title: Sample 59_Worm, 48h_1.25×10^-5g/L, rep4

city: San Diego

first: Goran

last: Bozinovic

address: 3030 Bunker Hill St

institute: Boz Life Science Research and Teaching Institute

description: pooled around 1000 worms

data_processing:
Base call was performed via NextSeq 1000/2000 Control Software Suite v1.4.1
Raw data from shallow RNA sequencing were demultiplexed by Scripps Research Genomics Core using BBTools (version 37.62) (Bushnell et al., 2017).
Unique Molecular Identifiers (UMI) were extracted from the sequencing results using UMI-tools (version 1.1.2) (Smith et al., 2017). Sequencing data quality was assessed in R (R Core Team, 2022. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/) via FastQCR (version 0.1.2) (Kassambara, 2019). SortMeRNA (version 4.3.4) was used to filter out rRNA with the sequence database smr_v4.3_sensitive_db.fasta (Kopylova et al., 2012). Trimmomatic (version 0.39) was used to remove the first 16 bases of each read, trim reads with a sliding-window of 4 bases and a minimum Phred score of 30, and remove resulting reads shorter than 18 bases (Bolger et al., 2014). The trimmed reads were aligned to C. elegans genome assembly WBcel235 with genome annotation WS283 via STAR (version 2.7.10a) (Dobin et al., 2013). PCR duplicates were removed using extracted UMI via UMI-tools. Gene counts based on reads mapping were generated through HTSeq (version 2.0.1) (Putri et al., 2022).
Identified genes with non-zero counts were selected from the raw output, and gene expression was normalized via trimmed mean of M-values (TMM) (Robinson & Oshlack, 2010). Average log counts per million (CPM) values were calculated via the aveLogCPM function of edgeR (version 3.36.0) (Robinson et al., 2010) and visualized via ggplot2 (version 3.3.5) (Wickham, 2016). Low count threshold was determined to be 16 counts per million, and low count filtering was performed via the filtered.data function of NOISeq (version 2.38.0) (Tarazona et al., 2015). Because RNA isolation was performed sequentially for the four replicates (batches) of the 15 experimental conditions, potential batch effects were removed via the ARSyNseq function of NOISeq.
Relative to the control condition, pairwise comparison to determine differentially expressed genes for each HFPO-DA exposure concentration was performed through NOISeq, with the probability of differential expression threshold, q, set to 0.99.
Assembly: WBcel235
Supplementary files format and content: worm_normalized.csv; contains the final normalized (as described in the data processing steps) gene counts that entered NOISeq analysis; column titles (Row 1) match the library names, and row titles (Column 1) are gene IDs

organism: Caenorhabditis elegans

source_name: Whole body

growth_protocol: The wild-type strain N2 (Bristol) of C. elegans was obtained from Caenorhabditis Genetics Center (CGC) at the University of Minnesota. Worms were maintained at 19 ± 1 ℃ in worm culturing media: S Medium [5.85 g/L NaCl, 1 g/L K2HPO4, 6 g/L KH2PO4, 5 mg/L cholesterol, 10mM potassium citrate (pH = 6.0), 1X N1010 Trace Metal Mix (United States Biological, Salem, Massachusetts, USA), 333 mg/L CaCl2, 361 mg/L MgSO4] with 100 mg/L streptomycin and 10 mg/L nystatin. Escherichia coli (E. coli) HB101 (CGC) were cultured at 37 ± 0.5 ℃ in lysogeny broth (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH = 7.0) with 100 mg/L streptomycin. Pelleted E. coli HB101 were resuspended in S Medium with a final concentration of 25 g/L as the food source for the worms. Worm cultures were maintained as 5-6 mL batches, with each in a cell culture flask (product number: 353109, Falcon®, Corning Inc., Corning, New York, USA), on a plate shaker at 180 rpm.

extract_protocol:
RNA isolation was performed on all 60 samples in the next two days by following TRIzol™ Reagent (catalog number: 15-596-018, Invitrogen™, Waltham, Massachusetts, USA) protocol with slight modifications. Briefly, TRIzol and five 2 mm borosilicate glass beads (product number: CG-1101-01, Chemglass Life Sciences, Vineland, New Jersey, USA) were added to each thawing worm pellet, and a Bead Mill 4 Homogenizer (catalog number: 15-340-164, Fisherbrand™) was used for homogenization. Each RNA sample was resuspended in 40 µL nuclease-free water. The quantity and quality of RNA samples were measured using a NanoDrop 2000 Spectrophotometer (catalog number: ND-2000, Thermo Scientific™, Thermo Fisher Scientific). On the following day, RNA samples were aliquoted and sent to Scripps Research Genomics Core (La Jolla, California, USA) for shallow RNA-sequencing.
cDNA library preparation and shallow RNA-Seq were performed at Scripps Research Genomics Core. Sample quality was assessed using a 2100 Bioanalyzer Instrument (part number: G2939BA, Agilent Technologies, Inc., Santa Clara, California, USA). Library preparation for RNA-Seq was performed via an early access kit, HTP RNA-Seq Library Prep, from iGenomX (now acquired by Twist Bioscience, South San Francisco, California, USA) with slight modifications. Briefly, PCR products 350–800 base pairs long were purified via 2% agarose gel electrophoresis, quantitated using Qubit dsDNA HS assay kit (catalog number: Q33231, Invitrogen™), and analyzed on a 4150 TapeStation System (part number: G2992AA, Agilent Technologies, Inc.).

treatment_protocol: Pre-treatment S Medium solutions with 0, 1.25×10-5, 6.25×10-5, 3.13×10-4, 1.56×10-3, 7.81×10-3, 1.56×10-2, 3.13×10-2, 6.25×10-2, 0.125, 0.25, 0.5, 1, 2, and 4 g/L HFPO-DA were prepared, and 1 ml of each solution was added to a well on 12-well plates. The placement of different concentrations was randomized, and a total of four replicates were prepared for each concentration. Plates with pre-treatment solutions were placed on a plate shaker at 180 rpm overnight. Exposure media with the same concentrations of HFPO-DA and age-synchronized L1 worms were prepared, and the pre-treatment solution in each well was replaced by 1 ml exposure media containing about 1000 worms. Exposure plates were placed on a plate shaker at 180 rpm for 48 hours. At 48 hours after exposures started, worms were pelleted (3000 rpm), washed twice with S Basal to remove residual E. coli HB101, and frozen at -80 ℃.

value: Whole body

tag: time

value: Developed 48 hours from synchronized first larval (L1) stage

tag: genotype

value: Wild type (N2, Bristol)

value: 48h exposure to 1.25×10^-5 g/L HFPO-DA


title: G10E05_wild_Benthic_1_run2_WGBS

status: Public on Jun 09 2022

type: SRA

city: Basel

first: Grégoire

middle: M

last: Vernaz

address: Vesalgasse 1

country: Switzerland

institute: University of Basel

description:
Total DNA from homogenized tissue
Benthic_wild_n3_mCG_50bpBIN_4x.bw
Bisulfite-Seq (whole genome)

library_source: genomic

data_processing:
RNAseq reads were mapped to MZ_UMD2a and transcript expression values (tpm) were calculated using kallisto (see methods)
WGBS and RRBS reads were mapped to MZ_UMD2a and methylation levels at CG site were called using Bismark after quality check and adaptor seq removeal using TrimGalore (see Methods).
Genome_build: GCF_000238955.4_M_zebra_UMD2a_genomic.fa
Supplementary_files_format_and_content: bw files containing mean mCG/CG levels averaged over 50bp non-overlapping bins and across samples for each group. Only CG with ≥4x coverage were used.
Supplementary_files_format_and_content: GeneQuant_tpm_Masoko_wild_liver.txt contains gene expression values (tpm) for all RNAseq samples.
Supplementary_files_format_and_content: Common_mCG_RRBS.txt file contains methylation values at common CG sites across all RRBS samples (min. 4x coverage in all CGs of all samples)

molecule: genomic DNA

organism: Astatotilapia calliptera

source_name: homogenized adult liver

extract_protocol:
Genomic DNA extracted using silica column based approach (see methods)
DNA strand-specific library using methylated adaptor (NEB) followed by bisulfite conversion (Imprint, Sigma), according to manusfacturer's instructions. WGBS libraries contain lambda genome spike-in control (0.2% w/w).

tag: ecomoprh

value: Benthic

tag: Sex

value: male

tag: type

value: wild caught

tag: tissue

value: liver


title: Bangor_Tank_Littoral_1_WGBS

description:
Total DNA from homogenized tissue
Littoral_tank_n2_mCG_50bpBIN_4x.bw
Bisulfite-Seq (whole genome)

value: Littoral

value: tank-reared


title: G08I06_wild_Littoral_1_run2_WGBS

description:
Total DNA from homogenized tissue
Littoral_wild_n2_mCG_50bpBIN_4x.bw
Bisulfite-Seq (whole genome)


title: G10E07_wild_Benthic_2_run1_WGBS


title: Sample 60_Worm, 48h_0g/L, rep4

state: CA

country: USA

description: pooled around 1000 worms

library_source: transcriptomic

molecule: polyA RNA

tag: treatment

value: control group (48h in 0 g/L HFPO-DA medium)


title: Bangor_Tank_Benthic_2_WGBS

description:
Total DNA from homogenized tissue
Benthic_tank_n2_mCG_50bpBIN_4x.bw
Bisulfite-Seq (whole genome)


title: G08I06_wild_Littoral_1_run1_WGBS


title: G10E05_wild_Benthic_1_run1_WGBS


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A8_S153]

city: Boston

first: Birgit

last: Knoechel

state: MA

address: 450 Brookline Ave

department: pediatric oncology

institute: Dana Farber Cancer Institute

description:
coculture24h_CART_NALM6_5to1_A8_S153
mRNA
052721_countmatrix_scRNAseq.csv

data_processing:
Adapter trimming was performed using trimmomatic version 0.36. Sequence quality was assessed using fastqc version 0.11.5. Sequencing reads were aligned to the human genome using STAR version 2.5.2b using following parameters ‘-- twopassMode Basic --alignIntronMax 100000 --alignMatesGapMax 100000 --alignSJDBoverhangMin 10 --alignSJstitchMismatchNmax 5 -1 5 5'.
Raw counts were obtained from aligned bam files using htseq version 0.9.1.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: scRNA-Seq: 052721_countmatrix_scRNAseq.csv: HTSeq count matrix with raw counts for all individual cells.
Supplementary_files_format_and_content: scATAC-Seq: 052721_countmatrix_scATACseq.csv: Fragments file with fragments for all individual cells; raw counts.

molecule: total RNA

organism: Homo sapiens

source_name: B-cell precursor leukemia cell line NALM-6

growth_protocol: NALM-6 cells were cultured in RPMI-1640 media with 10% of heat-inactivated fetal bovine serum, 100 μM penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1x NEAA, 20 mM HEPES and 0.5 mM 2-mercaptoethanol.

extract_protocol:
Single cells were directly sorted into 96 well plates containing TCL buffer.
Full-length single-cell RNA-seq libraries were prepared using the SMART-seq2 protocol. cDNA was fragmented using Nextera XT (Illumina) and amplified with indexed Nextera PCR primers.

treatment_protocol: Cells were treated for 24h with CAR T-cells/uninfected T-cells.

tag: cell line

value: CAR T-cells

tag: library strategy


title: G09E01_wild_Riverine_1_run2_WGBS

description:
Total DNA from homogenized tissue
Riverine_wild_n2_mCG_50bpBIN_4x.bw
Bisulfite-Seq (whole genome)

value: Riverine


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A8_S105]

description:
coculture24h_CART_NALM6_5to1_A8_S105
mRNA
052721_countmatrix_scRNAseq.csv


title: Bangor_Tank_Littoral_2_WGBS


title: Sample 58_Worm, 48h_6.25×10^-5g/L, rep4

description: pooled around 1000 worms

value: 48h exposure to 6.25×10^-5 g/L HFPO-DA


title: G09E01_wild_Riverine_1_run1_WGBS


title: Glucose RepB (Ctrl)

type: RNA

city: Saarbruecken

first: Michael

last: Kohlstedt

address: Campus A1.5

country: Germany

institute: Saarland University

hyb_protocol: Agilent hybridisation kit

scan_protocol: Agilent SureScan S2600D Microarray Scanner

data_processing:
GeneSpring 14.9 GC, Feature extraction,
Normalization: Shift to 75.0 percentile
Baseline Transformation: median of all samples
Statistical analysis (moderated t-test, Benjamini-Hochberg FDR, p_corr < 0.05, 2fold change), export

organism: Pseudomonas putida KT2440

source_name: cell biomass

label_protocol: Agilent labeling kit (one color)

growth_protocol: baffeled 1L shake flask, 100 mL E2 minimal medium (40 mM glucose), 30°C, 230 rpm, initial OD600 = 0.4

extract_protocol: mRNA extracted from harvested cell pellets (2 mg DCW) using Qiagen RNeasy kit and Turbo DNAfree kit

treatment_protocol: cells (2 mg) were harvested via centrifugation (30 sec, 4°C, 13.000 x g) and snap-frozen in liquid nitrogen

tag: treatment/time point

value: OD2 before catechol addition

tag: sample group

value: exponential growth


title: Glucose RepA (Ctrl)


