title: BRCO-1070

city: Guangzhou

first: Haoyu

address: Kai Yuan Avenue 190

institute: GIBH

data_processing:
Left read of paired-end reads contains the barcode information with 1-8bp to be the unique molecular identifier (UMI), 9-16bp to be the cell barcodes followed by polyT stretch. Barcode information from left reads were first extracted and added to the corresponding right reads using extract function from UMI-tools 1.0.0.
Right reads of read pairs were aligned to an artificial human genome (hg38) containing sequences of 92 ERCC spike-ins using STAR-2.7.0f with default settings. Aligned reads were assigned to genes using featureCount 1.6.4 with the following parameter, -g gene_id.
For each cell, the number of UMIs per gene was counted and the gene-cell matrixes were generated using count function from UMI-tools 1.0.0 with the following settings, --per-gene, --per-cell.
For the downstream analysis, gene-cell matrixes were merged per donor and imported into R. Seurat 3.0 was used for data filtering, normalization, scaling, clustering, marker identification and visualization following the standard process.
Briefly, Seurat objects were generated from the combined gene-cell matrixes per donor and cells that are not with the following criteria (1000< UMIs < 25000, mt%<50%) were filtered out. Then, UMIs were log normalized, and number of total UMIs and mitochondrial gene expression were regressed out during the data scaling.
Seurat objects of different donors were integrated using the functions FindIntegrationAnchors and IntegrateData from Seurat to minimize the biological effects. Pathway analysis was performed using Enrichr web tool with the significantly differentially expressed genes as input.
In addition, the previous published datasets by both Sampaziotis et al. (ArrayExpress: E-MTAB-8495) and Aizarani et al.13 (GEO accession number: GSE124395) were integrated within our own dataset using Seurat integration81 and analyzed as previously described.
Genome_build: hg38
Supplementary_files_format_and_content: Gene-cell count table
Supplementary_files_format_and_content: cell index information

source_name: human intrahepatic cholangiocyte organoids

growth_protocol: Huch et al. Cell 2015. For BRCO, branching organoids were created by switching culture conditions to non-canonical WNT stimulating conditions, consisting of William's E medium (WE, ThermoFisher Scientific) supplemented with 10 mM nicotinamide (Sigma), 17 mM sodium bicarbonate (Sigma), 0.2 mM 2-phospho-L-ascorbic acid trisodium salt (Sigma), 6.3 mM sodium pyruvate (Gibco), 14 mM glucose (Sigma), 20 mM HEPES (Fisher Scientific), ITS + premix (Life Technologies), 0.1 μM dexamethasone (Sigma), 2 mM Ultraglutamine (Fisher Scientific), 100 µg/mL penicillin/streptomycin (Fisher Scientific), 20 ng/mL EGF (Peprotech), 10% RSPO1 conditioned media (homemade) and 100 ng/mL DKK-1 (Abcam Ltd), after 3 passages.

extract_protocol:
Organoids (ICO and BRCO lines 6, 7 and 10) were incubated with Calcein for 45 min at 37⁰C before dissociation for live staining of the cells. To get single cells, organoids were then dissociated and digested by 0.05% Trypsin-EDTA following the online protocol. Single-cells were obtained using the flow cytometer FACS Aria (BD Biosciences) equipped with a 130 µm nozzle. Only GFP positive cells (living cells) were sorted into 384-well plates containing a unique CEL-seq primer and 5 µL mineral oil (Sigma, M5310) per well. Plates were stored at -80 ⁰C until they were processed.
Single-cell RNA libraries were prepared according to the mCEL-seq2 protocol. Briefly, plates were thawed on ice, and then 100 nL lysis buffer (0.2% Triton X-100, 2mM dNTPs, 1:50.000 ERCC ExFold RNA spike-in (Thermo, 4456739)) was dispensed per well using the Nanodrop Ns-2 Stage (BioNex). SuperScript™ II Reverse Transcriptase (Invitrogen, 18064-14) was used in reverse transcription reaction mix following the protocol, 4 ⁰C for 5 min; 25 ⁰C for 10 min; 42 ⁰C for 60 min; 70 ⁰C for 10 min. 960 nL second-strand reaction mix containing E. coli DNA ligase (NEB, M0205L), E. coli DNA polymerase I (NEB, M0209L) and Random hexamer (sigma, 11034731001) were used per well for second-strand synthesis with the following protocol, 16°C for 2 hours. Next, cDNA from one 384-well plate was pooled, and in vitro transcription was performed using the MEGAscript™ T7 Transcription Kit (Invitrogen, AMB 1334-5) by incubating the reaction at 37°C for 14 hours. Afterwards, the amplified RNA was fragmented and cDNA was converted using Superscript II  and the random octamer primer. Libraries were amplified for 9 cycles using Phusion® High-Fidelity PCR Master Mix (NEB, M0531S) with the Nextflex primers, and quantified on the bio-analyzer using DNA high-sensitivity kit. Barcoded libraries were sequenced on an Illumina NextSeq 500 with 38bp paired-end reads at a depth of 24.2-32 million reads per plate.
Cel-seq2

treatment_protocol: no treatment

tag: medium

value: cultured in branching medium


title: WD L-TBA_1

status: Public on May 04 2022

type: SRA

city: London

first: LMS

last: Bioinformatics Core

address: Hammersmith Hospital Campus, Du Cane Road

department: Bioinformatics Core

country: United Kingdom

institute: MRC London Institute of Medical Sciences

library_source: transcriptomic

data_processing:
Primary analysis RTA 1.17.21.3 and Secondary analysis CASAVA- 1.8.4
Sequencing was conducted on an Illumina HiSeq 2500 system with 50 base pair single-end reads
Sequencing reads were mapped to mm9 genome using Tophat 2.0.11
Gene based counting was performed using the Rsubread package.
Gene expression analysis was performed using the DESeq2 Bioconductor package (DESeq2_1.18.1 )
Genome_build: mm9
Supplementary_files_format_and_content: comma-delimited text file include raw counts for each Sample generated by  Rsubread.

molecule: total RNA

organism: Mus musculus

source_name: Liver

growth_protocol: H-TBA and L-TBA mice were fed a Western diet for 30 weeks.

extract_protocol:
Livers were flash frozen. RNA was then extracted by Trizol Reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: tissue

value: Liver

tag: strain

tag: treatment

value: Western diet for 30 weeks

tag: group

value: L-TBA


title: BRCO-1069

last: Wu

country: China

organism: Homo sapiens

tag: cell type

value: human intrahepatic cholangiocyte organoids


title: 2-cell mouse embryo derived mouse totipotent potential stem cells [scRNA-seq]

city: Beijing

first: Yiming

last: Sun

state: Beijing

address: NO.5, Yiheyuan Road, Haidian District

department: School of Life Sciences

institute: Peking University

library_source: transcriptomic single cell

data_processing:
The clean single-cell RNA-seq reads were mapped to mouse reference genome mm10 using Cell Ranger v3.1.0.
Assembly: mm10 (GRCm38)
Supplementary files format and content: Tar archives contain Cell Ranger output (barcodes.tsv, features.tsv, matrix.mtx).

molecule: polyA RNA

growth_protocol: Mouse TPS cells were cultured on feeder cells in CPEC condition. Mouse EPS cells were cultured on feeder cells in LCDM medium. Mouse ES cells were cultured on feeder cells in 2i+LIF condition.

extract_protocol:
Cells were washed and resuspended in 1X PBS (calcium and magnesium free) containing 10% FBS. Cell viability was determined by Count Star, and the density of living cells were adjusted  to 300-600 living cells per microliter. Cell suspension was loaded onto the Chromium single cell controller (10x Genomics). Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10x Genomics, 1000075) and Chromium Single Cell B Chip Kit (10x Genomics, 1000074) were used to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. In short, single cells were suspended in PBS containing 0.04% BSA. About 6,000 cells were added to each channel, and the target cells were recovered (about 3,000 cells). Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 min, followed by 85°Cfor 5 min, and hold at 4°C. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200(performed by CapitalBio Technology, Beijing).
Single-cell RNA-seq libraries were constructed using Single Cell 3’ Library and Gel Bead Kit V3.1 according to the manufacturer’s introduction. The libraries were finally sequenced using an IlluminaNovaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing).

value: totipotent potential stem cells


title: Joint_HAO_K_3

city: Glasgow

first: John

middle: Joseph

last: Cole

address: University Avenue

institute: University of Glasgow

data_processing:
A high read quality (mean score above 28) in all samples was confirmed using FastQC.
To remove adapters, ensure proper read pairing and trim reads with low start or end read quality the package Trim-Galore! was used under default settings.
Next reads were aligned to the mm10 genome using Tophat2, with the flags: --b2-sensitive, --no-coverage-search, and version 87 of the mouse transcriptome.
FPKMs were generated using cufflinks under default settings. All samples had a concordant pair alignment rate above 75% and at least 20 million aligned concordant read pairs.
Differential expression was called using the bioconductor package DESeq2 with adjusted p < 0.05 and absolute log2fold >1 considered significant.
The data was visualised using Searchlight2 and R .
Genome_build: mm10, v87
Supplementary_files_format_and_content: Tab delimited read count file.

source_name: Inflammed joint non-migrated CD45+ leukocytes

extract_protocol:
Ankle joints or ear pinnae were teased apart using forceps; excised LNs were passed through Nitex mesh (Cadisch Precision Meshes) and both tissues digested for 25 minutes at 37 °C with 2.64 mg/mL collagenase D (Roche). Joint tissue was then homogenized using the Miltenyi Tissue Dissociator and passed through a 70 μm cell strainer to obtain a single cell suspension. Joint and LN single cell suspensions were then washed in FACS buffer (PBS/ 2 % FCS/ 2mM EDTA) and stained for flow cytometry analysis. Cells stained for viability in PBS using Fixable Viability Dye (eFluor506; eBioscience) for 30 minutes at 4 °C. Cells were then washed in FACS buffer and subsequently incubated with FcR block for 10-15 minutes at 4 °C and then appropriate fluorochrome-conjugated antibodies (Supplemental Table 7) added and the cells stained for 30 minutes at 4 °C. Viable, single CD45+ Kaede Red+ cells were sorted using an Aria III (BD Biosciences).
RNA libraries were prepared for sequencing using standard Illumina protocols
Pair ended with a 100bp read length (Source Bioscience, Nottingham, UK).

treatment_protocol: CD4+ T cells from OT-II CD45.1 mice were polarized in vitro over 72 hours towards a Th1 phenotype in the presence of 1 μg/mL peptide OVA323-339 (pOVA) ((Sigma-Aldrich), 10 ng/mL rIL-12 and 2 μg/mL anti-IL-4 (Both R&D). Recipient mice received 3 x 106 in vitro polarized Vα2+Vβ5+ OT-II Th1 cells i.v. The following day, mice were immunization with 100 μg OVA (Worthingtons) in Complete Freuds Adjuvant (CFA) (Sigma Aldrich)in the scruff of the neck. Ten days later, mice were challenged in the footpad with PBS (Gibco) or 100 μg heat-aggregated ovalbumin (HAO), as previously described (10). Ear pinna inflammation was induced following the same protocol, except that the mice were challenged with either PBS or 100 μg HAO in the ear pinna.

