,000 cells/ml.
mRNA from single nuclei were tagged 3 rounds with barcoded primers, with in-cell ligations using T4 DNA ligase. Plates were incubated for 30 mins at 37 ºC with gentle sharking (50 rpm) to allow hybridization and ligation to occur. The ligation products were purified with Dynabeads MyOne Streptavidin C1 beads. After washing beads once with 10 mM Tris and 0.1% Tween-20 solution and once with water, beads were resuspended into a solution containing 110 μl of 2X Kapa HiFi HotStart Master Mix, 8.8 μl of 10 μM stocks of primers BC_0062 and BC_0108, and 92.4 μl of water. PCR thermocycling was performed as follows: 95 °C for 3 mins, then five cycles at 98 °C for 20 seconds, 65 °C for 45 seconds, 72 °C for 3 mins. After these five cycles, Dynabeads beads were removed from PCR solution and EvaGreen dye was added at a 1X concentration. Samples were again placed in a qPCR machine with the following thermocycling conditions: 95 °C for 3 mins, cycling at 98 °C for 20 seconds, 65 °C for 20 seconds, and then 72 °C for 3 mins, followed by a single 5 mins at 72 °C after cycling. Once the qPCR signal began to plateau, reactions were removed.

value: hippocampus

tag: disease

value: Control

tag: age


title: Specimen_34, _24, _25

value: mixed sample (non-demultiplexed)


title: PANC1 CID poly rep 2

value: Human pancreatic ductal adenocarcinoma cell line


title: Specimen_35

value: AD

value: 85 Yrs


title: mock 90 2


title: E20.3: Pik3ca gain of function mutation tongue sample 3

value: squamous epithelium


title: Bleo 90 3


title: Patient 1, Tumor, Right Side colon cancer

description: 10x Genomics

value: Colon,Right,Cecum


title: p53.3: p53 gain of function mutation tongue sample 3

source_name: Heterozygous LSL-p53+/R172H squamous epithelium from mouse tongue

value: Heterozygous LSL-p53+/R172H


title: p53.2: p53 gain of function mutation tongue sample 2


title: Specimen_30, _37, _33, _31

source_name: hippocampus


title: E20.1: Pik3ca gain of function mutation tongue sample 1


title: PANC1 DMSO poly rep 4

value: DMSO, 6hrs


title: PANC1 DMSO total rep 4

value: total cytosolic RNA


title: Specimen_29, _23, _32


title: mock 90 1


title: Specimen_36, _27, _26, _28


title: Bleo+HB 30 3

value: Treated for 30 min with Bleomycin collected 30 min after new medium with HB-EGF was added


title: PANC1 DMSO poly rep 3

value: polysome-associated mRNA


title: PANC1 DMSO total rep 3


title: Bleo+HB 30 2


title: p53.1: p53 gain of function mutation tongue sample 1


title: PANC1 DMSO poly rep 2


title: Specimen_8/3/4/19/42

data_processing:
Basecalls performed using BCL2fastq
RNAseq reads were aligned to the hg38 genome assembly using STAR 2.7.6a
Data processing was preprocessed using Drop-seq-1.13
Individual samples should be demutiplex using provided barcode sequence file.
Genome_build: hg38

extract_protocol:
Tissue were minced with a razor blade and homogenized using a tissue grinder in a 1 ml of HB buffer (1 mM DTT), 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor, 0.3% IGEPAL-630, 0.25 M sucrose, 25 mM MgCl2, 20 mM Tricine-KOH) for 5 to 10 strokes, then filtered through a 40 mm strainer, underlayer with a cushion buffer (0.5 mM MgCl2, 0.5 mM DTT, EDTA-free protease inhibitor, 0.88 M sucrose) and centrifuged at 2800g for 10 minutes in a swinging bucket centrifuge at 4 ºC. Nuclei were collected as pellets. Nuclei were then centrifuged for 3 mins at 500g at 4 ˚C. The pellet was resuspended in 1 ml of cold PBS-RI (1x PBS + 0.05U/μl RNase Inhibitor). The nuclei were passed through a 40 μm strainer. 3 ml of cold 1.33% formaldehyde solution was then added to 1 ml of cells. Nuclei were fixed for 10 mins before adding 160 μl of 5% Triton X-100. Nuclei were then permeabilized for 3 mins and centrifuged at 500g for 3 mins at 4 ˚C. Nuclei were resuspended in 500 l of PBS-RI before adding 500 μl of cold 100 mM Tris-HCl pH 8. Then, nuclei were spun down at 500g for 3 mins at 4 ˚C and resuspended in 300 μl of cold 0.5 X PBS-RI. Finally, nuclei were again passed through a 40 μm strainer and then counted on a hemocytometer, diluted to 1,000,000 cells/ml.
mRNA from single nuclei were tagged 3 rounds with barcoded primers, with in-cell ligations using T4 DNA ligase. Plates were incubated for 30 mins at 37 ºC with gentle sharking (50 rpm) to allow hybridization and ligation to occur. The ligation products were purified with Dynabeads MyOne Streptavidin C1 beads. After washing beads once with 10 mM Tris and 0.1% Tween-20 solution and once with water, beads were resuspended into a solution containing 110 μl of 2X Kapa HiFi HotStart Master Mix, 8.8 μl of 10 μM stocks of primers BC_0062 and BC_0108, and 92.4 μl of water. PCR thermocycling was performed as follows: 95 °C for 3 mins, then five cycles at 98 °C for 20 seconds, 65 °C for 45 seconds, 72 °C for 3 mins. After these five cycles, Dynabeads beads were removed from PCR solution and EvaGreen dye was added at a 1X concentration. Samples were again placed in a qPCR machine with the following thermocycling conditions: 95 °C for 3 mins, cycling at 98 °C for 20 seconds, 65 °C for 20 seconds, and then 72 °C for 3 mins, followed by a single 5 mins at 72 °C after cycling. Once the qPCR signal began to plateau, reactions were removed.
split-seq