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A7_S49]

description:
coculture24h_CART_NALM6_5to1_A7_S49
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 56_Worm, 48h_1.56×10^-3g/L, rep4

description: pooled around 1000 worms

value: 48h exposure to 1.56×10^-3 g/L HFPO-DA


title: Sample 57_Worm, 48h_3.13×10^-4g/L, rep4

description: pooled around 1000 worms

value: Whole body

value: 48h exposure to 3.13×10^-4 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A7_S269]

description:
coculture24h_CART_NALM6_5to1_A7_S269
mRNA
052721_countmatrix_scRNAseq.csv


title: TC_EU3

city: storrs

first: saurav

last: Ranjitkar

state: Connecticut (CT)

address: George White Building, Unit 4040, 17 Manter Road

department: Animal Science

institute: University of Connecticut

data_processing: Raw reads from Illumina sequencing were processed through TrimGalore/0.6.5 (Krueger, 2015) to remove the adapter sequences and to improve the read quality. High-quality reads were aligned with the latest assembly for M. bovis PG45 (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/183/385/GCF_000183385.1_ASM18338v1/), using bowtie2/2.3.5.1 (Langmead and Salzberg, 2012) with -very-sensitive-local flag (Supplementary Table 1). The average read counts and mapping rates were 4,547,069 and 99.17%, respectively. The resulting sam files were converted to bam files using samtools. The read counts were generated using the htseq/0.11.0 package (Anders, Pyl and Huber, 2015), normalized, and analyzed for differential expression using the DESeq2 (Nadler et al., 1997) package in R (R Team, 2021)

organism: Mycoplasmopsis bovis PG45

source_name: Mycoplama bovis PG45

growth_protocol: Mycoplama bovis strain PG45 from ATCC (Manassas, VA) was grown for 24 h at 37°C to 1 X 108 color changing units per milliliter in Fortified Commercial (FC) broth media (Hale et al., 1962) supplemented with 20% horse serum. The M. bovis culture was aliquoted to 1 ml stock and frozen at -80°C until further use.  On days of the treatments, one vial of the stock was thawed, added to 10 ml of culture medium, and cultured for 24 h at 37°C. This fresh culture was used directly in subsequent treatments.

extract_protocol:
RNA extraction from M. bovis samples was performed according to the standard TRIzol extraction protocol (Rio et al., 2010). To remove any potential DNA contamination, the extracted RNA was incubated with 7.5 U of DNase I (Qiagen, Hilden, Germany) for 15 min at room temperature. Then, RNA was precipitated again with 0.1 volume of 3M sodium acetate and 2.5 volumes of absolute ethanol, washed in 75% ethanol. The RNA was resuspended with 20 µl of RNase free-water and stored at -80°C until further analysis.
Total RNA samples (250 ng of Qubit-quantified total RNA input) were prepared for prokaryotic transcriptome sequencing using the Zymo-Seq RiboFree Total RNA library preparation kit (Zymo Research, Irvine, CA) following the manufacturer’s protocol. Libraries were quantified using the dsDNA High Sensitivity Assay for Qubit 3.0 (Life Technologies, Carlsbad, CA, USA), then the library quality were validated for fragment length and adapter dimer removal using the Agilent TapeStation 4200 D1000 High Sensitivity assay (Agilent Technologies, Santa Clara, CA, USA) Sample libraries were pooled and sequenced on illumina HiSeq 4000 platform with 75bp, pair-end read.

treatment_protocol: Mycoplama bovis was cultured with with 0.02% trans-cinnamaldehyde, or 0.04% carvacrol or 0.08% eugenol, or combination of two compund, or combination of all

value: 0.02% Trans-Cinnamaldehyde + 0.08% Eugenol


title: Triple1

value: 0.02% Trans-Cinnamaldehyde +0.04% Carvacrol + 0.08% Eugenol


title: Sample 53_Worm, 48h_3.13×10^-2g/L, rep4

description: pooled around 1000 worms

value: Developed 48 hours from synchronized first larval (L1) stage

value: Wild type (N2, Bristol)

value: 48h exposure to 3.13×10^-2 g/L HFPO-DA


title: G09D05_wild_Riverine_2_run1_WGBS

value: male

value: wild caught

value: liver


title: Triple3


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A7_S145]

description:
coculture24h_CART_NALM6_5to1_A7_S145
mRNA
052721_countmatrix_scRNAseq.csv


title: Triple2


title: Sample 55_Worm, 48h_7.81×10^-3g/L, rep4

description: pooled around 1000 worms

value: 48h exposure to 7.81×10^-3 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A7_S193]

description:
coculture24h_CART_NALM6_5to1_A7_S193
mRNA
052721_countmatrix_scRNAseq.csv

value: CAR T-cells


title: TC_CAR2

value: 0.02% Trans-Cinnamaldehyde + 0.04% Carvacrol


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A7_S25]

description:
coculture24h_CART_NALM6_5to1_A7_S25
mRNA
052721_countmatrix_scRNAseq.csv


title: TC_EU2


title: TC2

value: 0.02% Trans-Cinnamaldehyde


title: TC_CAR3


title: TC_EU1


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A6_S89]

description:
coculture24h_CART_NALM6_5to1_A6_S89
mRNA
052721_countmatrix_scRNAseq.csv


title: TC1


title: Sample 52_Worm, 48h_6.25×10^-2g/L, rep4

description: pooled around 1000 worms

value: 48h exposure to 6.25×10^-2 g/L HFPO-DA


title: EU_CAR2

value: 0.08% Eugenol +0.04% Carvacrol


title: Sample 54_Worm, 48h_1.56×10^-2g/L, rep4

description: pooled around 1000 worms

value: 48h exposure to 1.56×10^-2 g/L HFPO-DA


title: TC_CAR1


title: TC3


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A6_S329]

description:
coculture24h_CART_NALM6_5to1_A6_S329
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 49_Worm, 48h_0.5g/L, rep4

description: pooled around 1000 worms

value: Whole body

value: 48h exposure to 0.5 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A6_S21]

description:
coculture24h_CART_NALM6_5to1_A6_S21
mRNA
052721_countmatrix_scRNAseq.csv


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A5_S177]

description:
coculture24h_CART_NALM6_5to1_A5_S177
mRNA
052721_countmatrix_scRNAseq.csv


title: EU_CAR1


title: Sample 51_Worm, 48h_0.125g/L, rep4

description: pooled around 1000 worms

value: 48h exposure to 0.125 g/L HFPO-DA


title: EU_CAR3


title: Sample 50_Worm, 48h_0.25g/L, rep4

description: pooled around 1000 worms

value: 48h exposure to 0.25 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A5_S255]

description:
coculture24h_CART_NALM6_5to1_A5_S255
mRNA
052721_countmatrix_scRNAseq.csv


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A5_S81]

description:
coculture24h_CART_NALM6_5to1_A5_S81
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 48_Worm, 48h_1g/L, rep4

description: pooled around 1000 worms

value: Developed 48 hours from synchronized first larval (L1) stage

value: 48h exposure to 1 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A5_S129]

description:
coculture24h_CART_NALM6_5to1_A5_S129
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 46_Worm, 48h_4g/L, rep4

description: pooled around 1000 worms

value: Wild type (N2, Bristol)

value: 48h exposure to 4 g/L HFPO-DA


title: EU3

value: 0.08% Eugenol


title: Control3

value: No PDAs


title: EU2


title: Sample 47_Worm, 48h_2g/L, rep4

description: pooled around 1000 worms

value: 48h exposure to 2 g/L HFPO-DA


title: CAR3

value: 0.04% Carvacrol


title: EU1


title: Sample 45_Worm, 48h_0g/L, rep3

description: pooled around 1000 worms

value: control group (48h in 0 g/L HFPO-DA medium)


title: Control1


title: CAR2


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A4_S73]

description:
coculture24h_CART_NALM6_5to1_A4_S73
mRNA
052721_countmatrix_scRNAseq.csv

value: CAR T-cells


title: HER2_high_rep_2

city: LONDON

first: Ateequllah

last: Hayat

address: CRANMER TERRACE

department: IMBE

country: United Kingdom

institute: SGUL

data_processing:
Peak calling was perfomed using Bowtie2.
Illumina adapter trimming was done using Cutadapt; settings: Cutadapt -a CTGTCTCTTATACACATCT -A CTGTCTCTTATACACATCT -o out.1.fastq -p out.2.fastq.
ATAC-seq reads were aligned to the UCSC hg38 genome using Bowtie version 1.1.2. Only uniquely mapping reads with at most two mismatches were retained.
Peaks were called uing MACS v2.1.0 with the significance cut-off q-value <=0.01
bigWig files were generated using DeppTools. Score represents the normalised coverage of DNA fragments at a given genomic coordinate. narrowPeak files were generated using MACS with default settings.
Assembly: hg38
Supplementary files format and content: bigwig

source_name: Breast

growth_protocol: MCF10A cell medium consists of Dulbecco’s Modified Eagle’s Medium (DMEM/F12) (SIGMA #D8347) supplemented with 5% Horse Serum (SIGMA #H1138), 0.5 µg/mL Hydrocortisone (SIGMA #H0888), 20 ng/mL Epidermal Growth Factor (EGF) (SIGMA #E4127), 100 ng/mL Cholera Toxin (SIGMA #C8052), 10 µg/mL Insulin (SIGMA #i9278) and 1X Pen/Strep.

extract_protocol:
For 3D overlay cell cultures, cells were grown in chamber wells in a mixture of matrigel (CORNING #356230) and collagen (CORNING #11563550), which were mixed with 0.1M NaOH and 10X PBS, as previously described  (Xiang and Muthuswamy, 2006).  To collect cells from 3D cell cultures, cell recovery solution (CORNING #354253) was used at 4°C for 30 to 60 minutes according to the manufacturer’s instructions. Staining 3D acini were fixed with 4% paraformaldehyde (PFA). Acini were permeabilised with 0.5% Triton-X and blocked in 10% goat serum in PBS-Tween.
ATAC-seq libraries were prepared using Nextera DNA Library Preparation kit (15055289). We performed 10 initial PCR amplification cycles followed by direct purification of the transposed DNA, without performing qPCR to calculate the additional numbers of required cycles.

value: Breast

tag: cell type

value: Breast epithelial cells

value: Cell line

value: Doxycyline


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A4_S25]

description:
coculture24h_CART_NALM6_5to1_A4_S25
mRNA
052721_countmatrix_scRNAseq.csv


title: HER2_high_rep_3


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A4_S313]

description:
coculture24h_CART_NALM6_5to1_A4_S313
mRNA
052721_countmatrix_scRNAseq.csv


title: HER2_med_rep_2


title: Sample 44_Worm, 48h_1.25×10^-5g/L, rep3

description: pooled around 1000 worms

value: Whole body

value: 48h exposure to 1.25×10^-5 g/L HFPO-DA


title: HER2_med_rep_1


title: Sample 43_Worm, 48h_6.25×10^-5g/L, rep3

description: pooled around 1000 worms

value: 48h exposure to 6.25×10^-5 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A4_S169]

description:
coculture24h_CART_NALM6_5to1_A4_S169
mRNA
052721_countmatrix_scRNAseq.csv


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A4_S248]

description:
coculture24h_CART_NALM6_5to1_A4_S248
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 41_Worm, 48h_1.56×10^-3g/L, rep3

description: pooled around 1000 worms

value: Developed 48 hours from synchronized first larval (L1) stage

value: 48h exposure to 1.56×10^-3 g/L HFPO-DA


title: HER2_med_rep_3


title: CAR1


title: HER2_high_rep_1


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A4_S13]

description:
coculture24h_CART_NALM6_5to1_A4_S13
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 40_Worm, 48h_7.81×10^-3g/L, rep3

description: pooled around 1000 worms

value: Wild type (N2, Bristol)

value: 48h exposure to 7.81×10^-3 g/L HFPO-DA


title: HER2_low_rep_3

value: No doxycyline


title: Control2

value: No PDAs


title: HER2_pos_rep_1


title: Sample 38_Worm, 48h_3.13×10^-2g/L, rep3

description: pooled around 1000 worms

value: 48h exposure to 3.13×10^-2 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A3_S241]

description:
coculture24h_CART_NALM6_5to1_A3_S241
mRNA
052721_countmatrix_scRNAseq.csv


title: HER2_low_rep_2


title: HER2_low_rep_1


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A3_S9]

description:
coculture24h_CART_NALM6_5to1_A3_S9
mRNA
052721_countmatrix_scRNAseq.csv


title: HER2_pos_rep_3


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A3_S161]

description:
coculture24h_CART_NALM6_5to1_A3_S161
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 39_Worm, 48h_1.56×10^-2g/L, rep3

description: pooled around 1000 worms

value: 48h exposure to 1.56×10^-2 g/L HFPO-DA


title: ChIPseq_ASO1_R2

city: Lisboa

first: Rui

last: Sousa-Luís

address: Av. Professor Egas Moniz

country: Portugal

institute: Instituto de Medicina Molecular João Lobo Antunes

data_processing:
TrimGalore (v0.4.4) was used to remove possible sequencing adapters and filter sequences by Phred quality scores (“-q 20 --length 10 --stringency 1). Reads were further aligned with bwa mem (v0.7.15-r1140) against the ENSEMBLE GRCh38 and FlyBase dm6 genome assemblies.
Bitwise flags read paired (0x1) and read mapped in proper pair (0x2) were required using samtools (v1.11)
Duplicated reads were identified and removed with Picard MarkDuplicates (v2.24.0)
BedTools2 coverage (v2.29.2) was employed to reveal the number of reads overlapping each analysed feature.
Assembly: hg38 and dm6
Supplementary files format and content: Normalised read counts scaled to S2 probes