value: Kaede

value: joint

value: inflammed


title: USA300_CLASH_WT_LPM76_r3.2

city: Edinburgh

first: Sander

last: Granneman

address: Mayfield Road, Kings Buildings, Waddington building, room 3.06

department: Centre for Synthetic and Systems Biology

institute: University of Edinburgh

description:
RNA-RNA complexes immunoprecipitated untagged control, LPM pH 7.6 medium, third replicate, technical replicate 2
Intermolecular_hybrids_LPM_shift_LPM7_data.txt

data_processing:
CLASH was performed on JKD6009 rnc::HTF in biological triplicates, with a single biological replicate for the wild type control. The cDNA libraries were sequenced on Illumina HiSeq 2500 machine. For USA300, four biological replicates of rnc::HTF were performed and two replicates of the untagged wild type. These were sequenced on a NovaSeq 6000. Raw sequencing reads in fastq files were processed using the CLASH data analysis pipeline developed by Sander Granneman, which uses tools from the pyCRAC package (version 1.5.0) (Webb et al., 2014). This PreProcessForHyb.py script first uses flexbar to trim the 3' adaptor sequence from the data and remove low quality nucleotides (Phred score <23). Subsequently, pyBarcodeFilter.py demultiplexes the data from the in-read barcode sequences found in the 5’ adapter. Using the random nucleotide information present in the 5’ adaptor sequences, the reads are then collapsed to remove potential PCR duplicates using pyFastqDuplicateRemover.py. For RNAseq, the CRAC_pipeline_PE.py script was used from the pyCRAC package. This firstly demultiplexes the data using pyBarcodeFilter.py and the in-read barcode sequences found in the L5 5’ adapters. Flexbar then trims the reads to remove 3’-adapter sequences and poor-quality nucleotides (Phred score <23). Using the random nucleotide information present in the L5 5’ adaptor sequences, the reads are then collapsed to remove potential PCR duplicates using pyFastqDuplicateRemover.py. The reads were then mapped to the JKD6008 and USA300 genomes using Novoalign (www.novocraft.com).
For mapping to the USA300 genome, we generated an annotation file in the Gene Transfer Format (GTF). This file contained the start and end positions of each gene on the chromosome as well as its encoded product (e.g. sRNA, protein- coding, tRNA). To generate this file, we used the Rockhopper software (Tjaden, 2015) on USA300 rRNA-depleted total RNA-seq data (Stuart McKellar and Ivayla Ivanova, unpublished) and a minimal GTF file obtained from ENSEMBL (without UTR information). The resulting GTF file contained information not only on the coding sequences, but also more complete 5’ and 3’ UTR coordinates. Overlapping features, such as sRNAs that overlap with UTRs of protein-coding genes, were removed from the GTF file. For USA300, we utilised already published genome annotations (Howden et al, 2010).
The hyb pipeline (Travis et al, 2014) was then used to detect RNA-RNA interactions. This uses bowtie2 and BLAST to map each half of the RNA-RNA interaction to the genome. Afterwards, a statistical analysis pipeline (Waters et al, 2017) was used to filter for statistical signifiance and intermolecular interactions were extracted, annotated and filtered for minimum folding energy cut-off using custom scripts. Finally, interactions involving only 'bona fide' sRNAs (Lui et al, 2018) were filtered from the data. To calculate enriched RNA structures, the MFE of structures were calculated using UNAFold and compared to a randomly shuffled control. The structures were converted into Vienna dot-bracket notation and double-stranded structures between 5 and 10 base-pairs were extracted.
Normalization and differential gene expression analysis on the RNAtag-Seq data were done with  DESeq2  package (Love et al., 2014).
Genome_build: NCBI:txid451515 (USA300), NCBI:txid546343 (JKD6009)
Supplementary_files_format_and_content: CLASH data: Supplementary Tables 3 and 4 are Excel tables describing the RNA-RNA complexes. The .hyb files give the unique, annotated output from hyb. For RNAseq, bedgraph files are provided which detail peaks that can be visualised by a genome browser, and the pyReadCounters.gtf output detailing the hit tables.

organism: Staphylococcus aureus

source_name: Staphylococcus aureus

growth_protocol: S. aureus cells expressing HTF-tagged RNase III (or an untagged, wild type control) were grown to saturation in TSB at 37 °C with shaking. Cells were then diluted into 500 mL of fresh TSB to an OD600 value of 0.05 and grown to an OD600 of 3. Subsequently, cells were harvested by vacuum filtration and shifted to an equivalent volume of stress media, either RPMI or LPM. As as control, cells were shifted back into their original TSB medium. For CLASH, samples were taken after 15 minutes of incubation in the stressful media. For RNAtag-Seq, samples were taken after 5, 10, 15 and 30 minutes.

extract_protocol:
For CLASH, the cells were washed off the filters using 25mL of cold phosphate buffer saline and pelleted by centrifugation. Cells were subsequently resuspended in 2 volumes of TN150-lysostaphin buffer (50 mM Tris pH 7.8, 150 mM NaCl, 100 µg/mL lysostaphin, 0.1% NP-40, 0.5% Triton X-100) and transferred to 5 mL screw-cap tubes (Eppendorf). Sixty µL of DNase RQ1 and 10 µL of SUPERase-In was added and the mixture was incubated at 37°C for 30 min to lyse the cells. Afterwards, 3 volumes/cell weight of zirconia beads (Thistle Scientific; 0.1 µm) were added and the mixture was vortexed vigorously 5 times for one minute with incubation on ice between each samples. Two volumes/cell weight of cold TN150 anti-peptidase (50 mM Tris pH 7.8, 150 mM NaCl, 1 Roche EDTA-free mini pellet, 0.1% NP-40, 0.5% Triton X-100 and 10 mM EDTA) was added before centrifugation for 30 minutes at 10000 g at 4°C. Subsequently, 75 µL of anti-FLAG® M2 Magnetic Beads (pre-washed with TN150 buffer) was added and incubated with the lysate for 2 hours at 4°C. The beads were then washed three times for five minutes with 2 mL of TN1000 buffer (50 mM Tris pH 7.8, 1M NaCl, 0.1% NP-40, 0.5% Triton X-100) and three times with 2 mL of TN150 buffer (50 mM Tris pH 7.8, 150 mM NaCl, 0.1% NP-40, 0.5% Triton X-100) for 5 minutes. Beads were subsequently resuspended in 250 µL of TN150 and 10 µL of homemade GST-TEV protease was added. The samples were rotated for 2 hours at room temperature. TN150 was added to a final volume of 600 µL and 550 µL of the TEV eluates were incubated with RNace-it™ (Agilent Technologies, 1 µL of 1:100 dilution) for exactly seven minutes at 20°C. Subsequently, 500 µL of the mixture was added to a tube with 0.4 g guanidium-HCl and vortexed vigorously to inactivate the RNases. Sodium chloride and imidazole (pH 8.0) were added to a final concentration of 300 mM and 10 mM respectively, and the mixture was transferred to 50 µL of Ni-NTA agarose beads (QIAGEN), pre-equilibrated with wash buffer I (300 mM NaCl, 10 mM imidazole, 6M GuHCl, 50 mM Tris-HCl pH 7.8, 0.1% NP40, 5 mM β- mercaptoethanol and 0.5% Triton X-100) and incubated at 4°C overnight on a rotator. The next day the beads were transferred to Pierce snap-cap columns (Thermo Scientific) and washed twice with 500 µL wash buffer I and three times with 500 µL 1x NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% NP40, 5 mM βMe and 0.5% Triton X-100). For RNAtag-Seq, total RNA was extracted from the cells using standard guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski et al, 1987).
For CLASH, the isolated RNA-RNA complexes had 3' and 5' Illumina adaptors ligated on their respective ends as described previously (McKellar et al, 2020; Granneman et al, 2009). For RNAtag-Seq, the total RNA samples were processed as described previously (Shishkin et al, 2015).

treatment_protocol: Following the shift, cells were UV cross-linked through 254 nm UV (1000 mJ/cm2) using a Vari-X-Linker (UVO3 (McKellar et al, 2020; van Nues et al, 2017). Afterwards, cells were harvested onto filters by vaccuum filtration, flash-frozen in liquid nitrogen and stored at -80°C until their processing.

tag: media

value: LPM 7.6

value: Sample harvested after shifting cells to LPM pH 7.6 for 15 minutes


title: USA300_CLASH_RNase_III_LPM76_r3.2

description:
RNA-RNA complexes immunoprecipitated via RNase III, LPM pH 7.6 medium, third replicate, technical replicate 2
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_LPM7_data.txt

value: USA300 rnc:HTF


title: in vitro induced definitive endoderm 54h

type: genomic

city: Yokohama

first: Takahiro

last: Suzuki

state: Kanagawa

address: 1-7-22 Suehiro-cho, Tsurumi-ku

department: Center for Integrative Medical Sciences

country: Japan

institute: RIKEN

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

scan_protocol: Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting

data_processing: GenomeStudio software v2010.3

label: Cy5 and Cy3

molecule: genomic DNA

source_name: iPS cells (201B7)

label_protocol: Standard Illumina Protocol

growth_protocol: iPS cells (201B7) were mainteined in in Cellartis® DEF-CS™ Culture System (Takara Bio Inc., Shiga, Japan)

extract_protocol: Genomic DNA was extracted and purified using NucleoSpin Tissue (Macherey-Nagel) according to standard instructions

treatment_protocol: in vitro definitive endoderm differentiation was performed using  Cellartis® Definitive Endoderm Differentiation Kit (Takara Bio Inc.)