value: human hippocampus

tag: development stage

value: Child/Infant/Infant/Aging/Child


title: WT.3: wild type esophagus sample 3

source_name: Wild type  squamous epithelium from mouse esophagus

value: Wild type


title: PANC1 DMSO total rep 2


title: Bleo+HB 30 1


title: Bleo 30 3

value: Treated for 30 min with Bleomycin collected 30 min after new medium added


title: WT.2: wild type esophagus sample 2


title: PANC1 DMSO poly rep 1


title: Specimen_2/5/10/14/21

value: Infant/Child/Adolescent/Adult/Aging


title: Specimen_12/17/18/40/41

value: Adolescent/Adult/Aging/Adolescent/Aging


title: PANC1 CID total rep 1


title: prepupal_hemocyte_replicate3

city: Marburg

first: Katja

last: Rust

address: Emil-Mannkopff-Str. 2

country: Germany

institute: Phillips-University Marburg

data_processing:
Sequence images were transformed with Illumina software BaseCaller to BCL files.
Demultiplexing of fastq files was performed with bcl2fastq v2.20
Sequencing quality was asserted using FastQC (Andrews, S. (2014)
Alignment was performed using STAR (Dobin, Alexander, et al. "STAR: ultrafast universal RNA-seq aligner." Bioinformatics 29.1 (2013): 15-21.)(version 2.5) allowing for 2 mismatches within 50 bases.
Read counting was performed using featureCounts (Liao, Yang, Gordon K. Smyth, and Wei Shi. "featureCounts: an efficient general purpose program for assigning sequence reads to genomic features." Bioinformatics 30.7 (2013): 923-930.).
Read counts were analyzed in the R/Bioconductor environment (version 4.0.5) using the DESeq2 (Love, Michael I., Simon Anders, and Wolfgang Huber. "Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2." Genome biology 15.12 (2014): 550.) package version 1.28.1. Candidate genes were filtered using an absolute log2 fold-change >1 and FDR-corrected p-value <0.05.
Assembly: Drosophila melanogaster BDGP6.28
Supplementary files format and content: Aligned reads are provides in .bam files with a corresponding index (.bai) file
Supplementary files format and content: tab delimited text file (.txt) containing raw reads used as input for DESeq2 analysis

organism: Drosophila melanogaster

source_name: prepupal hemocyte

extract_protocol:
Larval and prepupal were isolated following the protocol published in https://doi.org/10.1007/978-1-4939-7701-7_17. In short, animals of the desired stage were washed and dissected in M3++ medium. Hemocytes were extracted by opening the sample and rinsing out blood cells. GFP positive cells were sorted using the S3e CELL SORTER 488/561-2L4D (Bio-Rad). Total RNA was extracted by Phenol:Chloroform extraction.
non stranded mRNA using the TruSeq RS-122-2001 according to manufacturer recommendation

value: prepupal hemocyte

value: hemocyte

tag: genotype

value: Hml-GAL4.delta, UAS-GFP

tag: developmental stage

value: white prepupae


title: WT.1: wild type esophagus sample 1

value: squamous epithelium


title: Specimen_6/11/16/22/39

value: Child/Adolescent/Adult/Aging/Infant


title: prepupal_hemocyte_replicate2


title: mock+HB 30 3

value: collected 30 min after new medium with HB-EGF  was added


title: Bleo 30 1


title: Specimen_20

data_processing:
Basecalls performed using BCL2fastq
RNAseq reads were aligned to the hg38 genome assembly using STAR 2.7.6a
Data processing was preprocessed using Drop-seq-1.13
Genome_build: hg38

value: Aging

value: 89 Yrs


title: mock+HB 30 2


title: Specimen_15

value: Adult


title: larval_hemocyte_replicate3

source_name: larval hemocyte

value: larval hemocyte

value: L3 wandering larvae


title: mock+HB 30 1


title: TCam-2 Dmab 4

city: Copenhagen

first: Lars

middle: Roed

last: Ingerslev

address: Blegdamsvej 3B

department: NNF Center for Basic Metabolic Research

country: Denmark

institute: Copenhagen University

data_processing:
Fastq files were generated using bcl2fastq2 v. 2.20.0,
Reads were aligned to the GRCh38 primary assembly using STAR v. 2.7.9a against a reference built using the ENSEMBL release 105 gene model.
Aligned reads were counted against the same gene model using featureCounts v. 1.6.2 counting only reads where both ends mapped to the same strand.
Genome_build: hg38
Supplementary_files_format_and_content: csv file with gene information and raw read counts

molecule: polyA RNA

source_name: pluripotent germ cell cancer cell line

growth_protocol: The EC-derived cell line NTera2 and the seminoma-derived cell line TCam-2 were grown under standard conditions at 37 °C in 5 % CO2 atmosphere in DMEM (NTera2) or RPMI 1640 (TCam-2) supplemented with 10 % fetal bovine serum, glutamine (58.5 mg/ml), penicillin (100 U/ml) and streptomycin (100 mg/ml). All reagents and cell media were purchased from Gibco (Invitrogen, Carlsbad, CA, US).

extract_protocol:
Total RNA was isolated using Nucleospin RNA II kit (Machery-Nagel, Düren, Germany) according to the manufacturer’s instructions.
Messenger RNA sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA protocol (Illumina). Poly-A containing mRNAs were purified by poly-T attached magnetic beads, fragmented, and cDNA was synthesized using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). cDNA was adenylated to prime for adapter ligation and after a clean-up using AMPure beads (Beckman coulter), DNA fragments were amplified using PCR followed by a final clean-up. Libraries were quality-controlled using a Fragment Analyzer instrument (Agilent Technologies) and subjected to 52-bp paired-end sequencing on a NovaSeq 6000 (Illumina).

treatment_protocol: NTera2 and TCam-2 cells were plated in 6 well plates and treated with 1 µg/ml RANKL, 1 µg/ml OPG, 1 µg/ml RANKL, or vehicle (D-PBS) for 48 hours. Cell pellets from four independent experiments were collected by trypsinization for analysis.

tag: celltype

value: Denosumab

tag: passage

value: Passage 122

tag: rin


title: NTera2 Vehicle 1

tag: treatment

value: Vehicle

value: Passage 27


title: NTera2 OPG 1

value: OPG


title: Specimen_13

value: human hippocampus

value: 40 Yrs


title: TCam-2 RANKL 4

value: RANKL


title: mock 30 3

value: collected 30 min after new medium added


title: PANC1 DMSO total rep 1

tag: cell type

value: Human pancreatic ductal adenocarcinoma cell line

value: DMSO, 6hrs

tag: replicate

value: total cytosolic RNA


title: larval_hemocyte_replicate2

tag: tissue

value: hemocyte

value: Hml-GAL4.delta, UAS-GFP


title: Specimen_9

value: Adolescent

value: 13 Yrs


title: NTera2 RANKL 1


title: Bleo 30 2

value: Treated for 30 min with Bleomycin collected 30 min after new medium added


title: mock 30 2


title: larval_hemocyte_replicate1

value: larval hemocyte

value: L3 wandering larvae


title: TCam-2 OPG 4

value: Passage 122


title: prepupal_hemocyte_replicate1

value: prepupal hemocyte

value: white prepupae


title: mock 30 1


title: Specimen_7

value: human hippocampus

value: Child

value: 4.3 Yrs


title: ECT2

city: Galveston

first: W.