source_name: embryonic kidney

extract_protocol:
HEK293T or S2 cells were treated for 10 min in 1% (v/v) formaldehyde at room temperature to crosslink protein-DNA complexes, and the crosslinking was then quenched with 125 mM glycine for 5 min. Cells were washed twice with cold PBS and resuspended in 600 µL of Cell Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40 and 1x complete) and incubated on ice for 10 min. They were then centrifuged at 2,400 rpm for 5 min and nuclear pellets were resuspended in 400 μL of Nuclear Lysis buffer (0.5% SDS, 5 mM EDTA, 25 mM Tris-HCl pH 8.0 and 1x complete) and incubated on ice for 10 min. Chromatin were sonicated for 10 min (high power, 30 s on-off repeats) to obtain an average size of 200-500 bp (Bioruptor, Diagenode). Soluble chromatin was obtained after sonicated nuclei were centrifuged at 13,000 rpm for 10 min. Thirty micrograms of HEK293T cells chromatin were mixed with 2 µg of S2 cells chromatin and diluted 10 times with IP dilution buffer (10 mM Tris-HCl pH8.0, 5 mM EDTA, 0.5% Triton X-100 and 0.15 M NaCl). Nucleosomes were isolated from supernatant by IP with 3 μg of H3K9me2 antibody overnight at 4C. Thirty microliter of washed Dynabeads™ M-280 Sheep Anti-Mouse IgG (11202D, Thermo Fisher) was added and samples were rotated for 2-3 hrs at 4C. IPed DNA/beads were washed with 1 mL of buffer A (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 0.05% SDS, 1% Triton X-100 and 0.165 M NaCl) once, 1 mL of buffer B (20 mM Tris-HCl pH8.0, 2 mM EDTA, 0.05% SDS, 1% Triton X-100 and 0.5 M NaCl) once, 1 mL of buffer C (10 mM Tris-HCl pH8.0, 1 mM EDTA, 1% NP-40, 1% Sodium Deoxycholate and 0.25 M LiCl) and then 1 mL of buffer D (10 mM Tris-HCl pH8.0 and 1 mM EDTA) twice. Next IPed beads were incubated with 0.01 mg/mL RNase A (Ambion) in 300 μL of buffer E (1% SDS, 0.1 M NaHCO3 and 0.5 M NaCl) at 65°C for at least 4 hr. After RNase treatment, 30 μL of 10x Proteinase K mixture (200 mM Tris-HCl pH 6.5, 150 mM EDTA and Proteinase K 0.3 mg/mL) were added and then incubated at 45°C for 2 hrs.
DNA libraries were made according to NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) manual, using 5 ng of DNA, and 10∼12 cycles of PCR were used to amplify the libraries

tag: cell-type

value: H3K9me2 ChIP-seq


title: ChIPseq_VPA_ASO1_R1


title: HER2_neg_rep_3


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A3_S65]

description:
coculture24h_CART_NALM6_5to1_A3_S65
mRNA
052721_countmatrix_scRNAseq.csv

value: CAR T-cells


title: Sample 42_Worm, 48h_3.13×10^-4g/L, rep3

description: pooled around 1000 worms

value: 48h exposure to 3.13×10^-4 g/L HFPO-DA


title: Sample 34_Worm, 48h_0.5g/L, rep3

description: pooled around 1000 worms

value: 48h exposure to 0.5 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A2_S57]

description:
coculture24h_CART_NALM6_5to1_A2_S57
mRNA
052721_countmatrix_scRNAseq.csv


title: HER2_neg_rep_2

value: Breast

value: Breast epithelial cells

value: Cell line


title: Sample 33_Worm, 48h_1g/L, rep3

description: pooled around 1000 worms

value: Whole body

value: 48h exposure to 1 g/L HFPO-DA


title: Sample 37_Worm, 48h_6.25×10^-2g/L, rep3

description: pooled around 1000 worms

value: Developed 48 hours from synchronized first larval (L1) stage

value: 48h exposure to 6.25×10^-2 g/L HFPO-DA


title: HER2_neg_rep_1


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A3_S17]

description:
coculture24h_CART_NALM6_5to1_A3_S17
mRNA
052721_countmatrix_scRNAseq.csv


title: ChIPseq_VPA_ASO1_R2


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A2_S297]

description:
coculture24h_CART_NALM6_5to1_A2_S297
mRNA
052721_countmatrix_scRNAseq.csv


title: ChIPseq_VPA_R2

value: VPA


title: Sample 35_Worm, 48h_0.25g/L, rep3

description: pooled around 1000 worms

value: Wild type (N2, Bristol)

value: 48h exposure to 0.25 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A2_S153]

description:
coculture24h_CART_NALM6_5to1_A2_S153
mRNA
052721_countmatrix_scRNAseq.csv


title: ChIPseq_VPA_R1


title: POINTseq_VPA

description: nascent RNA

value: POINT-seq


title: ChIPseq_InpCTR

value: untreated


title: ChIPseq_CTR_R2


title: POINTseq_CTR_VPA

description: nascent RNA


title: Sample 36_Worm, 48h_0.125g/L, rep3

description: pooled around 1000 worms

value: 48h exposure to 0.125 g/L HFPO-DA


title: Sample 29_Worm, 48h_1.25×10^-5g/L, rep2

description: pooled around 1000 worms

value: 48h exposure to 1.25×10^-5 g/L HFPO-DA


title: ChIPseq_InpVPA_ASO1

value: H3K9me2 ChIP-seq


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A2_S9]

description:
coculture24h_CART_NALM6_5to1_A2_S9
mRNA
052721_countmatrix_scRNAseq.csv

value: CAR T-cells


title: Sample 32_Worm, 48h_2g/L, rep3

description: pooled around 1000 worms

value: 48h exposure to 2 g/L HFPO-DA


title: POINTseq_CTR_ASO1

description: nascent RNA


title: ChIPseq_InpASO1


title: ChIPseq_ASO1_R1


title: Sample 28_Worm, 48h_6.25×10^-5g/L, rep2

description: pooled around 1000 worms

value: 48h exposure to 6.25×10^-5 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A1_S227]

description:
coculture24h_CART_NALM6_5to1_A1_S227
mRNA
052721_countmatrix_scRNAseq.csv


title: RNAseq_VPA_ASO1_R2

value: RNA-seq


title: Sample 27_Worm, 48h_3.13×10^-4g/L, rep2

description: pooled around 1000 worms

value: 48h exposure to 3.13×10^-4 g/L HFPO-DA


title: Sample 31_Worm, 48h_4g/L, rep3

description: pooled around 1000 worms

value: Whole body

value: 48h exposure to 4 g/L HFPO-DA


title: ChIPseq_InpVPA


title: ChIPseq_CTR_R1


title: Sample 30_Worm, 48h_0g/L, rep2

description: pooled around 1000 worms

value: Developed 48 hours from synchronized first larval (L1) stage

value: control group (48h in 0 g/L HFPO-DA medium)


title: RNAseq_ASO1_R3


title: RNAseq_VPA_ASO1_R3


title: RNAseq_ASO1_R2


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A2_S105]

description:
coculture24h_CART_NALM6_5to1_A2_S105
mRNA
052721_countmatrix_scRNAseq.csv


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A1_S145]

description:
coculture24h_CART_NALM6_5to1_A1_S145
mRNA
052721_countmatrix_scRNAseq.csv


title: POINTseq_ASO1

description: nascent RNA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A2_S234]

description:
coculture24h_CART_NALM6_5to1_A2_S234
mRNA
052721_countmatrix_scRNAseq.csv


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A1_S49]

description:
coculture24h_CART_NALM6_5to1_A1_S49
mRNA
052721_countmatrix_scRNAseq.csv


title: RNAseq_VPA_ASO1_R1


title: Sample 24_Worm, 48h_1.56×10^-2g/L, rep2

description: pooled around 1000 worms

value: 48h exposure to 1.56×10^-2 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A12_S89]

description:
coculture24h_CART_NALM6_5to1_A12_S89
mRNA
052721_countmatrix_scRNAseq.csv


title: RNAseq_CTR_R2


title: RNAseq_VPA_R2

value: VPA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A12_S377]

description:
coculture24h_CART_NALM6_5to1_A12_S377
mRNA
052721_countmatrix_scRNAseq.csv


title: RNAseq_ASO1_R1


title: Sample 26_Worm, 48h_1.56×10^-3g/L, rep2

description: pooled around 1000 worms

value: Wild type (N2, Bristol)

value: 48h exposure to 1.56×10^-3 g/L HFPO-DA


title: deltahd_zcchc8_rep3

city: New York

first: Christopher

middle: D

last: Lima

state: NY

address: 1275 York Ave RRL301A

department: Structural Biology

institute: Sloan Kettering Institute

data_processing:
The output data (FASTQ files) were mapped to human genome (hg19) using the 2-pass mapping method [Engström2013] with the rnaStar aligner [Dobin2012] with Gencode v18 annotations. Output SAM files were post-processed using PICARD tool AddOrReplaceReadGroups to add read groups, sort files and convert them to compressed BAM format.
Genome coverage normalized to reads per million was computed for each strand using rnaStar in run mode inputAlignmentsFromBAM and output in bedGraph format. Coverage was averaged across replicates in each sample group.
PROMPT regions were converted from GRCh38 to hg19 using UCSC liftOver. We used a custom R script to find all Gencode annotated protein coding and lincRNA transcription start sites (TSS) within 3kb downstream of any PROMPT. If multiple PROMPTs fell within the 3kb downstream region of a single TSS, only the closest one was kept for analysis. The region 3kb upstream and 3kb downstream of these PROMPT-proximal TSSs were saved in BED format. Coverage for these 6kb regions was extracted from bedGraph files of each sample group described above. Finally, Gencode annotated exon regions were converted to BED format and removed from TSS window coverage files using bedtools subtract. Figure was generated using R and shows total coverage of all PROMPTs on reverse strand and gene transcripts on forward strand, colored by sample group.
TSSs of eRNA regions were identified as the midpoint between their start and end coordinates and were filtered for those at least 2kb upstream or downstream of any Gencode annotated gene. As previously described, 6kb regions around each TSS were saved in BED format and coverage for these regions was extracted from bedGraph files of each sample group using bedtools intersect. Exon regions were removed using bedtools subtract. Figure was generated using R and shows total coverage around eRNA TSSs, separated by transcription direction and colored by sample group.
snRNA transcripts were filtered from Gencode annotations and regions extending from 1kb upstream to 1kb downstream of their 3' ends were saved in BED format. Coverage for these 2kb regions was extracted from bedGraph files for each sample group using bedtools intersect. Figure was generated using R and shows total coverage of all snRNA EAGs, colored by sample group.
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph

source_name: HAP1 cells

growth_protocol: Human HAP1 cells were grown in Iscove’s Modified Dulbecco’s medium (Thermo Fisher Scientific) supplemented with 10% v/v fetal calf serum (Sigma) at 37°C and 5% CO2.

extract_protocol:
Cell pellets were lysed in buffer with 50% isopropanol + 0.5% 2-Mercaptoethanol and RNA was extracted from the lysate using the MagMAX mirVana Total RNA Isolation Kit (ThermoFisher catalog # A27828) on the KingFisher Flex Magnetic Particle Processor (Thermo Scientific catalog # 5400630) according to the manufacturer’s protocol with 10^6 cells input. Samples were eluted in 38 µL elution buffer.
After RiboGreen quantification and quality control by Agilent BioAnalyzer, 1 µg of total RNA with an RNA integrity number varying from 9.3-10 underwent ribosomal depletion and library preparation using the TruSeq Stranded Total RNA LT Kit (Illumina catalog # RS-122-1202) according to instructions provided by the manufacturer with 8 cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 S2 Reagent Kit (200 Cycles) (Illumina).  On average, 56 million paired reads were generated per sample and 63% of the data mapped to the transcriptome.

treatment_protocol: Plasmid transfections were carried out using Lipofectamine 3000 (Thermo Fisher Scientific) following manufacturer’s recommendations. Stably transfected cells were produced by co-transfecting cells with piggybac-compatible plasmid constructs with the gene of interest under a tetracycline-inducible promoter, a plasmid containing a hyperactive piggybac transposase (pRP[Exp]-mCherry-CAG>hyPBase) (VectorBuilder), and a plasmid containing the rtTA gene and a puromycin resistance gene (PB-rtTA-Puro). Stable integrants were selected by treatment with 3-5 µg/ml puromycin for 1 week. Expression of gene constructs under the tetracycline-inducible promoter were induced by treatment with 20 ng/µl doxycycline for east 24 hours.