tag: differentiation time


title: mouse embryonic stem cells 1# [scRNA-seq]

source_name: mouse embryonic stem cells 1#

value: embryonic stem cells


title: mouse extended pluripotent stem cells 2# [scRNA-seq]

value: extended pluripotent stem cells


title: Ear KR_2

source_name: Inflammed ear non-migrated CD45+ leukocytes

value: ear


title: 17_KO_papain

city: San Diego

first: Ashu

last: Sethi

state: CA

address: 9420 Athena Circle

department: Health Sciences

country: USA

institute: La Jolla Institute for Immunology

data_processing:
Reads passing Illumina filters were further filtered for those mapping to tRNA, rRNA, adapters, and spike-in controls using Bowtie v1.0.0 (-S --fr)
Filtered reads were against the GRCm38.p4 reference genome and Gencode annotations using TopHat v 1.4.1
Reads mapping to the reference genome are further filtered to remove low-complexity reads with PRINSEQ v0.20.3 (-lc_method "dust" -lc_threshold 4)
Sequencing read coverage per gene was counted using htseq-count v 0.7.1 and features with 0 counts across all samples were removed
Genome_build: GRCm38.p4.gencode.rM9
Supplementary_files_format_and_content: read count matrix with rows as genes and columns as cells

source_name: Bronchiolar epithelial cells

extract_protocol:
Mouse bronchiolar epithelial cells were isolated and sorted using a FACS Aria and pooled from 3 mice per sample. RNA extraction was performed using RNeasy mini kit (Qiagen).
RNAseq libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v2 (TaKaRa)

treatment_protocol: 25 μg of papain or PBS was given intranasally, in a total volume of 40 μL on day 1, 2 and 3

value: Lung

tag: genotype

value: KO

value: papain-treated


title: BRCO-1071

value: cultured in branching medium


title: USA300_CLASH_WT_LPM54_r3.2

description:
RNA-RNA complexes immunoprecipitated untagged control, LPM pH 5.4 medium, third replicate, technical replicate 2
Intermolecular_hybrids_LPM_shift_LPM5_data.txt

value: LPM 5.4

value: Sample harvested after shifting cells to LPM pH 5.4 for 15 minutes


title: 16_KO_papain


title: USA300_CLASH_WT_LPM54_r3.1

description:
RNA-RNA complexes immunoprecipitated untagged control, LPM pH 5.4 medium, third replicate, technical replicate 1
Intermolecular_hybrids_LPM_shift_LPM5_data.txt


title: 18_WT_Resting

value: WT

value: untreated


title: USA300_CLASH_WT_LPM76_r3.1

description:
RNA-RNA complexes immunoprecipitated untagged control, LPM pH 7.6 medium, third replicate, technical replicate 1
Intermolecular_hybrids_LPM_shift_LPM7_data.txt


title: 15_WT_Papain


title: BRCO-1063

value: human intrahepatic cholangiocyte organoids


title: Ear KR_3

value: Kaede


title: USA300_CLASH_RNase_III_LPM76_r2.3

description:
RNA-RNA complexes immunoprecipitated via RNase III, LPM pH 7.6 medium, second replicate, technical replicate 3
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_LPM7_data.txt


title: USA300_CLASH_RNase_III_LPM54_r2.1

description:
RNA-RNA complexes immunoprecipitated via RNase III, LPM pH 5.4 medium, second replicate, technical replicate 1
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_LPM5_data.txt


title: BRCO-1062

value: cultured in expansion medium


title: 14_WT_Papain


title: L/Z_cKO_PD 52

city: Shenzhen

first: Chenxi

last: Guo

state: Guangdong

address: No.1 Haiyuan 1st Rd, Futian

department: Reproductive Medicine Center

institute: The University of Hong Kong-Shenzhen Hospital

data_processing:
Basecalls performed using CASAVA version 1.4
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads FRKM were calculated using a protocol
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: Spermatocytes

extract_protocol:
Spermatocytes were extracted from testis sample, flash frozen on dry ice, and RNA was harvested using Trizol reagent. NEBNext® Ultra™ RNA Library Prep Kit for Illumina® was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: developmental stage

value: PD 52

value: Mfn2 loxp/loxp; Stra8-Cre


title: 19_KO_resting


title: L/Z_WT_PD 52

value: Mfn2 loxp/loxp


title: Pachytene_cKO_PD 52


title: BRCO-983


title: USA300_CLASH_RNase_III_LPM54_r2.3

description:
RNA-RNA complexes immunoprecipitated via RNase III, LPM pH 5.4 medium, second replicate, technical replicate 3
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_LPM5_data.txt

value: USA300 rnc:HTF


title: BRCO-1061


title: 13_KO_resting


title: 11_WT_Resting


title: Pachytene_WT_PD 52


title: Pachytene_cKO_PD 24

value: PD 24


title: 10_KO_papain


title: BRCO-1060


title: USA300_CLASH_WT_LPM54_r2.2

description:
RNA-RNA complexes immunoprecipitated untagged control, LPM pH 5.4 medium, second replicate, technical replicate 2
Intermolecular_hybrids_LPM_shift_LPM5_data.txt


title: cAMP, RNA-seq

city: Ube

first: Haruka

last: Takagi

institute: yamaguchi university

description: cAMP treatment

data_processing:
To produce the raq bcl, or base call files, quality assessment and image analyses were performed using Next-seq packaging software Real Time Analysis
bcl2fastaq Conversion Software v2.19 was used for de-multiplexing of the samples
Reads with more than two ambiguous nucleotides and reads with quality scores less than 20 as calculated by the Phred program were removed using CLC Genomics Workbench software (ver.8.01, Qiagen)
Gene expression values were calculated as “fragments per kilobase of exon unit per million mapped reads” (FPKM).  We added 1 to the FPKM value before the following calculation.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...

source_name: endometrial stromal cells

growth_protocol: Cells were seeded at 10*5 cells/cm2 in 75 cm2 tissue culture flasks and incubated in Phenol Red-free DMEM containing glutamine, antibiotics, and 10% dextran-coated charcoal-stripped FBS at 37°C, 95% air and 5% CO2.

extract_protocol:
Total RNA was isolated from the cultured cells with an RNeasy® Mini Kit.  (Qiagen, Valencia, CA, USA).
Complementary DNA (cDNA) libraries for Illumina sequencing were generated with NEBNext Ultra II RNA library Prep kit (NEB) and NEBNextplex Oligos for Illumina.

treatment_protocol: To induce decidualization, ESCs were incubated with treatment medium (Phenol Red-free DMEM supplemented with glutamine, antibiotics and 2% dextran-coated charcoal-stripped FBS) containing cAMP (0.5 mM) for 4 days.

value: endometrial stromal cells

tag: transfection

value: Control

tag: agent

value: cAMP treatment


title: USA300_CLASH_WT_LPM54_r2.1

description:
RNA-RNA complexes immunoprecipitated untagged control, LPM pH 5.4 medium, second replicate, technical replicate 1
Intermolecular_hybrids_LPM_shift_LPM5_data.txt


title: 8_WT_Papain

value: Lung

value: papain-treated


title: cAMP_under_PGC-1a-knockdown, RNA-seq

description: cAMP treatment under PGC-1a-knockdown

value: PGC-1a siRNA


title: USA300_CLASH_RNase_III_LPM76_r2.1

description:
RNA-RNA complexes immunoprecipitated via RNase III, LPM pH 7.6 medium, second replicate, technical replicate 1
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_LPM7_data.txt

value: LPM 7.6

value: Sample harvested after shifting cells to LPM pH 7.6 for 15 minutes


title: Pachytene_WT_PD 24


title: BRCO-982


title: USA300_CLASH_WT_LPM76_r2.2

description:
RNA-RNA complexes immunoprecipitated untagged control, LPM pH 7.6 medium, second replicate, technical replicate 2
Intermolecular_hybrids_LPM_shift_LPM7_data.txt


title: 6_WT_Papain

value: WT


title: L/Z_cKO_PD 24


title: L/Z_WT_PD 24


title: miR-486 KO 3

city: Birmingham

first: Matthew

middle: S

last: Alexander

state: AL

address: 1900 University Blvd. THT 929 Box# 96

department: Pediatrics

institute: University of Alabama at Birmingham

data_processing:
Illumina CASAVA 1.8.2 was used for basecalling
Raw sequence reads were trimmed with Trim Galore version 0.4.2 using paramters --paired; --trim1
Trimmed sequence reads were aligned with STAR version 2.5.2b using parameters --outReadsUnmapped Fastx --outSAMtype BAM SoretedByCoordinate; --twopassMode Basic
Transcript abundances were calculated using HTSeq count version 0.6.1 using parameters -m union; -r pos; -t exon; -i gene_id; -a 10; -s no; -f bam
Genome_build: GRCme8 p4 Release M11 from Gencode
Supplementary_files_format_and_content: tab-delimited Excel file containing raw counts for each gene in each sample

source_name: Tibialis anterior

extract_protocol:
Tibialis anterior muscles were harvested from mice and flash frozen in liquid nitrogen and RNA was isolated using Trizol.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: FVB N/J

tag: age

value: 6 months

value: Tibialis anterior


title: 5_KO_resting

value: KO

value: untreated


title: 7_WT_Papain


title: miR-486 KO 4


title: S9T_S57

city: Houston

first: Randa

last: El-zein

address: 6670 Bertner Avenue

institute: Houston Methodist

description: Tumor.Squamus

data_processing:
Reads were mapped with Hisat2. Only uniquely mapping reads were allowed.
The mapped reads were then assembled into transcripts with Stringtie and the GENECODE gene model.
Gene expression (FPKM) was quantile normalized using the R statistical system.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include quantile normalized FPKM values for each Sample

extract_protocol:
Total RNA was purified using the RNeasy plus MiniKit (Qiagen) and the integrity of RNA was assessed using the RNA pico chip on Bioanalyzer 2100 (Agilent) and quantified with Qubit Fluorometer (Thermo Fisher). RNA Integrity Numbers (RINs) were used to evaluate the integrity of the RNA samples with > 7.0 considered intact and selected these samples for RNA library prep.
The RNA libraries were prepared and sequenced at the University of Houston Seq-N-Edit Core per standard protocols. RNA libraries were prepared with SMARTer Stranded Total RNA-Seq Kit v1 - Pico Input Mammalian Kit (Takara) using 10ng input RNA. RNA was fragmented, reverse transcribed into cDNA with random primers for the first strand and LNA-TSO switching oligo for the second stranded to give stranded information. Illumina adaptors were ligated to the cDNA at the 5’ and 3’ ends and rRNA was cleaved with ZapR enzyme. The uncleaved cDNA was amplified and size selection for libraries was performed using SPRIselect beads (Beckman Coulter). The purity of the libraries was analyzed using the DNA high sensitivity chip on Bioanalyzer 2100 (Agilent) and sequences were ~400bp in size. The indexed libraries were pooled and sequenced using NextSeq 500 (Illumina); generating ~20million 2×76 bp paired-end reads per samples.