middle: Samuel

last: Fagg

state: TX

address: 301 University Blvd, Department of Surgery, MRB5.150, L21875

department: Surgery/Biochemistry and Molecular Biology

institute: University of Texas Medical Branch

data_processing:
Illumina bcl2fastq2-v2.20 was used for base calling
Fastq files from 2 separate sequencing runs ( A and B)  with all pooled samples were combined
Alignment to known splicing events database vastdb.hsa.16.02.19 using Vast-tools version 2.0.2 align function with default parameters.   Irimia, M., Weatheritt, R.J., Ellis, J.D., Parikshak, N.N., Gonatopoulos-Pournatzis, T., Babor, M., Quesnel-Vallieres, M., Tapial, J., Raj, B., O'Hanlon, D., et al. (2014). A highly conserved program of neuronal microexons is misregulated in autistic brains. Cell 159, 1511-1523
Reads aligned to the human hg38 genome using STAR version 2.6.1c with the ENCODE consortium recommended parameters
Read counts obtained from the STAR quantMode GeneCounts feature using the Gencode v28 annotation file.
Genome_build: hg19, hg38
Supplementary_files_format_and_content: Inclusion levels tab delimited file is the Vast-tools full output reporting the number of reads in each sample supporting each alternative splicing event.
Supplementary_files_format_and_content: Tab delimited file of raw read counts output by the STAR aligner

source_name: H9-derived ectoderm; replicate 2

extract_protocol:
Total cellular RNA was crudely extracted using Trizol Reagent (Life Technologies, CA) then purified on a ReliaPrep Mini RNA Column (Promega, WI)
After rRNA removal with Lexogen RiboCop depletion kit, strand specific RNA libraries were prepared  using Illumina's True_Seq RNA kit using standard protocols

value: ECT

value: ECT for ECT (Smukler et al 2006/Tchieu et al 2017)


title: ECT3

source_name: H9-derived ectoderm; replicate 3


title: SCC152_Input_Histone

city: Buffalo

first: Satrajit

last: Sinha

address: 955 Main Street

institute: University at Buffalo

data_processing:
RNA-seq - Reads were mapped to the Homo sapiens genome (hg19 build) with HISAT2 (v2.1.0), reads aligned to the UCSC hg19 build were quanitifed with featureCounts (v1.5.0), which disregarded any read/read pair that aligned to more than one location, or more than one gene at a single location, DESeq1 (v.1.10.1) was used to normalize the read counts and derive TPM values
ChIP-seq - Reads were mapped to the Homo sapiens genome (hg19 build) with bowtie2 (v2.4.4), MACS2 was then used for peak calling to identify enriched areas in the genome
Genome_build: hg19
Supplementary_files_format_and_content: featureCounts output containing gene-level read count summaries for each RNA-seq sample, peak files for ChIP-seq results

extract_protocol:
RNA-seq - RNA was extracted from human cancer cell lines (SCC104 and SCC152) using the Direct-zol Mini-Prep kit, ChIP-seq - cells were fixed with formaldehyde for 10 mins and then chromatin was extracted
RNA-seq - Illumina TruSeq RNA sample Prep Kit was used with 1 ug of total RNA for the construction of sequencing libraries, ChIP-seq - libraries were prepared using ThruPLEX DNA-seq kits from Reubicon Genomics

tag: cell line

tag: antibody

value: HPV+ Head and Neck Squamous Cell Carcinoma

tag: shRNAs


title: NTera2 Dmab 1

value: Denosumab

value: Passage 27


title: TCam-2 Vehicle 4

value: Vehicle


title: SCC152_H3K27ac_R1

value: H3K27ac (Diagenode)


title: ECT1

source_name: H9-derived ectoderm; replicate 1


title: SCC152_H3K4Me3_R1

value: H3K4Me3 (Diagenode)


title: CM3

source_name: H9-derived cardiac mesoderm; replicate 3

value: CM

value: CM for CM (Loh et al 2016)


title: TCam-2 Dmab 3

value: Passage 121


title: SCC152_H3K4Me1_R1

value: H3K4Me1 (Diagenode)


title: NTera2 OPG 2

value: OPG

value: Passage 29


title: SCC104_Input_Histone


title: TCam-2 RANKL 3

value: RANKL


title: AsPC-1 cells, L72-3_S47

city: Los Angeles

first: David

last: Dawson

state: CA

address: 650 Charles E Young Dr, S

department: Pathology & Laboratory Medicine

institute: UCLA

data_processing:
Illumina Hi Seq 3000 using the Hi Seq 3000/4000 SBS kit (50 cycles)
Raw reads were aligned to the UCSC Human Genome hg19 using the Bowtie2 aligner (version 2.2.9)
Read count was performed by RSEM (version 1.2.25) with differential expression analysis performed with edgeR Bioconductor package
The gene fragments were counted using htseq-count from HTSeq package (version 0.5.3p9)
Genes with less than 10 fragments in all samples were excluded from differential expression analysis, which was determined using the DEseq2 pipeline (version 2.6.10) with a FDR cutoff of < 0.05 considered significant.
Gene set enrichment analysis (GSEA, v2.2.3) was performed on ranked lists of differentially expressed genes using the GSEAPreranked tool, Hallmark, C2:CP:KEGG gene set categories obtained from the Molecular Signatures Database.
Default GSEA parameters were used with the exception of the classic enrichment statistic.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited excel file includes base mean, FoldChange(log2), lfcSE, stat, p value, and p-adj. values for each gene.

growth_protocol: AsPC-1 PDAC cells were adapted for growth in ultra low attachment suspension conditions. Cells were routinely split and maintained in RPMI (Invitrogen, Carlsbad, CA) with 10% fetal bovine essence (VWR, Radnor, PA) and 1% Penicillin-Streptomycin Solution (Corning, Corning, NY) at moderate confluency.