value: HAP1 cells


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A1_S1]

description:
coculture24h_CART_NALM6_5to1_A1_S1
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 23_Worm, 48h_3.13×10^-2g/L, rep2

description: pooled around 1000 worms

value: 48h exposure to 3.13×10^-2 g/L HFPO-DA


title: RNAseq_VPA_R1


title: RNAseq_CTR_R3

value: untreated


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A12_S45]

description:
coculture24h_CART_NALM6_5to1_A12_S45
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 20_Worm, 48h_0.25g/L, rep2

description: pooled around 1000 worms

value: 48h exposure to 0.25 g/L HFPO-DA


title: Sample 21_Worm, 48h_0.125g/L, rep2

description: pooled around 1000 worms

value: 48h exposure to 0.125 g/L HFPO-DA


title: deltahd_zcchc8_rep1


title: NSC_Input

city: xiamen

first: Liu

last: Zhixiong

state: Fujian

address: Xiangan Nanlu 4221-120

department: School of Life Sciences

country: China

institute: Xiamen University

data_processing:
The image analysis and base calling were performed by using Illumina’s Genome Analysis pipeline.
The sequencing reads were aligned to the human genome UCSC build hg18 by using Bowtie2 alignment programs in two ways: only uniquely aligned reads were kept or both uniquely aligned reads and the sequencing reads that align to repetitive regions were kept for downstream analysis (if a read aligns to multiple genome locations, only one location is arbitrarily chosen).
The multiple reads were collapsed in order to reduce the PCR biases. The aligned reads were used for peak finding with HOMER
Peaks were identified by searching locations of high read density using a 200-bp sliding window. Regions of maximal density exceeding a given threshold were called as peaks, and we required adjacent peaks to be at least 500 bp away to avoid redundant detection
Peaks from separate experiments were considered equivalent/co-bound if their peak centers were located within 200 bp of each other. Read density heat maps were created by first using HOMER to generate read densities and then visualized using Java TreeView
Assembly: hg38
Supplementary files format and content: xls, bigWig (except for Input sample)

organism: Mus musculus

source_name: Brain

growth_protocol: Cortical mouse NPCs were cultured using P0 mouse cortex in laminin and poly-L-ornithine-coated plates and maintained in DMEM/F12 (11320033, Thermo) containing 1% penicillin/streptomycin, 1% l-glutamine, 1% N-2 supplement, 1% B27,1×Glutamax (10565-042, Life technologies)，5 mM Hepes, 0.06% Glucose, 50 μM beta-mercaptoethanol, 20 ng/ml EGFand 20 ng/ml bFGF

extract_protocol:
Cells were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin was sonicated using a M220 focused-ultrasonicator (Covaris).
ChIP-seq libraries were prepared using the  KAPA HyperPrep Kits (Roche, 07962347001) following manufacturer's protocols..

value: Brain

value: primary neural stem cell

value: wild type

value: growth medium


title: wt_zcchc8_rep2


title: RNAseq_CTR_R1

value: RNA-seq


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A12_S137]

description:
coculture24h_CART_NALM6_5to1_A12_S137
mRNA
052721_countmatrix_scRNAseq.csv

value: CAR T-cells


title: wt_zcchc8_rep1


title: Sample 22_Worm, 48h_6.25×10^-2g/L, rep2

description: pooled around 1000 worms

value: Whole body

value: Developed 48 hours from synchronized first larval (L1) stage

value: 48h exposure to 6.25×10^-2 g/L HFPO-DA


title: Sample 19_Worm, 48h_0.5g/L, rep2

description: pooled around 1000 worms

value: 48h exposure to 0.5 g/L HFPO-DA


title: deltahd_zcchc8_rep2


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A11_S81]

description:
coculture24h_CART_NALM6_5to1_A11_S81
mRNA
052721_countmatrix_scRNAseq.csv


title: zcchc8_ko_rep3


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A11_S369]

description:
coculture24h_CART_NALM6_5to1_A11_S369
mRNA
052721_countmatrix_scRNAseq.csv


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A12_S299]

description:
coculture24h_CART_NALM6_5to1_A12_S299
mRNA
052721_countmatrix_scRNAseq.csv


title: wt_zcchc8_rep3


title: zcchc8_ko_rep2


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A12_S233]

description:
coculture24h_CART_NALM6_5to1_A12_S233
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 16_Worm, 48h_4g/L, rep2

description: pooled around 1000 worms

value: Wild type (N2, Bristol)

value: 48h exposure to 4 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A11_S177]

description:
coculture24h_CART_NALM6_5to1_A11_S177
mRNA
052721_countmatrix_scRNAseq.csv


title: NSC_IgG_ChIP-Seq


title: Sample 13_Worm, 48h_6.25×10^-5g/L, rep1

description: pooled around 1000 worms

value: 48h exposure to 6.25×10^-5 g/L HFPO-DA


title: COVID-19 patient_9

city: Suita

first: Daisuke

last: Okuzaki

state: Osaka

address: Yamadaoka 3-1

department: Immunology Frontier Research Center

country: Japan

institute: Osaka univ.

description: PBMCs derived from COVID-19 patient_9

data_processing:
Sequencing was  performed on an Illumina NovaSeq 6000 platform in 101- base paired-end  mode.
The sequenced reads were mapped to the human reference genome  sequences (hg19) using TopHat, version 2.0.13, in combination with  Bowtie2, version 2.2.3, and SAMtools, version 0.1.19.
The fragments per kilobase of exon per million mapped fragments (FPKMs) were calculated using Cufflinks version 2.2.1.
Genome_build: hg19
Supplementary_files_format_and_content: FPKM processed value in csv file

source_name: COVID-19 patient_9

extract_protocol:
Total RNA isolation of leukocytes from ten COVID-19 patients on day1  and five healthy controls in the Osaka Covid-19 Cohort was performed  using the PAXgene™ Blood RNA System (BD Bioscience, San Jose, CA,  USA). The collection tubes were stored after blood collection until  further analysis at -30°C.
Library preparation was performed using a TruSeq stranded mRNA sample prep kit (Illumina, San Diego, CA) according to the manufacturer’s instructions.

value: peripheral blood mononuclear cells

tag: diagnosis


title: Sample 12_Worm, 48h_3.13×10^-4g/L, rep1

description: pooled around 1000 worms

value: 48h exposure to 3.13×10^-4 g/L HFPO-DA


title: COVID-19 patient_7

description: PBMCs derived from COVID-19 patient_7

source_name: COVID-19 patient_7


title: Sample 18_Worm, 48h_1g/L, rep2

description: pooled around 1000 worms

value: 48h exposure to 1 g/L HFPO-DA


title: NSC_Seh1_ChIP-Seq


title: COVID-19 patient_6

description: PBMCs derived from COVID-19 patient_6

source_name: COVID-19 patient_6


title: RNAseq_VPA_R3

value: VPA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A10_S169]

description:
coculture24h_CART_NALM6_5to1_A10_S169
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 10_Worm, 48h_7.81×10^-3g/L, rep1

description: pooled around 1000 worms

value: 48h exposure to 7.81×10^-3 g/L HFPO-DA


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A10_S121]

description:
coculture24h_CART_NALM6_5to1_A10_S121
mRNA
052721_countmatrix_scRNAseq.csv


title: COVID-19 patient_10

description: PBMCs derived from COVID-19 patient_10

source_name: COVID-19 patient_10


title: HER2_pos_rep_2

value: Breast

value: Breast epithelial cells

value: Cell line

value: Doxycyline


title: Sample 25_Worm, 48h_7.81×10^-3g/L, rep2

description: pooled around 1000 worms


title: RNA-seq_IPAH 4

city: Bad Nauheim

first: Carsten

last: Kuenne

address: Ludwigstrasse 43

department: Bioinformatics

institute: Max Planck Institute for Heart and Lung Research

data_processing:
Raw reads were assessed for quality, adapter content and duplication rates with FastQC.
Trimmomatic version 0.33 was employed to trim reads after a quality drop below a mean of Q18 in a window of 5 nucleotides. Only reads above 30 nucleotides were cleared for subsequent analyses.
Trimmed and filtered reads were aligned versus the Gencode human genome version hg19 (GRCh37) using STAR 2.4.0a with the parameter “--outFilterMismatchNoverLmax 0.1”.
The number of reads aligning to genes was counted with featureCounts 1.4.5-p1 tool from the Subread package. Only reads mapping at least partially inside exons were admitted and aggregated per gene. Reads overlapping multiple genes or aligning to multiple regions were excluded.
Differentially expressed genes were identified using DESeq2 version 1.6.2.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files contain DESeq normalized counts.

source_name: Pulmonary Artery

growth_protocol: PAAFs were maintained ex vivo in fibroblast growth medium (ScienCell Research Laboratories, Catalog: 2301) under normoxic conditions.

extract_protocol:
RNA was isolated from ex vivo isolated donor and PAH PAAFs using the miRNeasy mini kit (Qiagen) combined with on-column DNase digestion (DNase-Free DNase Set, Qiagen) to avoid contamination by genomic DNA.
RNA and library preparation integrity were verified with a BioAnalyzer 2100 (Agilent) or LabChip Gx Touch 24 (Perkin Elmer). Total RNA was used as input for Truseq Stranded mRNA Library preparation following the low sample protocol (Illumina). Sequencing was performed on the NextSeq500 instrument (Illumina) using v2 chemistry. For each run, 4-6 RNA-seq libraries were measured resulting minimum of 83M reads per library with 2x75bp paired end setup. Raw reads were assessed for quality, adaptor content and duplication rates with FastQC 0.10.1, trimmed by trimmoMatic and terminally aligned to the Ensemble human genome version hg38 (GRCh38) by STAR 2.4.0a (Dobin et al., 2013). Differentially expressed genes were identified using DESeq2 version 1.62 (Love et al., 2014).

tag: disease state

value: IPAH

value: Pulmonary Artery Adventital  Fibroblasts (PAAF)


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A11_S225]

description:
coculture24h_CART_NALM6_5to1_A11_S225
mRNA
052721_countmatrix_scRNAseq.csv

value: CAR T-cells


title: Sample 17_Worm, 48h_2g/L, rep2

description: pooled around 1000 worms

value: Whole body

value: Developed 48 hours from synchronized first larval (L1) stage

value: 48h exposure to 2 g/L HFPO-DA


title: RNA-seq_IPAH 3


title: zcchc8_ko_rep1

value: HAP1 cells


title: Sample 7_Worm, 48h_6.25×10^-2g/L, rep1

description: pooled around 1000 worms

value: Wild type (N2, Bristol)

value: 48h exposure to 6.25×10^-2 g/L HFPO-DA


title: COVID-19 patient_2

description: PBMCs derived from COVID-19 patient_2

source_name: COVID-19 patient_2

value: peripheral blood mononuclear cells


title: RNA-seq_IPAH 2


title: Sample 15_Worm, 48h_0g/L, rep1

description: pooled around 1000 worms

value: control group (48h in 0 g/L HFPO-DA medium)


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A11_S129]

description:
coculture24h_CART_NALM6_5to1_A11_S129
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 14_Worm, 48h_1.25×10^-5g/L, rep1

description: pooled around 1000 worms

value: 48h exposure to 1.25×10^-5 g/L HFPO-DA


title: Sample 6_Worm, 48h_0.125g/L, rep1

description: pooled around 1000 worms

value: 48h exposure to 0.125 g/L HFPO-DA


title: Model3

city: Chengdu

first: Xueyan

last: Wang

address: 37 Guoxue Alley

institute: Sichuan University

data_processing:
Raw sequencing data was first filtered by Trimmomatic (version 0.36), low-quality reads were discarded and the reads contaminated with adaptor sequences were trimmed.
Clean reads were first clustered according to the UMI sequences, in which reads with the same UMI sequence were grouped into the same cluster. Reads in the same cluster were compared to each other by pairwise alignment, and then reads with sequence identity over 95% were extracted to a new sub-cluster. After all sub-clusters were generated, multiple sequence alignment was performed to get one consensus sequence for each sub-cluster.
RNA-seq reads were aligned to the Ensembl mouse ge-nome built GRCm38 using STRA software (version 2.5.3a) with default parameters. Reads mapped to the exon regions of each gene were counted by feature Counts (Subread-1.5.1; Bioconductor) and then RPKM was calculated.
Genes differentially ex-pressed between groups were identified using the edgeR package (version 3.12.1). Sig-nificant differentially expressed genes were identified as such if the fold change was larger than 2.0 and the P-value was <0.05.
Enriched gene ontology (GO) for annotated differentially expressed genes and Enriched Kyoto encyclopedia of genes and genomes (KEGG) pathways were performed by KOBAS software (version: 2.1.1) with a P-value cutoff of 0.05 to judge statistically significant enrichment.
Genome_build: Mus_musculus.GRCm38
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

extract_protocol:
Total RNA was extracted from brain tissue using TRIzol reagent according to the methods by Chomczynski et al (DOI:10.1006/abio.1987.9999). DNA digestion was carried out after RNA extraction by DNaseI.
To prepare the library for RNA-seq, Ribo-off rRNA Depletion Kit (NO. MRZG12324) and KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina® (NO. DR08502) were used according to the manufacturer’s instruction.The kit eliminates duplication bias in PCR and sequencing steps, by using unique molecular identifier (UMI) of 8 random bases to label the pre-amplified cDNA mole-cules. The library products corresponding to 200-500 bps were enriched, quantified and finally sequenced on NovaSeq 6000 sequencer (Illumina) with PE150 model.

tag: strain

value: brain

value: ICH


title: Healthy control_5

description: PBMCs derived from healthy control_5

source_name: Healthy control_5

value: healthy


title: COVID-19 patient_8

description: PBMCs derived from COVID-19 patient_8

source_name: COVID-19 patient_8


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A10_S37]

description:
coculture24h_CART_NALM6_5to1_A10_S37
mRNA
052721_countmatrix_scRNAseq.csv