tag: patient diagnosis

value: Non-small cell lung cancer (NSCLC)

tag: cancer subtype

value: Squamus

value: lung

tag: subject id

tag: disease state

value: Tumor


title: USA300_CLASH_WT_LPM76_r2.1

description:
RNA-RNA complexes immunoprecipitated untagged control, LPM pH 7.6 medium, second replicate, technical replicate 1
Intermolecular_hybrids_LPM_shift_LPM7_data.txt


title: 4_KO_resting


title: USA300_CLASH_RNase_III_LPM54_r1.1

description:
RNA-RNA complexes immunoprecipitated via RNase III, LPM pH 5.4 medium, first replicate, technical replicate 1
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_LPM5_data.txt

value: USA300 rnc:HTF

value: LPM 5.4

value: Sample harvested after shifting cells to LPM pH 5.4 for 15 minutes


title: S8N_S53

description: Normal.Squamus

value: Normal


title: S8T_S54

description: Tumor.Squamus


title: 3_WT_Resting

value: Lung


title: Control, RNA-seq

description: no treatment

value: endometrial stromal cells

value: Control

value: no treatment


title: USA300_CLASH_RNase_III_LPM76_r1.2

description:
RNA-RNA complexes immunoprecipitated via RNase III, LPM pH 7.6 medium, first replicate, technical replicate 2
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_LPM7_data.txt

value: LPM 7.6

value: Sample harvested after shifting cells to LPM pH 7.6 for 15 minutes


title: USA300_CLASH_RNase_III_LPM76_r1.1

description:
RNA-RNA complexes immunoprecipitated via RNase III, LPM pH 7.6 medium, first replicate, technical replicate 1
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_LPM7_data.txt


title: WT 3

value: 6 months

value: wildtype

value: Tibialis anterior


title: miR-486 KO 2

value: FVB N/J


title: S7N_S51

description: Normal.Squamus

value: Non-small cell lung cancer (NSCLC)

value: Squamus


title: S6T_S50

description: Tumor.Squamus

value: lung

value: Tumor


title: S6N_S49

description: Normal.Squamus


title: 2_WT_Resting

value: WT

value: untreated


title: 1_WT_Resting


title: WT 1


title: USA300_CLASH_RNase_III_TSB_r1.2

description:
RNA-RNA complexes immunoprecipitated via RNase III, TSB medium, first replicate, technical replicate 2
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_TSB_data.txt

value: USA300 rnc:HTF

value: TSB

value: Sample harvested at OD600 = 3 after growth in TSB


title: USA300_CLASH_RNase_III_TSB_r1.1

description:
RNA-RNA complexes immunoprecipitated via RNase III, TSB medium, first replicate, technical replicate 1
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_TSB_data.txt


title: S7T_S52

description: Tumor.Squamus


title: Tumor, EV4

city: Hershey

first: Hong-Gang

last: Wang

state: PA

address: 500 University Drive, H085, Rm C7833B

institute: Penn State College of Medicine

data_processing:
Adaptors were trimmed from raw FASTQ files using the BBDuk tool.
The trimmed FASTQ files were mapped against the mm10 mouse reference genome using STAR aligner to generate bam files.
Merge bam files for each library sample using samtools merge.
The aligned reads were quantified with htseq-count.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include raw counts for each Sample

source_name: Tumor

growth_protocol: Tumors were isolated from female mice receiving 5 treatments of dinutuximab or dinutuximab plus sEVs.

extract_protocol:
Tumor tissue was flash frozen, and RNA was extracted using RNeasy Mini Kit (Qiagen, 74004).
RNA libraries were prepared for sequencing using Illumina stranded mRNA prep protocols. Each library was runned on two lines to generate enough reads.

treatment_protocol: Mice bearing subcutaneous 9464D-GD2 neuroblastoma tumors were dosed twice per week with 25ug dinutuximab or 25ug dinutuximab plus 20ug sEVs.

tag: Sex

value: female

value: Dinutuximab plus sEVs (sEVs derived from 9464D-GD2 cells)


title: USA000_CLASH_WT_RPMI_r2

description:
RNA-RNA complexes immunoprecipitated untagged control, USA300 strain, RPMI medium, second replicate
Intermolecular_hybrids_RPMI_shift_RPMI_data.txt

value: RPMI

value: Sample harvested after shifting cells to RPMI for 15 minutes


title: S5T_S48

description: Tumor.Squamus

tag: tissue


title: WT 2

tag: strain

value: 6 months


title: Tumor, EV3

tag: treatment

value: subcutaneous 9464D-GD2 neuroblastoma tumor


title: in vitro induced definitive endoderm 0h

value: 0 h


title: Adult Bone Marrow 4

type: RNA

city: Novosibirsk

first: Roman

middle: Yurievich

last: Perik-Zavodskii

state: Novosibirsk region

address: Yadrincevskaya 14

country: Russia

institute: "Research Institute of Fundamental and Clinical Immunology" (RIFCI)

description: We performed mRNA profiling for 579 target genes, for 14 housekeeping genes, for 8 negative and 6 positive spike-in controls

hyb_protocol: 50ng of total RNA was hybridized with NanoString Immunology V2 panel according to the manufacturer's protocols.

data_processing: We performed QC on the raw data, performed background thresholding using the geometric means of negative controls, normalized gene expression  using the 4 housekeeping genes (ALAS1, PPIA, POLR1B and SDHA), we used the prenormalized in basic analysis data for the advanced analysis.

source_name: Adult Bone Marrow

extract_protocol: We obtained adult bone marrow and fetal human liver mononucler cells (MNCs) using Ficoll-paque density gradient, then labeled the MNCs  with anti-CD71-biotin-magnetic bead antibody and sorted the MNCs using a magnetic separator.

value: 23 years

tag: gender

value: Adult Human Bone Marrow

value: CD71+ erythroid cells


title: Tumor, Dinu4

value: Dinutuximab


title: S5N_S47

description: Normal.Squamus

value: Non-small cell lung cancer (NSCLC)

value: Squamus

value: lung

value: Normal


title: S4T_S46

description: Tumor.Squamus

value: Tumor


title: Adult Bone Marrow 3

value: 25 years

value: male

tag: cell type


title: S4N_S44

description: Normal.Squamus


title: USA000_CLASH_WT_RPMI_r1

description:
RNA-RNA complexes immunoprecipitated untagged control, USA300 strain, RPMI medium, first replicate
Intermolecular_hybrids_RPMI_shift_RPMI_data.txt

tag: media


title: USA300_CLASH_WT_TSB_r1

description:
RNA-RNA complexes immunoprecipitated untagged control, USA300 strain, TSB medium, first replicate
Intermolecular_hybrids_LPM_shift_TSB_data.txt

value: TSB

value: Sample harvested at OD600 = 3 after growth in TSB


title: mouse extended pluripotent stem cell EPS #3-2

data_processing:
Basecalling using RTA v3.4.4
Sequenced reads were trimmed for adaptor sequence, and masked for low quality data by fastp v0.19.7, then mapped to mm10 whole genome using STAR v2.7.3a and annotated using gencode vm25 gtf file.
sam file generated by STAR was converted to bam and counts are generated using summarizeOverlaps function from GenomicAlignments R package v3.13.
raw counts were divided by gene width information provided by gencode vm25 gtf file, then normalized to TPM (transcript per million).
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample.

source_name: mouse extended pluripotent stem cells

growth_protocol: Mouse totipotent potential stem cells were cultured on feeder cells in VCED medium composed of 4C basic medium. Mouse extended pluripotent stem cells were cultured on feeder cells in LCDM medium.

extract_protocol:
Total RNA were extracted from mouse totipotent potential stem cells and extended pluripotent stem cells using the RNeasy Mini Kit (Qiagen, 74106)
RNA sequencing libraries were constructed by using magnetic beads with oligo (DT) enriched mRNA. After that, fragment buffer was added to break the mRNA into short segments. Using mRNA as template, a strand of cDNA was synthesized using six base random primers. Then buffer, dNTPs, DNA polymer I and RNase H were added to synthesize a two strand cDNAs, which were purified using AMPURE XP beads. The purified double stranded cDNAs were repaired, a-tailed and sequenced, and then the fragment size was selected by AMPURE XP beads. Finally, PCR amplification was carried out and PCR products were purified with AMPURE XP beads to obtain the final library. The fragmented and randomly primed 2 ×100-bp paired-end libraries were sequenced using an Illumina HiSeq 2500.

value: mouse extended pluripotent stem cells


title: Adult Bone Marrow 2

value: 26 years

value: female


title: mouse extended pluripotent stem cell EPS #3-1


title: USA000_CLASH_RNase_III_RPMI_r4

description:
RNA-RNA complexes immunoprecipitated via RNase III, USA300 strain, RPMI medium, fourth replicate
Supplementary_Table_4.xlsx
Intermolecular_hybrids_RPMI_shift_RPMI_data.txt

value: USA300 rnc:HTF

value: RPMI

value: Sample harvested after shifting cells to RPMI for 15 minutes


title: in vitro induced definitive endoderm 48h

tag: differentiation time


title: Adult Bone Marrow 1

value: Adult Human Bone Marrow

value: CD71+ erythroid cells


title: S3N_S42

description: Normal.Squamus

value: Non-small cell lung cancer (NSCLC)


title: USA300_CLASH_WT_TSB_r2

description:
RNA-RNA complexes immunoprecipitated untagged control, USA300 strain, TSB medium, second replicate
Intermolecular_hybrids_LPM_shift_TSB_data.txt


title: Fetal Human Liver 4

source_name: fetal liver

value: 20-22 weeks

value: not collected

value: Fetal Human Liver


title: mouse extended pluripotent stem cell EPS #2-2


title: USA300_CLASH_RNase_III_TSB_r4

description:
RNA-RNA complexes immunoprecipitated via RNase III, USA300 strain, TSB medium, fourth replicate
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_TSB_data.txt


title: USA000_CLASH_RNase_III_RPMI_r3

description:
RNA-RNA complexes immunoprecipitated via RNase III, USA300 strain, RPMI medium, third replicate
Supplementary_Table_4.xlsx
Intermolecular_hybrids_RPMI_shift_RPMI_data.txt


title: miR-486 KO 1

value: FVB N/J

value: 6 months

tag: genotype

value: Tibialis anterior


title: Fetal Human Liver 3


title: S2T_S41

description: Tumor.Squamus

value: Squamus

value: lung

value: Tumor


title: USA300_CLASH_RNase_III_TSB_r3

description:
RNA-RNA complexes immunoprecipitated via RNase III, USA300 strain, TSB medium, third replicate
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_TSB_data.txt

value: TSB

value: Sample harvested at OD600 = 3 after growth in TSB


title: mouse extended pluripotent stem cell EPS #2-1

value: mouse extended pluripotent stem cells


title: Fetal Human Liver 2


title: USA000_CLASH_RNase_III_RPMI_r2

description:
RNA-RNA complexes immunoprecipitated via RNase III, USA300 strain, RPMI medium, second replicate
Supplementary_Table_4.xlsx
Intermolecular_hybrids_RPMI_shift_RPMI_data.txt

value: USA300 rnc:HTF

value: RPMI


title: Fetal Human Liver 1


title: S3T_S43

description: Tumor.Squamus


title: Tumor, Dinu3

value: female

value: Dinutuximab

value: subcutaneous 9464D-GD2 neuroblastoma tumor


title: USA300_CcpA-HTF_biolrep2_techrep3

description: Cross-linked RNAs from CRAC on the USA300 CcpA-HTF strain, second biological replicate, third technical replicate