extract_protocol:
Cells were collected, washed with PBS, and snap frozen. RNA was isolated using Quick-RNA Miniprep (Zymo Research, Irvine, CA).
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: AsPC-1 cells were plated at 400,000 cells/well in 6-well ultra low attachment plates (Corning, Corning, NY) with 3 replicates were treatment group. Cells were treated as indicated for 4 hours, 24 hours, or 72 hours (with re-treatment every 24 hours) with 25 nM LGK974 (Cayman Chemical, Ann Arbor, MI) or equivalent DMSO vehicle control.

value: PDAC cells

value: Treated with LGK974

tag: treatment time

value: 72 hours

tag: concentration

value: 25 nM


title: CM2

source_name: H9-derived cardiac mesoderm; replicate 2


title: DE3

source_name: H9-derived definitive endoderm; replicate 3

value: DE

value: SR1 for DE (Loh et al 2014)

tag: sorting

value: DE was sorted for CXCR4+ cells by FACS using mouseIgG2a-anti-CD184, APC conjugate BioLegend Cat# 306510; clone 12G5


title: AsPC-1 cells, L72-2_S46


title: SCC104_H3K4Me3_R1

value: HPV+ Head and Neck Squamous Cell Carcinoma


title: SCC104_H3K27ac_R1

value: H3K27ac (Diagenode)


title: TCam-2 OPG 3


title: NTera2 RANKL 2


title: NTera2 Vehicle 2

value: Vehicle


title: NTera2 Dmab 2

value: Denosumab


title: AsPC-1 cells, L72-1_S45


title: SCC104_H3K4Me1_R1


title: CM1

source_name: H9-derived cardiac mesoderm; replicate 1

value: CM

value: CM for CM (Loh et al 2016)


title: TCam-2 Vehicle 3

value: Passage 121


title: AsPC-1 cells, V72-2_S43

value: Treated with vehicle control

value: control


title: AsPC-1 cells, V72-3_S44


title: AsPC-1 cells, V72-1_S42


title: TCam-2 Dmab 2

value: Passage 120


title: UD2

source_name: undifferentiated H9 hESC; replicate 2

value: UD

value: mTeSR1 for UD


title: DE2

source_name: H9-derived definitive endoderm; replicate 2


title: NTera2 Vehicle 3

value: Passage 30


title: UD1

source_name: undifferentiated H9 hESC; replicate 1


title: NTera2 OPG 3

value: OPG


title: DE1

source_name: H9-derived definitive endoderm; replicate 1

value: DE


title: AsPC-1 cells, LH24-3_S41

value: PDAC cells

value: Treated with LGK974

value: 24 hours

value: 1 uM


title: AsPC-1 cells, LH24-1_S39


title: SCC104_p63_4A4

value: p63 4A4 (Santa Cruz)

value: HPV+ Head and Neck Squamous Cell Carcinoma


title: AHA-11a total RNA-seq replicate 2

city: Boulder

first: John

last: Rinn

state: CO

address: 3415 Colorado Ave

department: Biochemistry

institute: CU Boulder

description:
AHA-11a mouse cells containing human chrX total RNA-seq
salmon_merged_gene_counts.csv
salmon_merged_gene_tpm.csv

data_processing:
Reads were aligned and quantified using nf-core/rnaseq v1.4.2 with the aligner parameter: `--pseudo_aligner salmon` and all other parameters default.
As a part of the nf-core/rnaseq pipeline, reads were quantified with Salmon v0.14.1 using Gencode M27 annotation with human gene annotations from chrX appended (Gencode v38)
Genome_build: mm39 with chrX from hg38
Supplementary_files_format_and_content: Comma separated table of gene counts and abundance estimates (TPM) across all samples calculated using Salmon

source_name: AHA-11a Human/Mouse somatic cell hybrid with human chromosome X

growth_protocol: Cultured in DMEM (Thermo Fisher Scientific, 11965092) supplemented with 10% FCS (MilliporeSigma, F8067), 1X GlutaMAX supplement (Thermo Fisher Scientific, 35050061), 4 x 10-7 M aminopterin (MilliporeSigma, A3411-10MG), 1 x 10-4 M hypoxanthine (MilliporeSigma, H9636-1G), and 1.6 x 10-5 M thymidine (MilliporeSigma, T1895-1G) at 37°C and 7.5% CO2

extract_protocol:
KAPA RNA HyperPrep with RiboErase kit (Roche)
Maxwell LEV SimplyRNA Purification kit (Promega, AS1280) with DNase I treatment

value: AHA-11a (Coriell, GM10324)

value: untreated


title: SCC104_p63_deltaNp63

value: deltaNp63-1.1 (gift from Collaborator, Nekulova M. Virchows Arch. 2013.)


title: SCC152_Input_TF


title: Mouse Treg WT1

city: Esch-sur-Alzette

first: Feng

middle: Q.

last: Hefeng

address: 29, rue Henri Koch

department: Department of Infection and Immunity

country: Luxembourg

institute: Luxembourg Institute of Health

description: Expression value derived using Expression Console software; Exon analysis summarized using PLIER

hyb_protocol: The fragmented/labeled single-strand cDNA (with hybridization controls) was hybridized to the Affymetrix Mouse Gene 2.0 ST Array at 45°C for 16h with rotation (60rpm). After the hybridization step, each of the arrays has been washed and stained on the GeneChip Fluidics Stations 450 (Affymetrix), using the GeneChip® Hybridization Wash and Stain Kit.

scan_protocol: The arrays were scanned using the GeneChip Scanner 3000 7G with GeneChip Command Console software (Affymetrix). The software generates the .CEL files containing the probe cell intensity data and enables sample and array registration, data management, instrument control as well as automatic and manual image gridding.

data_processing: The expression signal at exon level was summarized with Affymetrix PLIER algorithm with DABG and PM-GCBG options using the sketch-quantile normalization approach (Affymetrix Expression Console v1.4).The corresponding probesets were considered differentially expressed if they passed the following combinatory filters: (a) whether the change folds >= 2 between the means of DJ-1 KO and WT Tregs or Teffs; (b), whether the P-value, resulted from two-tailed unpaired Student T-test, <=0.05; (c),  whether the cross-hyb type of the probeset was equal to 1; (d), whether the probeset with the highest expression level is higher than 100 (with the median ~90 of each sample); (e), for a given group (e.g. WT) with higher mean intensity value of the probeset, whether the probeset in all the replicates of the given group was detected as ‘present’ according to the default setting of the Expression Console.