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A10_S287]

description:
coculture24h_CART_NALM6_5to1_A10_S287
mRNA
052721_countmatrix_scRNAseq.csv


title: NALM-6_CARTtreated_24h_SMARTseq2_0001 [A10_S217]

description:
coculture24h_CART_NALM6_5to1_A10_S217
mRNA
052721_countmatrix_scRNAseq.csv


title: Sample 11_Worm, 48h_1.56×10^-3g/L, rep1

description: pooled around 1000 worms

source_name: Whole body

value: 48h exposure to 1.56×10^-3 g/L HFPO-DA


title: Model1


title: COVID-19 patient_5

description: PBMCs derived from COVID-19 patient_5

source_name: COVID-19 patient_5


title: COVID-19 patient_4

description: PBMCs derived from COVID-19 patient_4

source_name: COVID-19 patient_4


title: Sample 9_Worm, 48h_1.56×10^-2g/L, rep1

description: pooled around 1000 worms

value: 48h exposure to 1.56×10^-2 g/L HFPO-DA


title: RNA-seq_Donor 1

value: Pulmonary Artery Adventital  Fibroblasts (PAAF)


title: F3

value: treated with Febuxostat before ICH


title: Sham1

value: untreated


title: COVID-19 patient_3

description: PBMCs derived from COVID-19 patient_3

source_name: COVID-19 patient_3


title: Sample 8_Worm, 48h_3.13×10^-2g/L, rep1

description: pooled around 1000 worms

value: Whole body

value: Developed 48 hours from synchronized first larval (L1) stage

value: 48h exposure to 3.13×10^-2 g/L HFPO-DA


title: F2


title: BE2C-HDAC2-dTAG-HDAC1oe cells, dTAG13 (500 nM), 24 h, biol rep 3

status: Public on Jun 08 2022

city: La Jolla

first: Yuxiang

last: Zhang

address: 10550 N. Torrey Pines Rd.

department: Chemistry

institute: The Scripps Research Institute

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
hdac1_oe_24h_dtag13_3

data_processing:
Raw basecall (or BCL) files from the NextSeq2000 sequencer were converted to sample fastqs using an in-house custom built wrapper around Illumina’s bcl2fastq conversion software v2.19.0.
SLAM-dunk was used for read alignment, CPM calculation, and conversion rate calculation.
For ERCC normalization, normalize.loess function of the affy v1.50.0 R package76 was used. Locally estimated scatterplot smoothing (LOESS) normalization was applied to all CPM values of transcripts with the model specified by the distribution of the SIRV-Set-3 spike-ins.
Unexpressed genes were filtered out with the cutoff of CPM < 3 in all 8-h and 24-h DMSO and 500 nM dTAG-13 treated samples.
Assembly: hg19
Supplementary files format and content: T-to-C conversion rates of all genes in each sample for SLAM-seq, ERCC-normalized CPMs of all genes in each sample for 3' end mRNA-seq

source_name: Neuroblastoma

growth_protocol: BE(2)-C cells were cultured in DMEM supplemented with 10% FBS and Gibco Antibiotic-Antimycotic.

extract_protocol:
RNA was extracted with RNeasy miniprep kit (Qiagen). For SLAM-seq, 5 µg RNA of 4sU laballed RNA for each sample was subjected to alkylation by 100 mM iodoacetamide.
1 µg of RNA of each sample was used for libarary synthesis with QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, #015) following manufacturer's protocols.
3' end mRNA-seq.

treatment_protocol: For SLAM-seq cells were treated with 100 µM 4-thiouracil (4sU) for 1 hour for newly synthesized mRNA labeling. For 3' end mRNA-seq 2.1 ng/mL of ERCC RNA Spike-In Control SIRV Set 3 (Lexogen, #051.0) per sample was added for ERCC normalization.

value: Neuroblastoma

value: endogenous HDAC2-dTAG, exogenous HDAC1 expression

tag: time point

value: 24 hours


title: BE2C-HDAC2-dTAG-HDAC1oe cells, dTAG13 (500 nM), 24 h, biol rep 2

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
hdac1_oe_24h_dtag13_2


title: Sample 3_Worm, 48h_1g/L, rep1

description: pooled around 1000 worms

tag: time

value: Wild type (N2, Bristol)

value: 48h exposure to 1 g/L HFPO-DA


title: BE2C-HDAC2-dTAG-HDAC1oe cells, dTAG13 (500 nM), 24 h, biol rep 1

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
hdac1_oe_24h_dtag13_1

tag: genotype


title: F1


title: BE2C-HDAC2-dTAG-HDAC1oe cells, DMSO (0.1%), 24 h, biol rep 2

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
hdac1_oe_24h_dmso_2

value: DMSO, 0.1percent


title: BE2C-HDAC2-dTAG-HDAC1oe cells, DMSO (0.1%), 24 h, biol rep 1

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
hdac1_oe_24h_dmso_1


title: COVID-19 patient_1

description: PBMCs derived from COVID-19 patient_1

source_name: COVID-19 patient_1

value: peripheral blood mononuclear cells


title: RNA-seq_IPAH 1

value: IPAH


title: BE2C-HDAC2-dTAG cells, ACY957 (5 µM), 24 h, biol rep 3

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
24h_acy957_3

value: endogenous HDAC2-dTAG

value: ACY-957, 5 microM


title: Sample 5_Worm, 48h_0.25g/L, rep1

description: pooled around 1000 worms

value: 48h exposure to 0.25 g/L HFPO-DA


title: BE2C-HDAC2-dTAG cells, DMSO (0.1%), 24 h, biol rep 3 [s3]

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
24h_dmso_3


title: BE2C-HDAC2-dTAG cells, ACY957 (5 µM), 24 h, biol rep 2

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
24h_acy957_2


title: BE2C-HDAC2-dTAG cells, ACY957 (5 µM), 24 h, biol rep 1

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
24h_acy957_1


title: RNA-seq_Donor 4

value: healthy

value: Pulmonary Artery Adventital  Fibroblasts (PAAF)


title: BE2C-HDAC2-dTAG cells, DMSO (0.1%), 24 h, biol rep 1 [s1]

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
24h_dmso_1


title: BE2C-HDAC2-dTAG cells, DMSO (0.1%), 24 h, biol rep 2 [s2]

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
24h_dmso_2


title: Healthy control_3

description: PBMCs derived from healthy control_3

source_name: Healthy control_3


title: RNA-seq_Donor 2


title: Model2

value: brain

value: ICH


title: BE2C-HDAC2-dTAG cells, DMSO (0.1%), 72 h, biol rep 1

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
72h_dmso_1

value: 72 hours


title: Sham2

value: untreated


title: Healthy control_2

description: PBMCs derived from healthy control_2

source_name: Healthy control_2


title: Sample 2_Worm, 48h_2g/L, rep1

description: pooled around 1000 worms

value: Whole body

value: Developed 48 hours from synchronized first larval (L1) stage

value: 48h exposure to 2 g/L HFPO-DA


title: Healthy control_1

description: PBMCs derived from healthy control_1

source_name: Healthy control_1


title: Sham3

source_name: Brain


title: Sample 1_Worm, 48h_4g/L, rep1

description: pooled around 1000 worms

value: Wild type (N2, Bristol)

value: 48h exposure to 4 g/L HFPO-DA


title: RNA-seq_Donor 3


title: Sample 4_Worm, 48h_0.5g/L, rep1

description: pooled around 1000 worms

value: 48h exposure to 0.5 g/L HFPO-DA


title: BE2C-HDAC2-dTAG, IgG control for HA

data_processing:
Raw basecall (or BCL) files from the NextSeq2000 sequencer were converted to sample fastqs with paired-end reads using an in-house custom built wrapper around Illumina’s bcl2fastq conversion software v2.19.0.
The sequencing reads were aligned to human genome build hg19 and RefSeq genes by Bowtie2.
The signal-enriched regions were identified by Model-based Analysis of ChIP-seq (MACS12) peak-finding algorithm (v1.4.1) with the P value threshold of 1e-9 using the IgG sample as background. Bed files and wiggle files were created by MACS.
Assembly: hg19
Supplementary files format and content: wiggle file, bed file (except for Input sample)
Library strategy: CUT&RUN

growth_protocol: BE(2)-C cells were cultured in DMEM supplemented with 10% FBS and Gibco Antibiotic-Antimycotic

extract_protocol:
Cells were permeablized with 0.02% digitonin. CUT&RUN fragments were extracted following the protocol based on Skene&Henikoff, Nat Protoc. 2018. and resuspended in 13 µL of H2O.
CUT&RUN libraries were prepared with Takara ThruPLEX DNA-Seq Kit and barcoded with Takara DNA Unique Dual Index Kit. 10 µL of input material was used for library preparation. The libraries were then amplified with 16 cycles of 98° C for 15 s and 60° C for 10 s on thermocycler to allow prefrential amplification of the small CUT&RUN fragments. Libraries were then purified with AMPure XP beads and validated on Agilent TapeStation.

treatment_protocol: Cells were treated with DMSO (0.1%) or dTAG-13 (500 nM) for 2 h.

value: Neuroblastoma

tag: chip antibody

value: IgG, CiteAb, ABIN101961


title: BE2C-HDAC2-dTAG, HA

value: HA, Cell Signaling Technology, #3724S


title: BE2C-HDAC2-dTAG, H3K9ac, dTAG-13 (500 nM), 2 h

value: H3K9ac, Cell Signaling Technology, #9649S


title: BE2C-HDAC2-dTAG-HDAC1oe cells, DMSO (0.1%), 24 h, biol rep 3

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
hdac1_oe_24h_dmso_3

value: endogenous HDAC2-dTAG, exogenous HDAC1 expression

value: DMSO, 0.1percent

value: 24 hours


title: BE2C-HDAC2-dTAG cells, DMSO (0.1%), 72 h, biol rep 3

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
72h_dmso_3

value: endogenous HDAC2-dTAG


title: Healthy control_4

description: PBMCs derived from healthy control_4

source_name: Healthy control_4

value: peripheral blood mononuclear cells

value: healthy


title: BE2C-HDAC2-dTAG cells, dTAG13 (500 nM), 72 h, biol rep 3

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
72h_dtag13_3

value: 72 hours


title: BE2C-HDAC2-dTAG cells, dTAG13 (500 nM), 72 h, biol rep 1

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
72h_dtag13_1


title: BE2C-HDAC2-dTAG cells, dTAG13 (500 nM), 72 h, biol rep 2

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
72h_dtag13_2


title: BE2C-WT, IgG control for HDAC2


title: BE2C-HDAC2-dTAG cells, DMSO (0.1%), 72 h, biol rep 2

description:
mRNA-seq_ERCC-normalized_CPM_all_treatments_all_replicates_all_genes.txt
72h_dmso_2


title: set77.Y336F-KI.PRO-seq.rep2

city: Baltimore

first: Weidong

state: MD

address: 251 Bayview Blvd

department: National Institute on Aging

institute: Shuaikun Su

description: sample 87 to 90 are in one group

data_processing:
For Ribo-seq, low-quality reads and the linker sequence (AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC) were removed by FASTX-Toolkit from FastQ files. Then reads less than 25 nt were also removed by FASTX-Toolkit. Align the remaining reads to an rRNA reference using the Bowtie short-read alignment program, discard the rRNA alignments and collect unaligned reads. For PRO-seq, adaptor sequences (ADAPT1=GATCGTCGGACTGTAGAACTCTGAA, ADAPT2=TGGAATTCTCGGGTGCCAAGG) were removed by cutadpt. For eCLIP-seq, adaptor sequences were also removed before mapping to the genome.
RNA-seq, Ribo-seq and eCLIP-seq reads were mapped to the hg38 human genome with HISAT2 or TopHat. PRO-seq reads were mapped to the genome using Bowtie2 allowing maximum of 2 mismatches.
For RNA-seq and Ribo-seq, the raw counts were generated using HTSeq-Counts. Then DESeq2 was used to identify the differentially expressed genes (DEGs) and generate the normalized counts. The genes with total counts (untreated and treated groups) less than 10 were filtered. The genes (Fold change > 1.5 and adjust P-value < 0.1) were identified as DEGs. The bedGraph files were generated by deepTools and normalized using RPKM. The bedGraph files were visualized using the Integrative Genomics Viewer (IGV). For PRO-seq, normalized reads at specific sub-regions were used to calculate the DEGs (fold change > 1.2-fold). eCLIP-seq reads were mapped to the genome using TopHat. Other steps were similar to the RNA-seq analysis pipeline. Genes with criterion (WT-IP_fpkm/WT-Input_fpkm > 1.5 and WT-IP_fpkm/TOP3B-KO-IP_fpkm > 1.5) in both biological replicates were selected as TOP3B eCLIP targets. RSeQC was used to analyze the read distribution of Ribo-seq, RNA-seq and eCLIP-seq on CDS, 5’UTR and 3’UTR.
Genome_build: hg38
Supplementary_files_format_and_content: bedGraph file

source_name: human colorectal carcinoma cell line

growth_protocol: HCT116 cells were cultured in  DMEM with 10% fetal bovine serum (HyClone) and 1% antibiotics (Penicillin-Streptomycin, Sigma).