data_processing:
Library strategy: CLIP-seq/CRAC
Two biological CcpA CRAC experiments were performed, each with three technical replicates. The cDNA libraries were sequenced on Illumina NovoSeq6000 machines (NovoGene) and Illumina MiniSeq systems. Raw sequencing reads in fastq files were processed using the paired-end CRAC data analysis pipeline developed by Sander Granneman, which uses tools from the pyCRAC package (version 1.5.0) (Webb et al., 2014). The CRAC_pipeline_PE.py script first demultiplexes the data using pyBarcodeFilter.py and the in-read barcode sequences found in the L5 5’ adapters. Flexbar then trims the reads to remove 3’-adapter sequences and poor-quality nucleotides (Phred score <23). Using the random nucleotide information present in the L5 5’ adaptor sequences, the reads are then collapsed to remove potential PCR duplicates using pyFastqDuplicateRemover.py. The reads were then mapped to the JKD6008 and USA300 genomes using Novoalign (www.novocraft.com).
To determine to which genes the reads mapped to, we generated an annotation file in the Gene Transfer Format (GTF). This file contains the start and end positions of each gene on the chromosome as well as what genomic features (i.e. sRNA, protein- coding, tRNA) it belongs to. To generate this file, we used the Rockhopper software (Tjaden, 2015) on USA300 rRNA-depleted total RNA-seq data (Stuart McKellar and Ivayla Ivanova, unpublished) and a minimal GTF file obtained from ENSEMBL (without UTR information). The resulting GTF file contained information not only on the coding sequences, but also more complete 5’ and 3’ UTR coordinates. Overlapping features, such as sRNAs that overlap with UTRs of protein-coding genes, were removed from the GTF file.
We then used pyReadCounters.py with Novoalign .novo or .sam output files as input and the GTF annotation file to count the total number of unique cDNAs that mapped to each gene. Reads that mapped to multiple features in the genome were randomly distributed over the features.
The individual tables generated by pyReadCounters were subsequently merged into a single table (WT_and_CcpA_raw_counts_rep1.txt and WT_and_CcpA_raw_counts_rep2.txt).
Because the technical replicates indicated high reproducibility, the raw counts of the technical replicates from the CcpA-HTF samples were subsequently merged to generate total coutns fro the two biological replicates. The negative control data were not further analysed as the counts for the total number of mapped reads for the parental control samples were extremely low compared to the CcpA-HTF data.
Genome_build: Staphylococcus_aureus_usa300_FPR3757
Supplementary_files_format_and_content: text, bedgraph, fasta and GTF files

organism: Staphylococcus aureus subsp. aureus USA300

source_name: CcpA-HTF cells

growth_protocol: S. aureus cells expressing HTF-tagged proteins were grown to saturation in TSB at 37 °C. Cells were then diluted in 100 ml fresh TSB to OD600 0.05, grown to an OD600 of roughly 3. Subsequently, cells were harvested by vacuum filtration and shifted to an equivalent volume of Low Phosphate Medium (LPM) for 15 minutes.

extract_protocol:
For the CRAC experiments, the cells were washed off the filters with 25ml of ice-cold Phosphate Buffer Saline (PBS) and pelleted by centrifugation. Cells (up to 0.5 grams) were subsequently resuspended in 2 volumes/cel weight of TN150-Lysostaphin buffer (50 mM Tris pH 7.8, 150 mM NaCl, 100 µg/mL Lysostaphin, 0.1% NP-40, 0.5% Triton X-100) and transferred to 5 mL screw-cap tubes (eppendofr). Sixty µL of RQ DNase 1 and 10 µL of SUPERase·In was added to the mixture and incubated at 37°C for 10 min to lyse the cells. 3V/cell weight of Zirconia beads (Thistle Scientific; 0.1 µm) were added and the mixture was vortexed vigorously 5 times for one minute with one-minute incubations on ice between each step. 2V/cell weight of cold TN150 anti-peptidase (50 mM Tris pH 7.8, 150 mM NaCl, 1 Roche EDTA-free mini pellet, 0.1% NP-40, 0.5% Triton X-100 and 10 mM EDTA) buffer before centrifugation for 30 minutes at 10000 g at 4°C. Subsequently, 75µl Anti-FLAG® M2 Magnetic Beads (pre-washed with 3 times 1 mL TN150 buffer) was added and incubated with the lysate for 2 hours at 4ºC. The beads were then washed three times five minutes with 2 mL TN1000 buffer (50 mM Tris pH 7.8, 1M NaCl, 0.1% NP-40, 0.5% Triton X-100) and three times with 2 mL TN150 buffer (50 mM Tris pH 7.8, 150 mM NaCl, 0.1% NP-40, 0.5% Triton X-100) for 5 minutes. Beads were subsequently resuspended in 250 µl of TN15, 10 µL of home-made GST-TEV protease was added and the samples were rotated for 1.5-2 hours at room temperature. TN150 was added to a final volume of 600 µL and 550 µL of the TEV eluate was incubated with RNace-it™ (Agilent Technologies1 µL of 1:100 dilution) for exactly five minutes at 37ºC. Subsequently, 500 µl of the mixture was added to a tube with 0.4 g Guanidium-HCl and vortexed vigorously to inactivate the RNases. NaCl and imidazole (pH 8.0) were added to a final concentration of 300 mM and 10 mM, respectively and the mixture was transferred to 50µl of Ni-NTA agarose beads (QIAGEN) equilibrated with Wash buffer I (300 mM NaCl, 10 mM imidazole, 6M GuHCl, 50 mM Tris-HCl pH 7.8, 0.1% NP40, 5 mM β- mercaptoethanol (βMe) and 0.5% Triton X-100) and incubated at 4ºC overnight on a rotator. The next day the beads were transferred to Pierce snap-cap columns (Thermo Scientific) and washed twice with 500 µL Wash buffer I and three times with 500 µL 1x NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% NP40, 5 mM βMe and 0.5% Triton X-100).
Radioactive labelling of 5’ ends, linker ligation and cDNA library preparation was performed as described previously (McKellar et al., 2020).

treatment_protocol: Cells were subsequently UV cross-linked (254 nm; 1000 mJ/cm2) in the Vari-X-linker (UVO3; (McKellar et al., 2020; Van Nues et al., 2017)), collected by vacuum filtration and flash-frozen on the filters and stored at -80ºC. As negative controls we either used UV irradiated cells from the USA300 parental strain.

value: USA300 LAC

value: CcpA-HTF

value: Grown to OD600 ~3.0 in TSB, shifted to Low Phosphate Medium (LPM) and UV cross-linked in the Vari-X-linker at 1000mJ/cm2. Harversed on filter paper by vacuum filtration and stored at -80ºC


title: mouse totipotent potential stem cell TPS #3-2

source_name: mouse totipotent potential stem cells

value: mouse totipotent potential stem cells


title: USA300_WT_biolrep2_techrep3

description: Cross-linked RNAs from CRAC on the parental strain USA300, second biological replicate, third technical replicate

source_name: WT cells

value: WT


title: S16N-12_S36

description: Tumor.Squamus

value: Non-small cell lung cancer (NSCLC)


title: JKD6009_CLASH_WT_RPMI_r1

description:
RNA-RNA complexes immunoprecipitated untagged control, JKD6009 strain, RPMI medium, first replicate
Intermolecular_hybrids_RPMI_shift_RPMI_data.txt

value: Sample harvested after shifting cells to RPMI for 15 minutes


title: USA300_CLASH_RNase_III_TSB_r1

description:
RNA-RNA complexes immunoprecipitated via RNase III, USA300 strain, TSB medium, first replicate
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_TSB_data.txt


title: mouse totipotent potential stem cell TPS #2-1


title: S15N_S33

description: Normal.Squamus

value: Squamus

value: lung

value: Normal


title: mouse extended pluripotent stem cell EPS #1-1


title: mouse totipotent potential stem cell TPS #1-2


title: JKD6009_CLASH_RNase_III_RPMI_r3

description:
RNA-RNA complexes immunoprecipitated via RNase III, JKD6009 strain, RPMI medium, third replicate
Supplementary_Table_3.xlsx
Intermolecular_hybrids_RPMI_shift_RPMI_data.txt

value: JKD6009 rnc::HTF


title: S14T_S32

description: Tumor.Squamus

value: Tumor


title: S16T-11_S37

description: Tumor.Squamus


title: Ear KR_1

value: Kaede

value: ear

value: inflammed


title: JKD6009_CLASH_RNase_III_TSB_r3

description:
RNA-RNA complexes immunoprecipitated via RNase III, JKD6009 strain, TSB medium, third replicate
Supplementary_Table_3.xlsx
Intermolecular_hybrids_LPM_shift_TSB_data.txt

value: TSB

value: Sample harvested at OD600 = 3 after growth in TSB


title: L4N

city: Galveston

first: Alexander

last: Bukreyev

state: Texas

address: 301 University Boulevard

institute: University of Texas Medical Branch at Galveston

description:
Golden Syrian Hamster
Animal source: Envigo
3-prime end polyA+ RNA sequencing

data_processing:
Base calling was using Illumina's bcl2fastq2-v2.20 converter.
Adapter and polyA sequences were trimmed with cutadapt version 1.15.
UMIs were removed from the reads and added to the read name with the extract_umi.py software provided by the Smart-3SEQ authors.
Reads were aligned to the Mesocricetus auratus NCBI assembly GCF_000349665.1 using STAR version 2.7.5c using the parameters recommended by the software authors for the Encode consortium.
The dedup.py Smart-3SEQ software was used to mark and remove duplicated reads.
FeatureCounts software was used to count reads per gene using the Mesocricetus auratus NCBI annotation release 102.
Genome_build: GCF_000349665.1 (MesAur1.0)
Supplementary_files_format_and_content: all.counts3.tsv: Tab-separated text file of raw read counts for all samples.

organism: Mesocricetus auratus

source_name: lung

extract_protocol:
Total RNA was isolated using Direct-zol™ RNA Microprep kit (Zymo Research) with on-column DNase digestion according to the manufacturer’s recommendations.
Smart-3SEQ protocol (PMID: 31519740, PMCID: PMC6836736, DOI: 10.1101/gr.234807.118).