label: biotin

source_name: old WT mouse 1

label_protocol: Single-strand cDNA was labeled and fragmented using the WT Terminal Labeling, polyA and hyb Controls Kit.

growth_protocol: All mice were maintained in our specific-pathogen-free (SPF) animal facility and all animal experimental procedures were performed following the approval of the Animal Warfare Society (AWS) of University of Luxembourg and Luxembourg Institute of Health.

extract_protocol: Freshly sorted T cells were first lysed with RLT buffer, supplemented with 1% beta-Mercaptoethanol and RNA were extracted using RNeasy Mini Kit following the manufacturer’s instructions including the digestion of genomic DNA with DNAse I.

treatment_protocol: No treatment. The cells were sorted from the spleen of homeostatic old mice (45-week old) under the SPF condition.

tag: Sex

value: Male

value: Treg


title: Mouse Treg KO2

source_name: old DJ-1 KO mouse 2


title: UD3

source_name: undifferentiated H9 hESC; replicate 3


title: AHA-11a total RNA-seq replicate 3


title: Mouse Treg WT2

source_name: old WT mouse 2


title: Mouse Treg KO3

source_name: old DJ-1 KO mouse 3


title: Mouse Treg WT3

source_name: old WT mouse 3


title: Mouse Treg KO1

source_name: old DJ-1 KO mouse 1


title: SCC152_p63_4A4


title: NTera2 RANKL 3

source_name: pluripotent germ cell cancer cell line

value: RANKL


title: SCC152_sh1_R2

value: TP63 sh1


title: NTera2 Dmab 3

value: Denosumab


title: SCC152_sh2_R2

value: TP63 sh2


title: SCC152_sh1_R1


title: TX1903LHZY2X

first: Bing

middle: Hua

last: Li

address: 280 Chongqing South Rd, Building #5, Room 602, Shanghai

institute: Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine

data_processing:
Illumina Bcl2fastq software used for basecalling.
CellRanger was used to analyze the original single cell RNA sequencing results.
Genome_build: mm10
Supplementary_files_format_and_content: text file, raw counts of sequencing reads for the genes of interest after filtering

source_name: colonic epithelial cells

extract_protocol:
Colonic epithelial cells were stained with EpCAM and live/dead cell kit on ice in dark for 30 min. The EpCAM+ live cells were sorted by flow cytometry
The single cell suspension of colonic epithelial cells were processed by 10x Genomics 3'RNASeq V2

tag: strain

value: primary colonic epithelial cells

value: Mettl14_ KO


title: AHA-11a polyA RNA-seq replicate 1

description:
AHA-11a mouse cells containing human chrX polyA selected RNA-seq
salmon_merged_gene_counts.csv
salmon_merged_gene_tpm.csv

extract_protocol:
KAPA mRNA HyperPrep kit (Roche)
Maxwell LEV SimplyRNA Purification kit (Promega, AS1280) with DNase I treatment


title: SCC104_Input_TF


title: SCC152_shCTL_R1

value: shCTL


title: AsPC-1 cells, L24-2_S37

value: PDAC cells

value: Treated with LGK974

value: 25 nM


title: AsPC-1 cells, L4-3_S32

value: 4 hours


title: AHA-11a total RNA-seq replicate 1


title: SCC152_sh2_R1

value: HPV+ Head and Neck Squamous Cell Carcinoma


title: AsPC-1 cells, V24-3_S35

value: Treated with vehicle control

value: 24 hours

value: control


title: AHA-11a polyA RNA-seq replicate 2

value: AHA-11a (Coriell, GM10324)

value: untreated


title: AHA-11a polyA RNA-seq replicate 3


title: TCam-2 OPG 2

value: OPG

value: Passage 120


title: AsPC-1 cells, L24-3_S38


title: NTera2 Vehicle 4

value: Vehicle

value: Passage 31


title: TCam-2 RANKL 2

value: RANKL


title: AsPC-1 cells, V24-1_S33


title: AsPC-1 cells, V24-2_S34


title: SCC152_shCTL_R2


title: TCam-2 Dmab 1

value: Denosumab

value: Passage 118


title: AsPC-1 cells, L24-1_S36

value: PDAC cells


title: AsPC-1 cells, LH24-2_S40

value: Treated with LGK974

value: 1 uM


title: TX1903LHZY1X

value: primary colonic epithelial cells

tag: genotype


title: TCam-2 Vehicle 2


title: SCC104_sh2_R2

value: HPV+ Head and Neck Squamous Cell Carcinoma

value: TP63 sh2


title: SCC104_sh1_R2

value: TP63 sh1


title: NTera2 OPG 4


title: NTera2 RANKL 4


title: AsPC-1 cells, L4-1_S30

value: 4 hours

value: 25 nM


title: TCam-2 RANKL 1


title: SCC104_sh1_R1


title: TCam-2 OPG 1


title: SCC104_sh2_R1


title: AsPC-1 cells, V4-3_S29

value: Treated with vehicle control

value: control


title: AsPC-1 cells, V4-2_S28


title: SCC104_shCTL_R2

value: shCTL


title: NTera2 Dmab 4


title: TCam-2 Vehicle 1

value: Vehicle


title: SCC104_shCTL_R1


title: AsPC-1 cells, V4-1_S27


title: CUGexp−

city: Suita

first: Masayuki

last: Nakamori

state: Osaka

address: 2-2, D-4, Yamadaoka

department: Neurology

country: Japan

institute: Osaka University Graduate School of Medicine

data_processing:
The libraries were sequenced on an Illumina Nova Seq 6000 (Illumina).
The raw data were evaluated by NGQC software (Novogene) and trimmed to remove adaptor contaminants and low-quality reads.
Clean reads were aligned to the reference genome sequence using TopHat v2.0.12 (Trapnell et al., 2009).
The estimated transcript abundance was calculated, and the count values were normalized to the upper quartile of the fragments per kilobase of transcript per million mapped reads using HTSeq v0.6.1 (Anders, S.et al, 2015).
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample.

source_name: fibroblasts

extract_protocol:
Total RNA was extracted from the DM1 model cells using an RNeasy Mini Kit (Qiagen). A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations.
Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina.