extract_protocol:
Different sequencing types have different protocols, please follow the protocols in the "Materials and Methods" part in the article. Briefly, for RNA-seq, total RNAs were extracted with TRizol. For Ribo-seq, ribosome protected fragments were collected through sucrose cushion and extracted with TRizol, then the 24 to 34 nt RNA fragments were collected through 15% TBE-Urea gel. For eCLIP-seq, protein-RNA complexes were isolated with antibody. For PRO-seq, nascent RNA fragments were extractd and made library.
For RNA-seq, library construction protocol can be found here (Y. Joo et al., Topoisomerase 3beta knockout mice show transcriptional and behavioural impairments associated with neurogenesis and synaptic plasticity. Nat Commun 11, 3143 (2020).). For Ribo-seq, library was constructed using NEBNext® Multiplex Small RNA Library Prep Set for Illumina (E7300S, New England BioLabs). For eCLIP-seq, library construction protocol can be found here (E. L. Van Nostrand et al., Robust, Cost-Effective Profiling of RNA Binding Protein Targets with Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP). Methods Mol Biol 1648, 177-200 (2017).). For PRO-seq, library construction protocol can be found here (D. B. Mahat et al., Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nature Protocols 11, 1455-1476 (2016).).
RNA-seq; Ribo-seq; eCLIP-seq; PRO-seq

treatment_protocol: For the Ribo-seq samples and the RNA-seq samples, cells were treated with 100µg/mL cycloheximide for 10min

tag: molecule

value: Nascent RNA fragments


title: BE2C-HDAC2-dTAG, IgG control for H3K9ac, H3K27ac


title: BE2C-HDAC2-dTAG, H3K27ac, DMSO (0.1%), 2 h

value: H3K27ac, Abcam, #ab4729


title: BE2C-HDAC2-dTAG, H3K27ac, dTAG-13 (500 nM), 2 h


title: BE2C-HDAC2-dTAG, H3K9ac, DMSO (0.1%), 2 h


title: set77.TOP3B-KO1.PRO-seq.rep2

description: sample 87 to 90 are in one group


title: set75.TDRD3-KO.PRO-seq.rep1

description: sample 83 to 86 are in one group


title: BE2C-WT, CHD4

value: CHD4, Cell Signaling Technology, #12011S


title: BE2C-WT, HDAC2

value: HDAC2, Active motif, #31505


title: BE2C-WT, MBD3

value: MBD3, Cell Signaling Technology, #99169S


title: set59.WT.2.Ribo-seq.rep3

description: sample 19,22,25,28,31,34 are in one group

value: Wild type

value: Cells were treated with 100µg/mL cycloheximide for 10min and then were used for Ribo-seq.

value: Ribosome protected RNA fragments


title: set58.FMR1-KO.Ribo-seq.rep2

description: sample 18,21,24,27,30,33 are in one group


title: set58.TDRD3-KO.Ribo-seq.rep2


title: set58.FMR1-KO.RNA-seq.rep2

value: Cells were treated with 100µg/mL cycloheximide for 10min and then lysed in polysome lysis buffer. After centrifugation, 100 µL cell lysates were used to extract RNA with TRIzol. Then 5~10 µg RNA was used to purify mRNAs with oligo dT beads. The purified mRNAs were used for RNA-seq library preparation.

value: polyA RNA


title: set59.WT.2.RNA-seq.rep3


title: set77.WT.3.PRO-seq.rep2

description: sample 87 to 90 are in one group


title: set77.TDRD3-KO.PRO-seq.rep2

description: sample 87 to 90 are in one group


title: set75.TOP3B-KO1.PRO-seq.rep1

description: sample 83 to 86 are in one group


title: HepG2.2.15 Fig5-HBV_mock_rep3

city: Cambridge

first: Joseph

last: Barry

address: 675 West Kendall St

institute: Alnylam Pharmaceuticals

data_processing:
Raw RNAseq reads were filtered with minimal mean quality scores of 25 and minimal remaining length of 36, using the ea-utils software fastq-mcf.
Filtered reads were aligned to the Rattus norvegicus genome (Rnor_6.0) [rat liver and primary hepatocyte samples], or combined Human (hgl9/GRCh37) and HBV (GenBank nucleotide ID U95551; NCBI GeneID: 7276) genomes using STAR (ultrafast universal RNAseq aligner) [HepG2.2.15 samples].
Differential gene expression analysis was performed by the R package DESeq2 v1.16.1 with the betaPrior parameter set to TRUE to shrink log2 fold-change estimates for noisy, low-count genes.
Assembly: Rnor_6.0, GRCh37

growth_protocol: HepG2.2.15 cells, a HepG2 derived cell line stably transfected with full genome HBV, were diluted with culture media to a final concentration of 187,500 cells/mL, and 80 µL was pipetted into 96-well collagen coated plates (BD Biocoat, Cat#356407) to give a final concentration of 15,000 cells/well.  RNAiMAX (ThermoFisher, Cat#13778150) was diluted with Opti-MEM (ThermoFisher Cat#31985062) at a concentration of 0.3 µL RNAiMAX/10 µL Opti-MEM and incubated for 5 minutes at room temperature.  After incubation, 10 µL/well was added to each well of 96-well collagen coated plates (BD Biocoat, Cat#356407), along with 10 µL/well of the appropriate dsRNA agent dilution (as appropriate in 1x PBS), mixed gently and incubated for 20 minutes at room temperature.  80 µL of prepared cell suspension was added to each well to give final cell density of 15,000 cells/well and final dsRNA agent concentrations of 10 nM.  Cells were incubated in a 37 °C incubator with 5% CO2 for 16-22 hours. Primary Rat Hepatocyte cells followed a similar protocol, but were incubated for 48 hours.

extract_protocol:
Rat livers collected at the end of the toxicity studies were snap-frozen, powdered, and RNA was extracted with the miRNeasy kit (Qiagen). HepG2.2.15 RNA was isolated using the ThermoFisher RNAqueous-96 Total RNA Isolation Kit, as per the protocol.
cDNA libraries were prepared with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on a NextSeq500 sequencer (Illumina), all according to manufacturers’ instructions.

tag: age

tag: Sex


title: HepG2.2.15 Fig5-HBV_mock_rep2


title: HepG2.2.15 Fig5-HBV_ALN-HBV02_10nM_rep2


title: HepG2.2.15 Fig5-HBV_ALN-HBV_10nM_rep4


title: HepG2.2.15 Fig5-HBV_ALN-HBV02_10nM_rep3


title: set59.FMR1-KO.Ribo-seq.rep3

tag: treatment


title: HepG2.2.15 Fig5-HBV_mock_rep1

tag: cell line


title: set75.WT.3.PRO-seq.rep1

description: sample 83 to 86 are in one group


title: set59.FMR1-KO.RNA-seq.rep3


title: set59.TDRD3-KO.Ribo-seq.rep3


title: set59.TDRD3-KO.RNA-seq.rep3


title: HepG2.2.15 Fig5-HBV_mock_rep4


title: set58.TDRD3-KO.RNA-seq.rep2


title: HepG2.2.15 Fig5-HBV_ALN-HBV_10nM_rep2


title: BE2C-WT, IgG control for MBD3, CHD4

value: Neuroblastoma

value: IgG, CiteAb, ABIN101961


title: BE2C-HDAC2-dTAG, ATAC-seq, DMSO (0.1%), 24 h, biol rep 2

data_processing:
Reads were processed with the ENCODE ATAC-seq pipeline (v1.8.0) and were aligned to the human genome build hg19.  Adapters were trimmed with Cutadapt (v3.0) and reads were aligned with Bowtie2 (v2.4.2). Blacklist regions excluded using data file wgEncodeDacMapabilityConsensusExcludable.bed.gz. Unmapped/low quality reads were filtered with Samtools (v1.11) and duplicated reads were removed with Picard Tools (v2.23.9).
Peaks were called by MACS2 (v2.2.7.1) with call-peak P value threshold of 0.01, maximum peaks of 5×105, size of smoothing window of 150, and IDR (irreproducible discovery rate) threshold of 0.05. GC bias computation was performed and TSS (transcription start site) enrichment was calculated on filtered reads.
Assembly: hg19
Supplementary files format and content: bigWig, narrowPeak

extract_protocol:
ATAC-seq fragmentes were extracted following the protocol based on Corces et al, Nat Methods. 2017.
ATAC-seq libraries were prepared following the protocol based on Corces et al, Nat Methods. 2017.

treatment_protocol: Cells were treated with DMSO (0.1%) or dTAG-13 (500 nM) for 2 h or 24 h

tag: agent

value: DMSO


title: set58.WT.2.RNA-seq.rep2


title: BE2C-HDAC2-dTAG, ATAC-seq, dTAG-13 (500 nM), 2 h, biol rep 2

value: 2 h


title: set75.Y336F-KI.PRO-seq.rep1

description: sample 83 to 86 are in one group


title: HepG2.2.15 Fig5-HBV_ALN-HBV02_10nM_rep4


title: HepG2.2.15 Fig5-HBV_ALN-HBV02_10nM_rep1


title: BE2C-HDAC2-dTAG, ATAC-seq, DMSO (0.1%), 2 h, biol rep 1


title: set58.WT.2.Ribo-seq.rep2


title: Jurkat non-activated

city: Seoul

first: Sangho

last: Lee

address: College of Medicine, Biomedical Science Building 202, 103 Daehakro, Jongro-gu

department: MMBS

country: South Korea

institute: Seoul National University

data_processing:
The raw image data was transformed by base calling into sequence data and stored in FASTQ format.
Reads were trimmed for adaptor sequences, low-quality sequences using ngsShoRT and aligned to the human genome (GRCh37/hg19 genome assembly) using STAR (v2.4.0.1) with the default settings.
The tag directory was created by Homer makeTagDirectory and visualized by Homer makeUCSCfile.
Genome_build: hg19
Supplementary_files_format_and_content: BedGraph; Chromosome name, start site, end site and data value were displayed in four columns.

source_name: acute T cell leukemia cell line

growth_protocol: Jurkat T cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), and antibiotics at 37°C in a humidified atmosphere of 5% CO2.

extract_protocol:
Total RNA was extracted using QIAzol Lysis Reagent (QIAGEN, Hilden, Germany).
Extracted RNA was used to prepare an mRNA sequencing library using TruSeq Stranded mRNA sample preparation kit (Illumina, San Diego, CA, USA).

treatment_protocol: Jurkat T cells were treated with 40 nM PMA and 1 μM Ionomycin for 8 hr. 0.5 μM FK506 was pretreated for 1hr before treatment with PMA and Ionomycin.

value: Jurkat, Clone E6-1

value: HA-mCherry expression

value: untreated


title: HepG2.2.15 Fig5-HBV_ALN-HBV_10nM_rep1


title: HepG2.2.15 Fig5-HBV_ALN-HBV_10nM_rep3


title: BE2C-HDAC2-dTAG, ATAC-seq, dTAG-13 (500 nM), 2 h, biol rep 3


title: BE2C-HDAC2-dTAG, ATAC-seq, dTAG-13 (500 nM), 24 h, biol rep 1


title: BE2C-HDAC2-dTAG, ATAC-seq, dTAG-13 (500 nM), 24 h, biol rep 3


title: BE2C-HDAC2-dTAG, ATAC-seq, dTAG-13 (500 nM), 24 h, biol rep 2


title: BE2C-HDAC2-dTAG, ATAC-seq, DMSO (0.1%), 24 h, biol rep 1


title: HR_RPF_NAT10_rep2

city: Bethesda

first: David

last: Sturgill

state: MARYLAND

address: Building 41, Rm B622

department: NCI

institute: NIH

data_processing:
Library strategy: Ribo-seq
Raw reads were pre-processed to remove low quality bases and adapter sequences using cutadapt
For Ribo-Seq: After trimming, reads were stringently filtered for the presence of ribosomal RNA using a two-step process.  First, we performed local alignments to the ribosomal repeating subunit (gb|U13369.1) in very sensitive mode using bowtie2 (v.2.3.5.1)). Second, we mapped remaining reads using Hisat2 (v2.2.1.0), explicitly allowing for mismatches at the first nucleotide position. Trimmed and filtered reads were then aligned to a reference composed of canonical transcript sequence from the UCSC genome browser in the hg19 assembly, using bowtie2 (v.2.3.5.1)
RedaC:T-seq reads were aligned to the human genome (hg19) and the human transcriptome (longest isoform from UCSC) using STAR (v 2.5.4.a) in local alignment mode, keeping uniquely aligned reads and allowing five mismatches per read. Separate alignments to 18S rRNA, 28S rRNA and tRNA sequences were performed to specifically analyze reads originating from these features. Variant calls were performed in pooled biological replicates using Samtools mpileup including a minimum base quality -Q 20, the BAQ (Base Alignment Quality) and -C50 parameters activated and supplying a -f indexed reference fasta file. mpileup results were then tabulated using the mpileup2readcounts script.
For Polysome sequencing: reads were aligned to the human genome (hg19 assembly) containing the Luciferase Spike-in sequence using Hisat2 (v2.2.1.0). Gene counts within each fraction were obtained using StringTie2, normalized to the spike-in probe and converted to Transcripts per million (TPM)
Genome_build: hg19
Supplementary_files_format_and_content: HR_riboseq.txt contains P-sites called from Harringtonine-treated Ribo-seq
Supplementary_files_format_and_content: RedaCTseq_ac4C_sites.txt contains data for ac4C sites called from RedaCT-Seq
Supplementary_files_format_and_content: Counts text files contain raw gene abundance values from Stringtie2

source_name: HeLa cells

growth_protocol: Parental HeLa (Human cervix carcinoma, female, ATCC, Cat. #: CCL-2) and NAT10-/- cells were cultured in Dulbeccos’ Modified Eagle Medium (DMEM, ThermoFisher Scientific, Cat. #: 10313021) containing 25 mM glucose, 1 mM sodium pyruvate and supplemented with 2 mM L-glutamine (ThermoFisher Scientific, Cat. #: 25030149) and 10% bovine calf serum (BCS, HyClone, Cat. #: SH30073.03), in the absence of antibiotics.