value: Lung

value: Female

tag: vaccination status

value: Unvaccinated

tag: infection status

value: Uninfected

tag: experiment group

value: A


title: USA300_WT_biolrep2_techrep2

description: Cross-linked RNAs from CRAC on the parental strain USA300, second biological replicate, second technical replicate

value: USA300 LAC

value: WT

value: Grown to OD600 ~3.0 in TSB, shifted to Low Phosphate Medium (LPM) and UV cross-linked in the Vari-X-linker at 1000mJ/cm2. Harversed on filter paper by vacuum filtration and stored at -80ºC


title: L3N


title: mouse extended pluripotent stem cell EPS #1-2

value: mouse extended pluripotent stem cells


title: USA300_CLASH_RNase_III_TSB_r2

description:
RNA-RNA complexes immunoprecipitated via RNase III, USA300 strain, TSB medium, second replicate
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_TSB_data.txt

value: USA300 rnc:HTF


title: USA300_CcpA-HTF_biolrep2_techrep2

description: Cross-linked RNAs from CRAC on the USA300 CcpA-HTF strain, second biological replicate, second technical replicate

value: CcpA-HTF


title: S2N_S40

description: Normal.Squamus

value: Non-small cell lung cancer (NSCLC)

value: Squamus

value: lung

value: Normal


title: S17N-4_S38

description: Tumor.Squamus

value: Tumor


title: mouse totipotent potential stem cell TPS #2-2

value: mouse totipotent potential stem cells


title: S13T_S30

description: Tumor.Squamus


title: USA000_CLASH_RNase_III_RPMI_r1

description:
RNA-RNA complexes immunoprecipitated via RNase III, USA300 strain, RPMI medium, first replicate
Supplementary_Table_4.xlsx
Intermolecular_hybrids_RPMI_shift_RPMI_data.txt

value: RPMI

value: Sample harvested after shifting cells to RPMI for 15 minutes


title: S15T_S35

description: Tumor.Squamus


title: L2N

value: Lung

value: Female

value: Unvaccinated


title: S17T-3_S39

description: Tumor.Squamus


title: USA300_WT_biolrep2_techrep1

description: Cross-linked RNAs from CRAC on the parental strain USA300, second biological replicate, first technical replicate

value: USA300 LAC


title: JKD6009_CLASH_RNase_III_TSB_r2

description:
RNA-RNA complexes immunoprecipitated via RNase III, JKD6009 strain, TSB medium, second replicate
Supplementary_Table_3.xlsx
Intermolecular_hybrids_LPM_shift_TSB_data.txt

value: JKD6009 rnc::HTF

value: TSB

value: Sample harvested at OD600 = 3 after growth in TSB


title: mouse totipotent potential stem cell TPS #3-1


title: JKD6009_CLASH_WT_TSB_r1

description:
RNA-RNA complexes immunoprecipitated untagged control, JKD6009 strain, TSB medium, first replicate
Intermolecular_hybrids_LPM_shift_TSB_data.txt


title: USA300_CcpA-HTF_biolrep2_techrep1

description: Cross-linked RNAs from CRAC on the USA300 CcpA-HTF strain, second biological replicate, first technical replicate

value: Grown to OD600 ~3.0 in TSB, shifted to Low Phosphate Medium (LPM) and UV cross-linked in the Vari-X-linker at 1000mJ/cm2. Harversed on filter paper by vacuum filtration and stored at -80ºC


title: L5A

value: Mock-vaccinated

value: SARS-CoV2 infected

value: A


title: C1D_L_Her2

city: Memphis

first: Jiyang

last: Yu

state: TN

address: 262 Danny Thomas Pl

department: Department of Computational Biology

institute: St Jude Children's Research Hospital

data_processing:
After the removal of adapter and trimming of low quality bases, the reads were used for expression quantification using Salmon (v0.9.1). Expression values were quantified by counts in gene level.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include counts and TPM

growth_protocol: Cell lines were obtained from the American Type Culture Collection. Comma1-D cells were grown in DMEM:F12 (Gibco) supplemented with 2% FCS, 5 ng/mL murine EGF (Sigma), 10 μg/mL human insulin (Sigma), and 50 μg/mL gentamicin (Gibco).

extract_protocol:
Total RNA was isolated using either RNeasy Micro Kit (Qiagen) or miRVana isolation kit (Ambion). RNA quality was checked at Bioanalyser before library preparation.
Stranded RNA libraries were prepared with 200ng total RNA using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche)

treatment_protocol: Cells were transduced with Her2 activated oncogene or MYC activated oncogene as described in materials and methods

value: Comma1D, Sca-1(L), EpCAM (H)

tag: Stage

value: tranduced with Her2


title: JKD6009_CLASH_RNase_III_RPMI_r2

description:
RNA-RNA complexes immunoprecipitated via RNase III, JKD6009 strain, RPMI medium, second replicate
Supplementary_Table_3.xlsx
Intermolecular_hybrids_RPMI_shift_RPMI_data.txt


title: C1D_bi_Her2

value: Comma1D, Sca-1(H), EpCAM (H)


title: S14N_S31

description: Normal.Squamus

tag: patient diagnosis

value: Non-small cell lung cancer (NSCLC)

value: Squamus

value: lung

value: Normal


title: mouse totipotent potential stem cell TPS #1-1

value: mouse totipotent potential stem cells


title: C1D_bi_MYC

value: tranduced with MYC


title: L4A

value: Lung

value: Female


title: TH-totalRNA_r2

city: Taipei

first: Ming-Jung

last: Liu

address: 128 Sec. 2, Academia Rd, Nankang

country: Taiwan

institute: Academia sinica

description: Processed data_read_counts

data_processing:
Adaptor sequences (CTGTAGGCACCATCAAT) were trimmed from the sequence reads.
Genome_build: The reference genome sequences (SL3.0) and gene annotation (ITAG3.2) of  Solanum Lycopersicum were retrieved from Sol Genomics Network
Supplementary_files_format_and_content: The processed data contians the number of reads of each nucleotide on the genome for 3 samples (LTM, CHX and mRNA) in 2 biological repeats. The "viral" and "complementary"" indicated the strand direction.

organism: Solanum lycopersicum

source_name: total RNAs from TYLCTHV-infected leaf samples

growth_protocol: The tomato S. lycopersicum was grown in a growth chamber under 12-h light (8:00 to 20:00, 150 μmol m-2 s-1)/12-h dark cycle at 28ºC.

extract_protocol:
The purification of the ribosome-protected fragments (RPFs) samples for generating cycloheximide  (CHX) datasets, the RPF smaples for generating lactimidomycin (LTM), the total mRNA samples were based a reported protocol (Li and Liu, Genome REsearch, 2020).
The purified RNA fragments was used to construct a library for single 75-bp single-end  sequencing following the protocol described previously (Li and Liu., Genome Research, 2020).

treatment_protocol: True leaves of two~three weeks old plants inoculated with Agrobacterrium tumefaciens LBA4404 containing the TYLCTHV infectious clones were collected.

tag: sample type

value: Three-week-old leaves

tag: infection

value: TYLCTHV

tag: molecule type

value: Total RNA


title: S13N_S29

description: Normal.Squamus

tag: cancer subtype

tag: subject id

tag: disease state


title: C1D_bi_CTRL

value: control


title: C1D_L_CTRL


title: TH-totalRNA_r1

description: Processed data_read_counts


title: C1D_L_MYC


title: L3A

tag: tissue

tag: Sex

value: Mock-vaccinated

value: SARS-CoV2 infected


title: S10N_S25

description: Normal.Squamus


title: L1N

value: Unvaccinated

value: Uninfected

value: A


title: JKD6009_CLASH_RNase_III_RPMI_r1

description:
RNA-RNA complexes immunoprecipitated via RNase III, JKD6009 strain, RPMI medium, first replicate
Supplementary_Table_3.xlsx
Intermolecular_hybrids_RPMI_shift_RPMI_data.txt

source_name: Staphylococcus aureus

tag: strain

value: JKD6009 rnc::HTF

value: RPMI

tag: treatment

value: Sample harvested after shifting cells to RPMI for 15 minutes


title: USA300_CLASH_RNase_III_LPM54_r1.2

description:
RNA-RNA complexes immunoprecipitated via RNase III, LPM pH 5.4 medium, first replicate, technical replicate 2
Supplementary_Table_4.xlsx
Intermolecular_hybrids_LPM_shift_LPM5_data.txt

value: USA300 rnc:HTF

tag: media

value: LPM 5.4

value: Sample harvested after shifting cells to LPM pH 5.4 for 15 minutes


title: L1A

value: Lung

value: B


title: S12T_S28

description: Tumor.Squamus

value: Non-small cell lung cancer (NSCLC)

value: Squamus

value: lung

value: Tumor


title: S12N_S27

description: Normal.Squamus

value: Normal


title: C1D_B_MYC

tag: cell type

value: Comma1D, Sca-1(H), EpCAM (L)

value: tranduced with MYC


title: JKD6009_CLASH_RNase_III_TSB_r1

description:
RNA-RNA complexes immunoprecipitated via RNase III, JKD6009 strain, TSB medium, first replicate
Supplementary_Table_3.xlsx
Intermolecular_hybrids_LPM_shift_TSB_data.txt

value: TSB

value: Sample harvested at OD600 = 3 after growth in TSB


title: A9T_S24

description: Tumor.Adenoma

value: Adenoma


title: S10T_S26

description: Tumor.Squamus


title: TH-CHX_r1

description: Processed data_read_counts

data_processing:
Library strategy: Ribo-seq
Adaptor sequences (CTGTAGGCACCATCAAT) were trimmed from the sequence reads.
Genome_build: The reference genome sequences (SL3.0) and gene annotation (ITAG3.2) of  Solanum Lycopersicum were retrieved from Sol Genomics Network
Supplementary_files_format_and_content: The processed data contians the number of reads of each nucleotide on the genome for 3 samples (LTM, CHX and mRNA) in 2 biological repeats. The "viral" and "complementary"" indicated the strand direction.