value: Human fetal lung fibroblasts

tag: expression

value: No CUGexp expression


title: ML14 (WAS-B)

city: San Diego

first: April

middle: Elizabeth

last: Williams

address: 10010 N Torrey Pines Rd

department: IGC

institute: Salk Institute for Biological Studies

description:
WASBcor_vs_WASBun_gene_exp.diff
WASBcor_vs_WASBun_isoform_exp.diff

data_processing:
FastQC v0.11.5 with default parameter was used for quality test.
RNA-Seq reads were mapped to hg19 reference genome by STAR v2.5.1 with default parameters. RNA-Seq gene expression (RNA-Seq_fpkm_WAS.txt) was performed by Homer that reads uniquely mapped to the exon region of each RefSeq gene were counted and normalized into FPKM value. Gene and isoform level differential expression analysis was performed by cuffdiff v2.2.1 with parameter -b -u --library-type fr-firststrand.
Genome_build: hg19
Supplementary_files_format_and_content: tab delimited text file for RNA-Seq analysis

source_name: hu-iPSC-macrophage, WAS-B

growth_protocol: Human fibroblasts from a Wiskott-Aldrich syndrome patient with the c.1271dupG mutation (in the WAS gene were purchased from the Coriell institute (Camden, NJ). Human fibroblasts from a Wiskott-Aldrich syndrome patient with the c.107_108del mutation and from the mother of the patient (unaffected) were purchased from the Cell Line and DNA Bank from Patients Affected by Genetic Diseases (Genova, Italy). The B-lymphocyte cell lines ID00003 (WAS: c.431G>A) and GM11518 (wild type) were purchase from the Coriell institute (Camden, NJ). iPSCs cultured on MEF feeders in human ESC medium were manually dissociated and plated on 8 days old overgrown OP9 feeders to start differentiation. Cells were co-cultured with the OP9 feeders for 12-14 days in OP9 differentiation medium (Alpha MEM containing 10% FBS, 100 μM monothioglycerol and 50 μg/ml ascorbic acid) as described by Vodyanik, M. A. & Slukvin, II. (Curr Protoc Cell Biol 2007). Differentiated cells were dissociated with collagenase and Accutase (Innovative Cell Technologies) and subjected to further differentiation into macrophages using GM-CSF and M-CSF. Human B cells, T cells and monocytes were sorted on a FACSAria II cell sorter using CD19, CD4 and CD14 antibodies, respectively. Macrophages were derived from PBMCs monocyte in the presence of M-CSF.

extract_protocol:
RNA was isolated by Qiagen RNeasy micro kit with in column DNase digestion.
The Agilent TapeStation or Bioanalyzer was used to determine RNA integrity (RIN) numbers prior to library preparation. Stranded mRNA-Seq libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit according to the manufacturer’s instructions (Illumina). Briefly, RNA with polyA tail was isolated using poly-T oligos conjugated to magnetic beads. mRNA was then fragmented and reverse-transcribed into cDNA. dUTPs were incorporated, followed by second strand cDNA synthesis. dUTP-incorporated second strand was not amplified. cDNA was then end-repaired, index adapter-ligated and PCR amplified. Purification of nucleic acids after each enzymatic steps was achieved by using AMPure XP beads (Beckman Coulter).

value: WAS-B

value: macrophage


title: Allograft Kidney Biopsy #1

first: Pavel

last: Morozov

department: Tuschl Lab

institute: Rockefeller University

description: Biopsy obtained at the time of core needle biopsy of the allograft.

data_processing:
Drop-seq core computational protocol V1.2, http://mccarrolllab.com/dropseq/
Read 1 (R1) consists of cell barcode (positions 1-16) and UMI (positions 17-27), read 2 (R2) originates from transcript
Genome_build: hg38
Supplementary_files_format_and_content: digital gene expression matrix, UMI count per barcoge for each gene

source_name: Kidney biopsy

extract_protocol:
Needle biopsy
Chromium Single Cell 3’ Reagent Kit V2 (10x Genomics) according to the manufacturer’s instructions.

tag: time, transplant to prior biopsy

value: 26 month

tag: relation of the donor to recipient

value: Cousin

tag: prior biopsy findings

value: Severe microvascular inflammation, severe transplant glomerulopathy


title: R19009515_101-ko-C-2W

city: Shanghai

data_processing:
FastQC (version 0.11.2) (Andrews.2010)  and Skewer (v0.2.2) (Jiang H, el at. 2014) software were performed for quality control.
STAR (v2.4.2a) (Dobin A, et al. 2013) software were used for sequence alignment.
Expression estimation of gene and transcript was performed by using RSEM (V1.2.29) (Li B, et al. 2011)
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

extract_protocol:
mRNA was extracted from total RNA using magnetic beads with oligo-dT then were fragmented randomly.
 The first strand of cDNA and the second strand are then synthesized using reverse transcriptase. The reverse transcription product is subjected to terminal repair, followed by addition of an A base at the 3' end. Subsequently, the fragments were ligated with a Illumina universal adapter. After PCR amplified, the ligation products were purified in order to remove the incompletely ligated fragment and the self-ligated adapter. Library sizes were detected using an Agilent 2100 Bioanalyzer and library concentrations were detected using Qubit 2.0. The DNA sequencing was performed according to Illumina's standard protocol.

value: Mettl14 _KO

value: primary colonic epithelial cells


title: FibroA

city: Solna

first: Yizhou

last: Hu

state: Stockholm

address: C6 , Biomedicum Karolinska, Solnavägen 9,

institute: Karolinska Institute

data_processing:
Count data were subsequently generated using CellRanger
10x demux pipeline was used to demultiplex the flowcells, and Fastq files were demultiplexed using mkfastq included in CellRanger.
Quality control of the processed expression matrix were performed by evaluating detected transcripts, detected counts, cell doublets, mito-ratio
Genome_build: GENCODE: GRCh38.p12
Supplementary_files_format_and_content: tab-delimited text files include raw counts for each Sample
Supplementary_files_format_and_content: HDF5 Loom file, raw counts

source_name: Genetically modified human brain vascular primary fibroblasts

growth_protocol: Human brain vascular fibroblast primary cells were cultured in fibroblast medium following the manufacturer’s instruction. After genetical modification, cells were cultured in selection medium following the manufacturer’s instruction and then cultured in Neurocult NS-a basal medium with addition of 1% B27,0.5% N2 , 10 ng/ml each of EGF and FGF2 and plated on laminin-coated Primaria dishes as half-adherent cells.