extract_protocol:
450 μL of each lysate was treated with 0.3 U/μL RNase I for 40 min at room temperature, while the remaining lysate was flash frozen in liquid nitrogen for later total RNA isolation. Following RNase I digestion (ThermoFisher Scientific, Cat. #: EN0601), 5 μL of Superase-In (ThermoFisher Scientific, Cat. #: AM2694) was added to each sample, and OD260 was measured in the Nanodrop 1000. ~300 µL of lysates was loaded on a 10%–45% sucrose gradient previously prepared in gradient buffer (100 mM Tris-HCl [pH 7.5], 75 mM KCl, 1.5 mM MgCl2) using a BioComp Gradient Master (Short Run, Sucr 10-45% SW41, wt15t). Gradients were spun at 37,000 rpm in a SW 41 Ti rotor for 2 hr at 4°C. Fractions were collected using a Brandel Density Gradient Fractionation system. Ribosome footprinted RNA was precipitated from fractions containing 80S monosomes using 2 volumes of 100% ethanol followed by extraction using TRIzol Reagent. Ribosomal RNA was depleted with the human/mouse/rat Ribo-Zero Gold rRNA removal kit (Illumina).
Sequencing libraries were constructed with the Illumina TruSeq Stranded Total RNA Library Prep Kit (RS-122-2201).

treatment_protocol: HeLa cells (2 x 106 cells) were seeded in 10 cm tissue culture dishes in 10 mL of complete DMEM medium and grown for 48 hrs at 37°C, 5% CO2. Harringtonine (HR, Abcam Cat. #: ab141941, dissolved at 2 mg/mL in DMSO) was added to the medium at a final concentration of 2 µg/ml. Cells were incubated for 10 min at 37°C, 5% CO2 in HR containing medium. Control cells were incubated with the same amount of DMSO for 10 min at 37°C, 5% CO2. After each incubation period, CHX was added to the medium at a final concentration of 100 µg/mL, culture medium was immediately aspirated, plates were placed on top of liquid nitrogen and transferred to dry ice. Lysis buffer (500 μL, 10 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2 mM DTT, 100 μg/mL CHX) was added to plates and cells were scraped into lysis buffer. Cell lysates were passed through a 26-G needle 10 times and clarified by spinning at 20,000 x g for 10 min at 4 °C. OD260 was measured in the Nanodrop-1000 and ~250 µL of lysates were loaded on a 10%–45% sucrose gradient, previously prepared in gradient buffer (100 mM Tris-HCl [pH 7.5], 75 mM KCl, 1.5 mM MgCl2) using a BioComp Gradient Master (Short Run, Sucr 10-45% SW41, wt15t). Gradients were spun at 37,000 rpm in a SW 41 Ti rotor for 2 hr at 4°C. Absorbance at 260 nm was recorded using a BioComp Fractionation System and a Triax Flow Cell model FC-1 for 260 nm scans.


title: BE2C-HDAC2-dTAG, ATAC-seq, DMSO (0.1%), 24 h, biol rep 3


title: HR_Cntrl_WT_rep2

extract_protocol:
Input RNA was obtained from 130 μL aliquots of lysate (without RNase I digestion) using TRIzol Reagent. Total RNA was treated with Turbo DNase I (ThermoFisher, 30 min at 37°C). RNA Spike-In mix (ThermoFisher Scientific, Cat. #: 4456740) was added to 5 μg of DNase I-treated total RNA according to the manufacturer’s instructions, and ribosomal RNA was depleted using the human/mouse/rat Ribo-Zero Gold rRNA removal kit. Next, total RNA samples were fragmented in alkaline fragmentation buffer (1 mM EDTA, 50 mM Na2CO3, 50 mM NaHCO3, [pH 9.2] for 40 min at 95°C, as described previously. Fragments between 26 and 34 nucleotides in size were gel purified from input and footprinted RNA samples, and used for library preparation as described previously.
Sequencing libraries were constructed with the Illumina TruSeq Stranded Total RNA Library Prep Kit (RS-122-2201).

treatment_protocol: HeLa cells (2 x 106 cells) were seeded in 10 cm tissue culture dishes in 10 mL of complete DMEM medium and grown for 48 hrs at 37°C, 5% CO2.  Control cells were incubated with the same amount of DMSO for 10 min at 37°C, 5% CO2. After each incubation period, CHX was added to the medium at a final concentration of 100 µg/mL, culture medium was immediately aspirated, plates were placed on top of liquid nitrogen and transferred to dry ice. Lysis buffer (500 μL, 10 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2 mM DTT, 100 μg/mL CHX) was added to plates and cells were scraped into lysis buffer. Cell lysates were passed through a 26-G needle 10 times and clarified by spinning at 20,000 x g for 10 min at 4 °C. OD260 was measured in the Nanodrop-1000 and ~250 µL of lysates were loaded on a 10%–45% sucrose gradient, previously prepared in gradient buffer (100 mM Tris-HCl [pH 7.5], 75 mM KCl, 1.5 mM MgCl2) using a BioComp Gradient Master (Short Run, Sucr 10-45% SW41, wt15t). Gradients were spun at 37,000 rpm in a SW 41 Ti rotor for 2 hr at 4°C. Absorbance at 260 nm was recorded using a BioComp Fractionation System and a Triax Flow Cell model FC-1 for 260 nm scans.

value: WT


title: HR_Cntrl_NAT10_rep1


title: BE2C-HDAC2-dTAG, ATAC-seq, DMSO (0.1%), 2 h, biol rep 3


title: Jurkat activated - FK506 treated

value: pretreated with FK506 for 1 hr before treatment with PMA and Ionomycin for 8 hr


title: Jurkat activated - CABIN1 peptide expressed

value: HA-mCherry-CABIN1 peptide expression

value: treated with PMA and Ionomycin for 8 hr


title: BE2C-HDAC2-dTAG, ATAC-seq, DMSO (0.1%), 2 h, biol rep 2


title: BE2C-HDAC2-dTAG, ATAC-seq, dTAG-13 (500 nM), 2 h, biol rep 1


title: Jurkat activated


title: MDA-MB-468 cells, Input

city: Padova

first: Mattia

last: Forcato

address: Via Ugo Bassi, 58/B

country: Italy

institute: University of Padova

data_processing:
Library strategy: ChIRP-seq
Paired-end reads were aligned using STAR version 2.7.3a to build version hg38 of the human genome.
Reads aligning on ribosomal RNA were discarded using RSeQC.
Strand specific coverage files (bigwig) were generated from the aligned reads using deepTools.
Genome_build: hg38
Supplementary_files_format_and_content: Strand specific (fwd and rev) CPM normalized coverage files in bigwig format

source_name: Triple-negative breast cancer cell line

growth_protocol: MDA-MB-468 cells were maintained in DMEM high glucose containing 10% FBS, L-Glutamine and penicillin/streptomycin.

extract_protocol:
After the washing steps, the whole recovered material was used for RNA purification with TRIzol and cleaned-up using RNA Clean & Concentrator TM-5 columns (Zymo Research, Cat. N°R1015). RNA was eluted in 20 µl of H2O and 4µl were used for reverse transcription using SuperScript™ IV VILO™ Master Mix (Invitrogen) to check enrichment for circ_0076611 vs. LacZ and Input samples. RNAseq was performed on DNase-treated RNA recovered from ChIRP experiments, and from Input samples as well, on the Illumina platform (NextSeq instrument). The RNA of the ChIRP experiments was controlled on a Bioanalyzer with the Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA) to verify the suitable size distribution and to simultaneously quantify the low RNA amounts.
The RNA libraries for the RNA sequencing were prepared using the SMARTer Stranded Total RNA Sample Prep Kit - Low Input Mammalian (Takara Bio. USA, Inc). Each ChIRP-RNA-Seq was conducted combining two independent biological replicates. For the input we used 60ng total RNA and performed ribosomal depletion with RiboGone (Takara Bio. USA, Inc) prior to cDNA and library preparation. For the RNA deriving from the ChIRP experiments we used 1200pg for cDNA and library preparation following the manufacturer’s instructions. The quality of the resulting libraries was controlled on a Bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA). The quantification of the libraries was performed by qPCR. Sequencing was carried out on a NextSeq 500 instrument (Illumina Inc., San Diego, CA, USA), sequencing in paired-end mode 76bp.
ChIRP followed by RNAseq

treatment_protocol: To perform ChIRP experiments 70% confluent MDA-MB-468 cells were fixed with 1% glutaraldehyde for 10min at RT and quenched with 125 mM Glycine solution for 5min at RT. ChIRP was performed as described [Chu C et al. Chromatin isolation by RNA purification (ChIRP). J Vis Exp. 2012 Mar 25;(61):3912. doi: 10.3791/3912]. 20 million cells were used for each condition. 1/100 of lysate volume was used for Input sample preparation. The biotinylated oligonucleotides directed to circ_0076611 and to control LacZ are: Bio_circ_0076611, CAGGGACTGCAAGTAC/3’BioTEG; Bio_LacZ_1, GTCATATGCATAAAGCGTTG/3’BioTEG; Bio_LacZ_2, TTAACGCCGCAGTGGTAGAA/3’BioTEG. Biotinylated oligonucleotides were recovered after hybridization step using Dynabeads® MyOne™ Streptavidin C1 (Invitrogen).

value: MDA-MB-468 (ATCC HTB-132)

value: Triple-negative breast cancer cells

tag: chirp target

value: Input


title: MDA-MB-468 cells, circVEGF


title: HR_RPF_WT_rep2


title: HR_RPF_WT_rep1


title: CTX-HFHSdiet-4

city: Neuherberg

first: Dominik

last: Lutter

address: Ingolstädter Landstraße 1

department: Institute for Diabetes and Obesity

institute: Helmholtz Zentrum München

description: Salmon quantification of transcript expression

data_processing:
The raw transcriptomics sequencing reads were first evaluated using FastQC (Andrews, 2010), and after passing quality control, the expression of the transcripts was aligned and quantified against the reference index created based on the GRCm38 genome using Salmon (Patro et al., 2017), with default parameters.
Assembly: GRCm38
Supplementary files format and content: tsv text file

source_name: Brain, cortex

growth_protocol: Mice were housed in groups under a 12-h light/12-h dark cycle at 22 +/- 2°C. All animals had free access to water and food.

extract_protocol:
Hypothalami, hippocampi and half cortexes were dissected and pooled together in distinct tubes prefilled with cold D-PBS. Tissue dissociation was performed following manufacturer’s instructions (Adult Brain Dissociation Kit, Miltenyi BioTec; #130-107-677). The resulting cell suspensions were incubated with magnetic microbeads coupled with an anti-ACSA2 antibody (Anti-ACSA-2 MicroBead Kit, Miltenyi Bi-oTec; #130-097-678), and labeled cells were sorted via MACS, according to commercially available manufacturer’s protocol (Anti-ACSA-2 MicroBead Kit, Miltenyi BioTec; #130-097-678). In particular, for hypothalamic and hippocampal samples (pre-labeled number of cells < 10^7), standard reagent volumes and MS columns were used, while for cortical samples (pre-labeled number of cells > 10^7), reagent volumes were scaled up accordingly and LS columns were used. The resulting ACSA2+ and ACSA2- fractions were collected in cold PBS and centrifuged at 3000 x g at 4°C for 10 min, and the pelleted cells stored at -80°C until use. The RNA extraction from ACSA2+ cells was performed (RNeasy Micro Kit; Qiagen; #74004) following the manufacturer’s protocol. Next, SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Takara; #634891) was used to generate and amplify cDNA, according to the instructions provided with the kit.
The cDNA samples were sheared by the Covaris system, and then the sheared fragments end-repaired, A-tailed, and ligated to sequencing adaptors. A size selection of about 200 bp is performed before the PCR enrichment. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to 2 ng/µl before checking the insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR (Q-PCR) (library activity >2 nM) (from Novogene Co., Ltd).

treatment_protocol: At 8-9 weeks of age, one group of mice received SC diet (SC diet- control), and the second group was fed with HFHS diet (HFHS diet) for 4 months.