source_name: ribosome protected fragments from CHX-treated TYLCTHV-infected leaf samples

value: Three-week-old leaves

value: TYLCTHV

value: CHX-treated

value: Ribosome-protected fragments


title: L15B

value: Female

tag: vaccination status

value: Vaccinated

value: SARS-CoV2 infected

value: A


title: L14B

value: Lung

tag: infection status


title: TH-CHX_r2

description: Processed data_read_counts


title: L2A

value: Mock-vaccinated

tag: experiment group

value: B


title: C1D_B_CTRL

tag: Stage

value: control


title: TH-LTM_r1

description: Processed data_read_counts

source_name: ribosome protected fragments from LTM-treated TYLCTHV-infected leaf samples

value: LTM-treated


title: A9N_S22

description: Normal.Adenoma

tag: patient diagnosis

value: Non-small cell lung cancer (NSCLC)

value: lung

value: Normal


title: A7T_S19

description: Tumor.Adenoma

value: Adenoma

value: Tumor


title: 152_input_ALKo_R1

description: Input KO

data_processing:
Demultiplexing: Remove UMIs from the sequencing reads and append to the read name for PCR deduplication further downstream. umi_tools extract –-stdin=0096_IP_1_S3.fastq.gz --bc-pattern=NNNNNNNNNN --log=0096_IP_1_S3.processed.log --stdout 0096_IP_1_S3.demultiplex.fastq.gz
Fastqc round 1: Run and examine by eye to make sure libraries look alright. fastqc 0096_IP_1_S3.demultiplex.fastq.gz
Cutadapt round 1: Takes output from demultiplexed files. Run to trim off 3’ adapters. cutadapt --match-read-wildcards --times 1 -e 0.1 -O 1 --quality-cutoff 6 -m 18 -a AGATCGGAAGAGCAC -a GATCGGAAGAGCACA -a ATCGGAAGAGCACAC -a TCGGAAGAGCACACG -a CGGAAGAGCACACGT -a GGAAGAGCACACGTC -a GAAGAGCACACGTCT -a AAGAGCACACGTCTG -a AGAGCACACGTCTGA -a GAGCACACGTCTGAA -a AGCACACGTCTGAAC -a GCACACGTCTGAACT -a CACACGTCTGAACTC -a ACACGTCTGAACTCC -a CACGTCTGAACTCCA -a ACGTCTGAACTCCAG -a CGTCTGAACTCCAGT -a GTCTGAACTCCAGTC -a TCTGAACTCCAGTCA -a CTGAACTCCAGTCAC -o 0096_IP_1_S3.adapterTrim.fastq.gz 0096_IP_1_S3.demultiplex.fastq.gz > 0096_IP_1_S3.adapterTrim.metrics
Cutadapt round 2: Takes output from cutadapt round 1. Run to trim off the 3’ adapters, to control for double ligation events. cutadapt --match-read-wildcards --times 1 -e 0.1 -O 5 --quality-cutoff 6 -m 18 -a AGATCGGAAGAGCAC -a GATCGGAAGAGCACA -a ATCGGAAGAGCACAC -a TCGGAAGAGCACACG -a CGGAAGAGCACACGT -a GGAAGAGCACACGTC -a GAAGAGCACACGTCT -a AAGAGCACACGTCTG -a AGAGCACACGTCTGA -a GAGCACACGTCTGAA -a AGCACACGTCTGAAC -a GCACACGTCTGAACT -a CACACGTCTGAACTC -a ACACGTCTGAACTCC -a CACGTCTGAACTCCA -a ACGTCTGAACTCCAG -a CGTCTGAACTCCAGT -a GTCTGAACTCCAGTC -a TCTGAACTCCAGTCA -a CTGAACTCCAGTCAC -o 0096_IP_1_S3.adapterTrim.round2.fastq.gz 0096_IP_1_S3.adapterTrim.fastq.gz > 0096_IP_1_S3.adapterTrim.round2.metrics
Trim 3' UMI: For IP samples only, trim the 9 nucleotide UMI from the 3' end of the read. cutadapt -u -9 -o 0096_IP_1_S3.trimN9.fastq.gz 0096_IP_1_S3.adapterTrim.round2.fastq.gz > 0096_IP_1_S3.trimN9.metrics
Trim miRNA: For miR-486-enriched samples only, trim the miRNA primer sequence from the 5' end of the read. cutadapt -g file:0096_miRNA_W_S25.miRNA_seq.fa --match-read-wildcards -O 10 -m 18 -e 0.05 -o 0096_miRNA_W_S25.miRNATrim.fastq.gz  0096_miRNA_W_S25.trimN9.fastq.gz> 0096_miRNA_W_S25.miRNATrim.metrics
STAR rmRep: Takes output from trimming 3' UMIs, or from trimming the miRNA primer for miR-486-enriched samples. Maps to a repetitive element database which is used to remove repetitive elements, helps control for spurious artifacts from rRNA (& other) repetitive reads STAR --runMode alignReads --runThreadN 8 --genomeDir /path/to/repetitive/elements/genome/ --readFilesIn 0096_IP_1_S3.adapterTrim.round2.fastq.gz --outSAMunmapped Within --outFilterMultimapNmax 30 --outFilterMultimapScoreRange 1 --outFileNamePrefix 0096_IP_1_S3.adapterTrim.round2.rep.bam --outSAMattributes All --readFilesCommand gunzip -c --outStd BAM_Unsorted --outSAMtype BAM Unsorted --outFilterType BySJout --outReadsUnmapped Fastx --outFilterScoreMin 10 --outSAMattrRGline ID:foo --alignEndsType EndToEnd > 0096_IP_1_S3.adapterTrim.round2.rep.bam
Samtools view and count_aligned_from_sam: Takes output from STAR rmRep. Counts the number of reads mapping to each repetitive element samtools view 0096_IP_1_S3.adapterTrim.round2.rep.bam | count_aligned_from_sam.py > 0096_IP_1_S3.adapterTrim.round2.rmRep.metrics
Fastqc round 2: Takes output from STAR rmRep. Runs a second round of fastqc to verify that after read grooming the data still is usable fastqc 0096_IP_1_S3.adapterTrim.round2.rep.bamUnmapped.out.mate1
STAR genome mapping: Takes output from STAR rmRep. Maps unique reads to the appropriate genome STAR --runMode alignReads --runThreadN 8 --genomeDir /path/to/genome/ --readFilesIn 0096_IP_1_S3.adapterTrim.round2.rep.bamUnmapped.out.mate1 --outSAMunmapped Within --outFilterMultimapNmax 1 --outFilterMultimapScoreRange 1 --outFileNamePrefix 0096_IP_1_S3.adapterTrim.round2.rmRep.bam --outSAMattributes All --outStd BAM_Unsorted --outSAMtype BAM Unsorted --outFilterType BySJout --outReadsUnmapped Fastx --outFilterScoreMin 10 --outSAMattrRGline ID:foo --alignEndsType EndToEnd > 0096_IP_1_S3.adapterTrim.round2.rmRep.bam
Sort bam file: Takes output uniquely mapped genome data and sorts and indexes the results samtools sort 0096_IP_1_S3.adapterTrim.round2.rmRep.bam -o 0096_IP_1_S3.adapterTrim.round2.rmRep.sorted.bam
Samtools index: Takes output from sort bam, makes bam index for use downstream samtools index 0096_IP_1_S3.adapterTrim.round2.rmRep.sorted.bam
Umi_tools dedup: Takes output from STAR genome mapping, performs PCR duplicate removal umi_tools dedup --method=unique -I 0096_IP_1_S3.adapterTrim.round2.rmRep.sorted.bam --log= 0096_IP_1_S3.dedup.log -S 0096_IP_1_S3.adapterTrim.round2.rmRep.sorted.rmDup.bam
Sort bam file: Takes output uniquely mapped human genome data and sorts and indexes the results samtools sort 0096_IP_1_S3.adapterTrim.round2.rmRep.sorted.rmDup.bam -o 0096_IP_1_S3.adapterTrim.round2.rmRep.sorted.rmDup.sorted.bam
Samtools index: Takes output from sort bam, makes bam index for use downstream samtools index 0096_IP_1_S3.adapterTrim.round2.rmRep.sorted.rmDup.sorted.bam
Make_bigwig_files.py: Takes input from samtools view. Makes bw files to be uploaded to the genome browser or for other visualization python make_bigwig_files.py --bam 0096_IP_1_S3.adapterTrim.round2.rmRep.sorted.rmDup.sorted.bam --genome /path/to/chrom/sizes/file --bw_pos 0096_IP_1_S3.CombinedID.merged.r2.norm.pos.bw --bw_neg 0096_IP_1_S3.CombinedID.merged.r2.norm.neg.bw
Clipper: Takes results from sort bam. Calls peaks on those files clipper -b 0096_IP_1_S3.adapterTrim.round2.rmRep.sorted.rmDup.sorted.bam -s {species} -o 0096_IP_1_S3 .adapterTrim.round2.rmRep.sorted.rmDup.sorted.peaks.bed --save-pickle
Fix_scores.py: Takes input from clipper: Fixes p-values to be bed compatible python fix_scores.py --bed 0096_IP_1_S3.adapterTrim.round2.rmRep.sorted.rmDup.sorted.peaks.bed --out_file 0096_IP_1_S3.CombinedID.merged.r2.peaks.fixed.bed
bedToBigBed: Converts bed file to bigBed file for uploading to the genomeBrowser bedToBigBed 0096_IP_1_S3.CombinedID.merged.r2.peaks.fixed.bed /path/to/chrom/sizes/file 0096_IP_1_S3.CombinedID.merged.r2.peaks.fixed.bb -type=bed6+4
Peak normalization vs paired SMInput datasets is run as a second processing pipeline (Peak_input_normalization_wrapper.py). Input files for the normalization pipeline include .bam and .peak.bed files (generated through the pipeline above), as well as a manifest file pairing eCLIP datasets with their paired SMInput datasets as follows: uID \t RBP \t Cell line \t CLIP_rep1 \t CLIP_rep2 \t INPUT 001 RBP1 HepG2 /full/path/to/{sample_rep1}.adapterTrim.round2.rmRep.sorted.rmDup.sorted.bam /full/path/to/{sample_rep2}.adapterTrim.round2.rmRep.sorted.rmDup.sorted.bam /full/path/to/{sample_input}.adapterTrim.round2.rmRep.sorted.rmDup.sorted.bam
Final output for this pipeline is an input normalized peak file (in bed format): /full/path/to/desired_output_directory/{uID_Rep}.basedon_{uID_Rep}.peaks.l2inpu tnormnew.bed.compressed.bed
For total chimeric IP samples, miRNA sequences were mapped to reads, and the best aligned miRNA sequences were trimmed from each read. The remainder of the read was then mapped to the genome and PCR duplicates were removed. Chimeric reads overlapping with AGO2 peaks were counted.
Genome_build: UCSC version GRCm38/mm10
Supplementary_files_format_and_content: Formatted as follows: Chr \t start \t stop \t log10(p-value eCLIP vs SMInput) \t log2(fold-enrichment in eCLIP vs SMInput) \t strand
Supplementary_files_format_and_content: Output file has bed format: Chr \t start \t stop \t log10(p-value eCLIP vs SMInput) \t log2(fold-enrichment in eCLIP vs SMInput) \t strand

source_name: Skeletal muscle (Tibialis anterior)

extract_protocol:
Tibialis anterior muscles were harvested from 6 month mice. RNA was isolated using Trizol.
mRNA libraries were prepared for sequencing using standard Illumina protocols
eCLIP-Seq. Sequencing was performed as PE100 or PE150 on the NovaSeq platform.
GENCODE Release M24 (GRCm38.p6)

value: FVB N/J

tag: age

value: 6 months

tag: genotype


title: 152_input_WTAL_R1

description: Input WT

value: wildtype


title: USA300_CcpA-HTF_biolrep1_techrep3

description: Cross-linked RNAs from CRAC on the USA300 CcpA-HTF strain, first biological replicate, third technical replicate