extract_protocol:
Cells were treated with trypsin till fully detached and immediately washed with culture medium, and centrifuged and washed with PBS for twice, then 50-100 microlitre of cell suspension (approx. 1000 cells/microlitre) were used for further 10x droplet construction.
single cell RNA-Seq: 10x genomics (V3) and NovaSeq6000 sequencing

tag: tissue source

value: Brain

tag: age

tag: treatment


title: R19009511_97-wt-C-2W

value: Mettl14 _WT

tag: cell type


title: Native Kidney Biopsy

description: Biopsy obtained from the healthy kidney donor at the time of transplantation.

value: Sister


title: AsPC-1 cells, L4-2_S31

tag: cell line

value: PDAC cells

value: Treated with LGK974

value: 4 hours

value: 25 nM


title: R19009509_91-wt-C-2W


title: CUGexp+

value: CUGexp expression


title: R19009517_105-ko-C-2W


title: CXCR3pos2

status: Public on Apr 18 2022

city: Syracuse

first: Krista

middle: L

last: Newell

address: 766 Irving Ave.

department: Microbiology & Immunology

institute: SUNY Upstate Medical University

description: Biological replicate 2

data_processing:
Sample demultiplexing was performed within the BaseSpace Sequence Hub (Illumina, CA) according to the manufacturer's recommendations using the default settings.
Gene expression pseudoalignment was performed using Kallisto following quality assessment using FastQC and MultiQC. Transcript pseudoalignment was performed against a mouse reference library using Kallisto and the Ensembl GRCm39 cDNA FASTA.
Data normalization, visualization, and differential gene expression were performed using the statistical computing environment R version 3, RStudio version 1.3.1093 and Bioconductor. Transcript-level counts and abundance were summarized to genes using the TxImport package.
Data were filtered and normalized using the Trimmed Mean of M-values (TMM) method in EdgeR package .
Differential gene expression analysis was performed using linear modeling and Bayesian statistics in the R package Limma with Benjamini-Hochberg correction for multiple testing.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample

source_name: Peritoneal cavity wash

growth_protocol: Mice were infected i.p. with 50k copies of E. muris and peritoneal cavity cells harvested on day 8.

extract_protocol:
Total RNA was extracted using the standard Illumina preparation kit according to the manufacturer’s recommendations, and quality and quantity assessed on an Agilent Bioanalyzer (Agilent, Santa Clara, CA).
Library preparation was performed using the Illumina Library Preparation kit according to the manufacturer's protocol.

treatment_protocol: CXCR3+ and CXCR3-negative live singlet IgM+ IgD-neg CD19+ CD138-neg peritoneal cavity B cells were FACS sorted directly into RLT+ buffer (Qiagen, 1053393) on a BD FACS Aria II, and snap-frozen on dry ice.

tag: tissue

value: peritoneal cavity

value: wild type


title: CXCR3pos3

description: Biological replicate 3


title: CXCR3neg2

description: Biological replicate 2


title: CXCR3pos1

description: Biological replicate 1


title: CXCR3neg3

description: Biological replicate 3


title: CXCR3neg1

description: Biological replicate 1


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A09_6_0.030000uM_TC00000652_P22

city: RTP

first: Logan

middle: J

last: Everett

state: North Carolina

address: 109 T.W. Alexander Dr

department: Office of Research and Development

institute: U.S. Environmental Protection Agency

data_processing:
Read alignment:
Each FASTQ file was aligned to the probe sequences in the hWTv2 assay using HISAT2 v2.1.0 with spliced alignment disabled.
Probe counts:
Aligned reads in SAM format were processed with SAMtools v1.9 to compute the number of uniquely aligned reads for each probe. Probe counts and associated meta-data for each well were stored for analysis using MongoDB v3.6.14.
Assembly: hWTv2
Supplementary files format and content: Processed data provided as a single table of counts for all probes x all samples

source_name: U-2 OS

growth_protocol: U-2 OS human osteosarcoma cells (HTB-96®, Lot: 64048673, ATCC) were cultured as described in Nyffeler et al. (2020). Briefly, cells were cultured in media consisting of Dulbecco’s Modified Eagle Medium (DMEM) with 10% heat-inactivated fetal bovine serum and penicillin/streptomycin/glutamine, passaged using TrypLE Select and maintained at 37 C in a humidified atmosphere of 5% CO2 in air.   A cryo stock of internal passage number 6 (P6) was generated as described in Nyffeler et al. (2020); experiments were performed using U-2 OS cells from P7 to P10. Cells were counted using a Countess II automated cell counter and seeded into CellCarrier-384 Ultra microplates at 3000 cells/well in 40 ul media per well.

extract_protocol:
Twentyfour hours after chemical treatment, HTTr assay plates were removed from the incubator and media in each assay well was drained to a residual volume of 10 µL using MultiFlo FX microdispenser equipped with a vacuum-driven aspiration manifold. To create cell lysates, 10 µL volumes of 2X BioSpyder lysis buffer were dispensed into each assay well using the same MultiFlo FX instrument equipped with 1 µL peristaltic pump dispensing cassette. Plates were temporarily sealed with an adhesive film, shaken for 30 s on the stage of MultiFlow FX microplate dispenser (BioTek, Winooski, VT) and then placed in a hybridization oven (37°C) for 10 min. The adhesive films were removed, plates were re-sealed with NuncTM aluminum acrylate sealing tape and stored at -80°C. Prior to shipment, the tape was removed and reference samples (reference RNAs, bulk lysates and lysis buffer blank) (Harrill et al., 2021) were added in duplicate to each plate while keeping the plates frozen. The plates were then resealed with new NuncTM aluminum acrylate sealing tape.
384-well plates containing cell lysates were shipped to BioSpyder, Inc. frozen (on dry ice) using overnight priority shipping. Cell lysates were then analyzed by BioSpyder using the TempO-Seq human whole transcriptome version 2 (hWTv2) assay, which includes 22,537 probes covering 19,703 genes. Lysates were processed as described in. In brief, 2 µL of each lysate was hybridized with 2 µL of detector oligos from the hWTv2 assay using the following thermal cycler protocol: 10 min at 70°C, followed by gradual decrease to 45°C over 49 min, terminating with 45°C incubation for 16-24 h. Excess oligos were then removed via nuclease digestion (90 min at 37°C), and hybridized detector oligos were ligated (1 h at 37°C) following respective additions of 24 µL TempO-Seq nuclease and ligation mixes. RNA/DNA duplexes were then heat-denatured, and 10 µL of each ligation product were transferred to an amplification microplate containing 10 µL of PCR master mix per well. Ligation products were then uniquely labeled during product amplification with well coordinate-specific “barcoded” primer pairs containing universal adaptors for sequencing. Samples were pooled into sequencing libraries, distributed across multiple lanes of a HiSeq dual flow cell and analyzed on a HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina, San Diego, CA).