tag: tissue

value: Brain, cortex

value: Astrocytes

value: 8-9 weeks old

value: male

tag: diet

value: High-fat high-sugar (HFHS) diet

value: 4 months


title: CTX-HFHSdiet-5

tag: cell type


title: CTX-HFHSdiet-1


title: HR_Cntrl_WT_rep1


title: HR_Cntrl_NAT10_rep2


title: CTX-HFHSdiet-6


title: CTX-SCdiet-1

value: Standard chow (SC) diet


title: Hippo-HFHSdiet-7

source_name: Brain, hippocampus

value: Brain, hippocampus


title: Hippo-HFHSdiet-4


title: Hippo-HFHSdiet-6


title: Hippo-HFHSdiet-2


title: Hippo-SCdiet-4


title: CTX-HFHSdiet-2


title: CTX-HFHSdiet-3


title: HR_RPF_NAT10_rep1


title: CTX-SCdiet-6


title: CTX-SCdiet-4


title: HPT-SCdiet-5

source_name: Brain, hypothalamus

value: Brain, hypothalamus


title: HPT-HFHSdiet-3


title: HPT-HFHSdiet-2


title: HPT-SCdiet-4


title: CTX-SCdiet-2


title: CTX-SCdiet-3


title: Hippo-HFHSdiet-1


title: 7799

city: Asheville

first: Melissa

middle: Marie

last: Heintz

state: NC

address: 31 College Place, Suite B118

institute: ToxStrategies

data_processing:
no adapter trimming for TempO-Seq fastqs, default settings for clusters passing filters
bcl2fastq v2.17.1.14 or v2.18.0.12 for demultiplexing
fastqs were aligned to a pseudo transcriptome using Bowtie allowing up to 2 mismatches
Assembly: probe-based targeted sequencing
Supplementary files format and content: comma-separated text files include raw count values for each gene_probe

source_name: liver

extract_protocol:
Mounted, unstained FFPE liver sections from HFPO-DA-exposed male and female mice (n = 5 per dose group per sex, total of 40 samples) were scraped from the slides and processed according to the TempO-Seq® protocol by BioSypder Technologies
TempO-Seq® protocol by BioSypder Technologies

treatment_protocol: Mice were administered 0, 0.1, 0.5 or 5 mg/kg HFPO-DA by oral gavage once daily for a total of 84 – 85 or 53 – 65 doses in male or female mice, respectively. Male mice (F0) were dosed 70 days prior to mating through completion of mating period (14 days). Female mice (F0) were dosed 14 days prior to mating through lactation day 20 or 21. Following the last treatment, mice were euthanized by CO2 anesthesia and exsanguination. Livers were fixed in 10% neutral-buffered formalin, embedded in paraffin (FFPE), and sections approximately 4 – 6 µm in thickness were mounted to glass slides.

value: liver

value: 0.5 mg/kg HFPO-DA


title: Hippo-HFHSdiet-5


title: Hippo-SCdiet-5


title: Hippo-HFHSdiet-3


title: 7772

value: WT

value: 0 mg/kg HFPO-DA


title: Hippo-SCdiet-2


title: 7768

value: 0.1 mg/kg HFPO-DA


title: Hippo-SCdiet-3


title: 7787


title: Hippo-SCdiet-1


title: CTX-SCdiet-5


title: 7767


title: HPT-HFHSdiet-4


title: HPT-SCdiet-1


title: HPT-HFHSdiet-5


title: HPT-HFHSdiet-1


title: HPT-SCdiet-6


title: HPT-SCdiet-3


title: 7723


title: HPT-SCdiet-2


title: 7779


title: 7720


title: 7798


title: 5070

value: female


title: 5066


title: 5071


title: 7743


title: 7751

value: 5 mg/kg HFPO-DA


title: 7739


title: 7746


title: 5056


title: 7825


title: 7738


title: 7731


title: 7717


title: 5023


title: 4997


title: 7712


title: 5020


title: 5077


title: 7711


title: 4962


title: 5047


title: Riboseq_invivo_Lgr5_WT_m1

city: Amsterdam

first: William

last: Faller

address: Plesmanlaan 121

department: Oncogenomics

country: Netherlands

institute: Netherlands Cancer Institute

data_processing:
Library strategy: Ribo-seq
For Riboseq samples, adapters were removed with cutadapt version 1.18.
Transcript quantifications were done with salmon version 1.1.0 using Gencode vM21 annotation.
For invitro Ribo-seq transcript quantifications, UTRs were discarded from transcripts.
Differential Translation Efficiency analysis was performed with RiboDiff version 0.2.1.
Genome_build: mm10
Supplementary_files_format_and_content: "quant.sf" files are tab-seperated salmon outputs (includes transcript id, transcript length, effective length, TPM and raw_reads as columns).
Supplementary_files_format_and_content: *TE.RiboDiff.tsv: Differential Translation Efficiency measurements

source_name: small intestine

extract_protocol:
Briefly, for in vitro analyses, organoids were generated from VillinCre.ERT2.RPL22.HA and VillinCre.ERT2Rptor.fl/fl.RPL22.HA animals. Around 120-150 plugs of 30 μl BME were used for each replicate. Cells were treated with 100 μg/ml cycloheximide for 3-5 min at 37°C and immediately incubated on ice for the remainder of the experiment. After collecting the cells, pellets were washed twice with cold PBS supplemented with 100µg/mL cycloheximide, resuspended in ice-cold lysis buffer (20mM Tris HCl pH 7.4, 10mM MgCl2, 150mM KCl, 1% NP-40, 100µg/mL cycloheximide and 1x EDTA-free proteinase inhibitor cocktail (Roche)) and incubated for 20 min on ice. Lysates were then centrifuged at max speed for 20 min at 4°C and the supernatants were collected. For in vivo analyses, Lgr5CreERT2RPL22.HA and Lgr5CreERT2Rptor fl/flRPL22.HA animals were euthanized by CO2 and small intestines were immediately dissected, flushed with cold PBS supplemented with 100 µg/mL of cycloheximide and snap frozen using liquid nitrogen. Frozen samples were then homogenized by pestle and mortar while submerged in liquid nitrogen and the resulting powder was resuspended in ice-cold lysis buffer and incubated on ice for 30 min. Finally, lysates were centrifuged at max speed for 20 min at 4°C and the supernatants were collected. For Riboseq, HA pulldown was performed followingly. All lysates were incubated with Pierce™ Control Agarose Matrix (ThermoFisher) for 20min at 4’C in order to remove any non-specific binding, following by an incubation with pre-washed 425 AntiHA.11 Epitope Tag Affinity Matrix (BioLegend) for 4 hours (in vitro samples) or overnight (in vivo samples) at 4’C. Tagged ribosomes were then eluted from the beads by incubating with 200µg/mL HA peptide (ThermoFisher Scientifc) for 15 min at 30’C with constant agitation. Non-protected RNA was digested with 10 ul of RNase I (ThermoFisher Scientific) for 40min at 25’C and the reaction was stopped by adding 13ul of SUPERASE (ThermoFisher Scientific). Ribosome protected fragments (RPFs) were finally purified using miRNeasy minikit (Qiagen) accoridng to manufacturer’s instructions.
Riboseq library preparation was performed as previously described (Loayza-Puch et al., 2013) with some modifications. To discard undigested RNA fragments, RPFs were run in a 10% TBE-Urea polyacrylamide gel and size selected between 19 nt and 32 nt using marker RNAs. 3’ ends were dephosphorylated using T4 polynucleotide kinase (PNK) (NEB) and 1.5xMES buffer (150mM MES-NaOH, 15mM MgCl2, 15mM β-mercaptoethanol and 450mM NaCl, pH 5.5) and incubated at 37’C for 4h. After purifying RNAs with Trizol, 3’adpaters were ligated with T4 RNA ligase I (NEB) and incubated overnight at 24’C. After size-selecting the ligated products, 5’ ends were phosporylated with T4 PNK and incubated at 37’C during 30min. 5’ adapters were then ligated with T4 RNA ligase I and incubated 37’C for 2,5 hours. The RPFs containing both adapters were then size-selected one more time and rRNA depletion was performed by first denaturing the samples at 100’C for 1min followed by incubation with 10uM biotinylated oligos (5’-UGAUCUGAUAAAUGCACGCAUCCCCCC-3’; 5’-CGUGCGAUCGGCCCGAGGUUAUCUAGAGUCACCA-3’; 5’-AUUCCAUUAUUCCUAGCUGCGGUAUCCAGGCGGCUC-3’; 5’-GGGCCUCGAUCAGAAGGACUUGGGCCCCCCACGA-3’; 5’-UGGCUUCCUCGGCCCCGGGAUUCGGCGAAAGC-3’; 5’-ACGGACGCUUGGCGCCAGAAGCGAGAGCCCCUCGGG-3’; 5’-ACCCGGCUAUCCGGGGCCAACCGAGGCUCCUUCGGCG-3’; 5’-ACCGACGCUCAGACAGGCGUAGCCCCGGGAGGA-3’; 5’-GGCGGACGGGGGGAGAGGGAGAGCGC-3’; 5’-GGCGAGACGGGCCGGUGGUGCGCCCUCGGC-3’; 5’-CCAGAAGCAGGUCGUCUACGAAUGGUUUAG-3’; 5’-AUCCCCGAUCCCCAUCACGAAUGGGGUUCA-3’; for the in vivo samples also added the following: 5’-GCUCUGCUACGUACGAAACCCCGACC-3’; 5’-GUGUCGAGGGCUGACUUUCAAUAGAUCG-3’; 5’-AGAUCCAACUACGAGCUUUUUAACUG-3’; 5’-AACGCCACUUGUCCCUCUAAGAAGU-3’; 5’-CAAGUGCGUUCGAAGUGUCGAUGAUC-3’) in 2xSSC buffer (ThermoFisher Scientific) for 15min at 37’C. Pre-washed MyOne Streptavidin C1 DynaBeads (ThermoFisher Scientific) were then incubated with samples in wash/bind buffer (2M NaCl, 1mM EDTA, 5mM Tris and 0.2% Triton X-100) for 30min at 37’C with agitation. Supernatants were collected and RPFs were purified and re-suspended in 8µL of RNase-free water. cDNA was synthesized with SuperScript III (ThermoFisher Scientific) according to manufacturer’s instructions and using the RTP primer (seq). After purification with G50 columns (Merck), cDNA was amplified using Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific) for 18 cycles, with primers containing different indexes to allow for sequencing (see table). PCR products were purified using the QIAquick PCR purification kit (Qiagen) followed by a final size selection with a E-Gel SizeSelect II 2%, (ThermoFisher Scientific). All samples were accessed with the Agilent 2100 Bioanalyzer to assure high quality and quantify the molarity and the libraries were sequenced on the Illumina HiSeq2500.

treatment_protocol: To generate stem cell enriched (SCe) cultures, organoids were grown in ENR media supplemented with 10 μM of CHIR 99021 (Cayman Chemical) and 1.5 mM of valproic acid. To promote differentiation, stem cell depleted (SCd) cultures were achieved by growing the organoids in normal ENR media for the first 2 days, and removing R-spondin from the media for the last 2 days.

value: small intestine

value: untreated


title: Riboseq_invivo_Lgr5_Rptorflfl_m2


title: Riboseq_invivo_Lgr5_Rptorflfl_m1


title: Riboseq_invivo_Lgr5_Rptorflfl_m3


title: Riboseq_invitro_Rptorflfl_org1

source_name: intestinal organoid

value: intestinal organoid


title: Riboseq_invitro_Rptorflfl_org3


title: 5034


title: RNAseq_invitro_Rptorflfl_org2_rep1

data_processing:
For Riboseq samples, adapters were removed with cutadapt version 1.18.
Transcript quantifications were done with salmon version 1.1.0 using Gencode vM21 annotation.
For invitro Ribo-seq transcript quantifications, UTRs were discarded from transcripts.
Differential Translation Efficiency analysis was performed with RiboDiff version 0.2.1.
Genome_build: mm10
Supplementary_files_format_and_content: "quant.sf" files are tab-seperated salmon outputs (includes transcript id, transcript length, effective length, TPM and raw_reads as columns).
Supplementary_files_format_and_content: *TE.RiboDiff.tsv: Differential Translation Efficiency measurements


title: Riboseq_invivo_Lgr5_WT_m2


title: 4982


title: RNAseq_invitro_SCe_org3

value: stem cell enrichment


title: RNAseq_invitro_WT_org2_rep1


title: 4979


title: Riboseq_invivo_Lgr5_WT_m3


title: 4990


title: 4984


title: 4967


title: set36.WT.4.Input.rep2

description: sample 95 to 98 are in one group

value: Wild type

value: RNA fragments


title: Riboseq_invitro_SCe_org3


title: Riboseq_invitro_Rptorflfl_org2


title: Riboseq_invitro_SCe_org2


title: RNAseq_invitro_WT_org2_rep2


title: 5030


title: set33.TOP3B-KO1.Input.rep1

description: sample 91 to 94 are in one group


title: Riboseq_invitro_SCe_org1


title: set36.WT.TOP3B_eCLIP-seq.rep2

description: sample 95 to 98 are in one group


title: set33.TOP3B-KO1.TOP3B_eCLIP-seq.rep1

description: sample 91 to 94 are in one group


title: RNAseq_invitro_Rptorflfl_org1_rep1


title: RNAseq_invitro_SCe_org2


title: set24.WT.1.Ribo-seq.rep2

description: sample 2, 5, 8,10,13,16 are in one group

value: Cells were treated with 100µg/mL cycloheximide for 10min and then were used for Ribo-seq.

value: Ribosome protected RNA fragments


title: set20.WT.1.Ribo-seq.rep3

description: sample 3,6,11,14 are in one group


title: RNAseq_invitro_WT_org1_rep2


title: set20.TOP3B-KO1.Ribo-seq.rep3

description: sample 3,6,11,14 are in one group


title: RNAseq_invitro_SCe_org1


title: set24.WT.1.Ribo-seq.rep1

description: sample 1,4,7,9,12,15 are in one group


title: set24.TOP3B-KO2.Ribo-seq.rep2


title: RNAseq_invitro_Rptorflfl_org2_rep2


title: RNAseq_invitro_WT_org1_rep1


title: set36.TOP3B-KO1.Input.rep2

description: sample 95 to 98 are in one group


title: set36.TOP3B-KO1.TOP3B_eCLIP-seq.rep2

description: sample 95 to 98 are in one group