treatment_protocol: DMSO-solubilized chemical stock solutions were received frozen from the US EPA ToxCast chemical inventory management contractor (EvoTec, Princeton, NJ) and stored at -80°C prior to dose plate preparation. A seven-point dilution series (1/2 log10 spacing) of test chemicals was prepared in a LabCyte Echo-qualified 384-well polypropylene (384PP) plate at 200x the desired nominal test concentration for screening. Duplicated wells of dosing solutions for each test chemical concentration were arrayed in columns 2 through 24 of the dose plate (Figure 1A). Transcriptomics reference chemicals (genistein, trichostatin A, sirolimus) were solubilized as 200x dosing solutions corresponding to nominal test concentrations of 10, 1 and 0.1 µM, respectively. At 24 h post-plating, 200 nL of 200x dosing solutions were transferred to the assay plates using a LabCyte Echo 550 acoustic dispenser. The final concentration of DMSO in all treatment, reference treatment and vehicle control wells was 0.5%.  Well coordinates on each assay plate were uniquely randomized with respect to treatment so that any potential edge-well effects were distributed in an unbiased manner across all possible treatment conditions.

value: U-2 OS

value: 0.03 uM Cucurbitacin I

tag: chemical name

value: Cucurbitacin I

tag: chemical id

tag: chemical concentration

tag: dose level

tag: assay plate

tag: assay plate well


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A07_5_0.100000uM_TC00000652_P20

value: 0.1 uM Rapamycin

value: Rapamycin


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A03_2_0.030000uM_TC00000652_P23

value: 0.03 uM Cycloheximide

value: Cycloheximide


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A09_3_0.001000uM_TC00000652_P24

value: 0.001 uM Cucurbitacin I


title: Allograft Kidney Biopsy #2

value: 76 month

value: Brother

value: Active antibody-mediated rejection, no transplant glomerulopathy


title: DMSO_TC00000652_P21

description: DMSO

value: 0.5% DMSO vehicle control

value: DMSO

value: 0 uM


title: FibroB

source_name: Human brain vascular primary fibroblasts

value: Human brain vascular fibroblast primary cells

value: Brain


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A06_2_0.003000uM_TC00000652_P18

value: 0.003 uM Dexamethasone

value: Dexamethasone


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A07_1_0.001000uM_TC00000652_P17

value: U-2 OS

value: 0.001 uM Rapamycin


title: DMSO_TC00000652_P16

description: DMSO


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A03_6_3.000000uM_TC00000652_P13

value: 3 uM Cycloheximide

value: 3 uM


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A06_7_1.000000uM_TC00000652_P15

value: 1 uM Dexamethasone

value: 1 uM


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A05_2_0.003000uM_TC00000652_P14

value: 0.003 uM Retinoic acid

value: Retinoic acid


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A02_5_1.000000uM_TC00000652_P12

value: 1 uM Etoposide

value: Etoposide


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A07_2_0.003000uM_TC00000652_P09

value: 0.003 uM Rapamycin

value: Rapamycin


title: DMSO_TC00000652_P10

description: DMSO

value: 0.5% DMSO vehicle control

value: DMSO

value: 0 uM


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A07_2_0.003000uM_TC00000652_P08


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A06_2_0.003000uM_TC00000652_P11

value: 0.003 uM Dexamethasone


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A02_6_3.000000uM_TC00000652_P07

value: 3 uM Etoposide


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A01_5_10.000000uM_TC00000652_P06

value: U-2 OS

value: 10 uM Berberine chloride

value: Berberine chloride

value: 10 uM


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A03_5_1.000000uM_TC00000652_P05

value: 1 uM Cycloheximide

value: Cycloheximide


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A11_3_0.000300uM_TC00000652_P03

value: 3e-04 uM Docetaxel

value: Docetaxel


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A09_7_0.100000uM_TC00000652_P04

value: 0.1 uM Cucurbitacin I

value: Cucurbitacin I


title: DMSO_TC00000652_P19

description: DMSO


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A10_4_0.003000uM_TC00000652_N21

value: 0.003 uM Actinomycin D

value: Actinomycin D


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A08_5_0.300000uM_TC00000652_N22

value: 0.3 uM Trichostatin A

value: Trichostatin A


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A10_3_0.001000uM_TC00000652_N23

value: 0.001 uM Actinomycin D


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A01_5_10.000000uM_TC00000652_N19


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A04_1_0.003000uM_TC00000652_N20


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A05_4_0.030000uM_TC00000652_N17

value: 0.03 uM Retinoic acid

value: Retinoic acid


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A07_7_1.000000uM_TC00000652_N16

value: U-2 OS

value: 1 uM Rapamycin

value: Rapamycin

value: 1 uM


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A11_2_0.000100uM_TC00000652_N15

value: 1e-04 uM Docetaxel


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A05_1_0.001000uM_TC00000652_N13

value: 0.001 uM Retinoic acid


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A10_1_0.000100uM_TC00000652_N18

value: 1e-04 uM Actinomycin D


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A10_7_0.100000uM_TC00000652_N14

value: 0.1 uM Actinomycin D


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A01_1_0.100000uM_TC00000652_N12

value: 0.1 uM Berberine chloride

value: Berberine chloride


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A04_4_0.100000uM_TC00000652_N11


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title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A09_6_0.030000uM_TC00000651_O22

value: U-2 OS

value: 0.03 uM Cucurbitacin I


title: DMSO_TC00000651_O19

description: DMSO

value: 0.5% DMSO vehicle control

value: DMSO

value: 0 uM


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A05_7_1.000000uM_TC00000651_O23

value: 1 uM Retinoic acid

value: Retinoic acid


title: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A10_2_0.000300uM_TC00000651_O21

value: 3e-04 uM Actinomycin D

value: Actinomycin D