value: USA300 LAC

value: CcpA-HTF

value: Grown to OD600 ~3.0 in TSB, shifted to Low Phosphate Medium (LPM) and UV cross-linked in the Vari-X-linker at 1000mJ/cm2. Harversed on filter paper by vacuum filtration and stored at -80ºC


title: A8N_S20

description: Normal.Adenoma


title: L13B

value: Female

value: Vaccinated

value: SARS-CoV2 infected


title: L12B

value: Lung

value: A


title: USA300_CcpA-HTF_biolrep1_techrep2

description: Cross-linked RNAs from CRAC on the USA300 CcpA-HTF strain, first biological replicate, second technical replicate


title: 152_ALKo_miR_486a

description: miR-486-enriched IP KO


title: L11B


title: A7N_S18

description: Normal.Adenoma


title: 152_WtAL_miR_486a

description: miR-486-enriched IP WT


title: MIAPaCa-2 sh-CTL + PLK1i

city: Suita

first: Shotaro

last: Tatekawa

state: Osaka

address: 2-2(D10), Yamadaoka, Suita-city

department: Departments of Radiation Oncology,

institute: The University of Osaka Graduate School of Medicine

data_processing:
After checking the quality of sequencing using FastQC(v0.11.9) , and reads were aligned to human reference genomes and transcriptomes using STAR(v2.7.10) . We calculated the reads using RSEM(v1.3.3).
After the read count step, we performed expression analysis with iDEP and GSEA.
Assembly: hg19
Supplementary files format and content: Processed_Data.csv

source_name: pancreas

growth_protocol: All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
ISOGEN (NIPPON GENE)
RNA libraries were constructed following manufacturer instructions using the TruSeq stranded mRNA Library Prep Kit (Illumina, USA).

treatment_protocol: NMS-P937 for 24hr at 80 nM

value: pancreas

tag: cell line

value: epithelial cell

value: sh-CTL

value: NMS-P937 for 24hr at 80 nM


title: 152_AlKo

description: Total chimeric IP KO


title: USA300_WT_biolrep1_techrep3

description: Cross-linked RNAs from CRAC on the parental strain USA300, first biological replicate, third technical replicate

value: WT


title: 152_WtAl

description: Total chimeric IP WT


title: A8T_S21

description: Tumor.Adenoma

value: Non-small cell lung cancer (NSCLC)

tag: cancer subtype

value: Adenoma

value: lung

value: Tumor


title: A6N_S16

description: Normal.Adenoma

tag: subject id

value: Normal


title: A65T_S15

description: Tumor.Adenoma

tag: disease state


title: MIAPaCa-2 sh-METTL3

tag: tissue


title: TH-LTM_r2

description: Processed data_read_counts

tag: sample type

value: Three-week-old leaves

tag: infection

value: TYLCTHV

value: LTM-treated

tag: molecule type

value: Ribosome-protected fragments


title: USA300_WT_biolrep1_techrep2

description: Cross-linked RNAs from CRAC on the parental strain USA300, first biological replicate, second technical replicate

tag: strain

value: USA300 LAC

tag: treatment

value: Grown to OD600 ~3.0 in TSB, shifted to Low Phosphate Medium (LPM) and UV cross-linked in the Vari-X-linker at 1000mJ/cm2. Harversed on filter paper by vacuum filtration and stored at -80ºC


title: C1D_B_Her2

tag: cell type

value: Comma1D, Sca-1(H), EpCAM (L)

value: tranduced with Her2


title: 0096_miRNA_W_S25

description: miR-486a-enriched IP WT

value: 6 months

value: wildtype


title: MIAPaCa-2 sh-CTL

value: pancreas

value: epithelial cell

value: sh-CTL


title: USA300_WT_biolrep1_techrep1

description: Cross-linked RNAs from CRAC on the parental strain USA300, first biological replicate, first technical replicate

value: WT


title: A64N_S14

description: Normal.Adenoma

value: Non-small cell lung cancer (NSCLC)

value: Adenoma

value: lung


title: 0096_miRNA_K2_S27

description: miR-486a-enriched IP KO

value: FVB N/J


title: HepG2, CUT&Tag, BG4

city: Guangzhou

first: Lijun

last: Xiang

address: No.55, West of Zhongshan Avenue

department: Guangzhou Key Laboratory of Insect Development Regulation and Application Research

institute: South China Normal University

description: G4 CUT&Tag analysis with Flag-BG4 antibody

library_source: genomic

data_processing:
The CUT&Tag reads were aligned to the human genome (UCSC hg38) with Bowtie2. The aligned reads were removed with Picard MarkDuplicates, and the deduplicated BAM files were normalized to the total aligned reads (reads per million, RPM) with the bamCoverage command from deepTools 3.3.0.
Assembly: Homo sapiens .hg38
Supplementary files format and content: bigwig
Library strategy: CUT&Tag

source_name: Liver

growth_protocol: HepG2 cells were purchased from ATCC (American Type Culture Collection, VA, USA) and grown in DMEM (Thermo Scientific, MA, USA) supplemented with 10% FBS and 100 U/mL penicillin–streptomycin (Thermo Scientific, MA, USA) at 37 °C at 5% CO2.

extract_protocol:
CUT&Tag was performed essentially as described by Kaiwei Liang et al. 2021, with minor modifications according the Hyperactive Universal CUT&Tag Assay Kit protocol (Vazyme, Nanjing, China).One hundred thousand HepG2 cells were used for the CUT&Tag experiment, and cell counting was performed by using a Cellometer Mini (Nexcelom Bioscience, Boston, MA, USA). pA-Tn5 transposase was used to cut the genome and add a special adaptor sequence to build a library.
Libraries were prepared according to Hyperactive Universal CUT&Tag Assay Kit protocol (Vazyme, Nanjing, China).

value: Liver

tag: chip antibody

value: BG4(Sigma-Aldrich, MABE917), DYKDDDDK Tag Antibody(CST, #2368)


title: USA300_CcpA-HTF_biolrep1_techrep1

description: Cross-linked RNAs from CRAC on the USA300 CcpA-HTF strain, first biological replicate, first technical replicate

value: CcpA-HTF


title: A5N_S11

description: Normal.Adenoma

value: Normal


title: nSi and ion dissolution products hMSCs nSi 2

city: Bryan

first: Irtisha

last: Singh

state: TX

address: 8447 Riverside Pkwy Medical Research and Education Building II

institute: Texas A&M Health Science Center

description:
hMSCs nSi 2
nSi treatment

data_processing:
Sequence cluster identification, quality prefiltering, base calling and uncertainty assessment were done in real time using Illumina's NCS 1.0.2 and RFV 1.0.2 software with default parameter settings. Sequencer .cbcl basecall files were demultiplexed and formatted into .fastq files using bcl2fastq 2 2.19.0 script configureBclToFastq.pl.
Sequenced reads were trimmed for adaptor sequence and aligned by STAR aligner
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons (excluding 5UTR and 3UTR) of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library (only reads mapping to annotated regions of the genome were used for the total libraray size).
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for all samples

source_name: Mesenchymal stem cells

growth_protocol: hMSCs were cultured under media conditions consisting of α-minimal essential media (alpha-MEM, Hyclone, GE Sciences) with 16.5% fetal bovine serum (Atlanta Biologicals, USA) supplemented with 10 mM β-glycerophosphate (Sigma-Aldrich) and 50 µM ascorbic acid (BDH Chemicals)) and 1% penicillin/streptomycin (100 U/100 µg/mL, Gibco). After every 2-3 days, half of culture media was exchanged for fresh media. Cells were passaged with 0.5% trypsin-EDTA upon reaching confluency of ~70% and seeded at ~2500 cells/cm2. All experiments were completed with cell populations under P5.

extract_protocol:
At the end of 21 days of culture, total RNA was extracted (Zymogen, High Purity RNA Isolation kit), and then converted into cDNA (>1 μg).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: hMSCs were cultured and treated with/without and the various ion concentrations (4.49 µg/mL Si, 0.067 µg/mL Li, or 1.59 µg/mL Mg) or nSi (100µg/mL), or the combination of all ions (4.49 µg/mL Si, 0.067 µg/mL Li, and 1.59 µg/mL Mg)

value: Bone marrow derived mesenchymal stem cells


title: A6T_S17

description: Tumor.Adenoma

value: Tumor


title: OERipAB_1h_RNAseq_OERipAB_control_rep3

city: Wuhan

first: mengling

last: Huang

state: Hubei

address: shizisan

department: College of Plant Science and Technology

institute: Huazhong Agricultural Universityhuazong

data_processing:
RNA-seq library preparation and sequencing were performed on an Illunima HiSeq X-ten (Beijing, China). RNA-seq reads with low sequencing quality or reads with sequencing adaptors were filtered from the raw data. The resulting clean reads were then aligned to the Arabidopsis reference genome (TAIR10) using Hisat2  with default parameters. A GFF (general feature format) formatted gene model annotation file was provided for reads alignment. Following the alignments, transcriptome quantification based on gene level was performed by FeatureCounts  and then used DESeq2  to find the significant differential gene expression. Genes with expression fold change > 2 and Padj < 0.05 were considered as significantly different between samples with and without flg22 treatment.
Genome_build: TAIR 10
Supplementary_files_format_and_content: tab-delimited text files include raw gene counts for every gene and every sample

organism: Arabidopsis thaliana

source_name: seedlings

growth_protocol: Seedlings were germinated on half-strength Murashige and Skoog (½MS) plates containing 1% sucrose and 0.8% agar, and grown in a growth room at 21°C, 45% humidity, and 75 μE m-2 s-1 light with a 12-h-light/12-h-dark photoperiod.

extract_protocol:
Total RNA was extracted from ten-day-old seedlings by TRNzol reagent.
Libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: Seedlings were treated  with or without  500 μM INA  for 1 h.

value: OERipAB

value: with 500 μM INA for 1 h


title: nSi and ion dissolution products hMSCs All Ions 2

description:
hMSCs All Ions 2
All ions treatment


title: A4N_S9

description: Normal.Adenoma


title: OERipAB_1h_RNAseq_OERipAB_control_rep1


title: A5T_S13

description: Tumor.Adenoma


title: nSi and ion dissolution products hMSCs nSi 1

description:
hMSCs nSi 1
nSi treatment


title: USA300_TSB_medium_Nanopore

description: Ribosome-depleted total RNA

data_processing:
Basecalling, demultiplexing and quality analysis were done with gruppy. Reads with   mean quality above 7 were kept for further analysis. Next, the orientation of the reads was determined with cdna.claasifier.py (pychopper, https://github.com/nanoporetech/pychopper.git) and mapped to the USA300 genome with minimap2 (H. Li, Bioinformatics 2018). After mapping, sam files were converted into sorted bam files and then to bedgraphs with normalized coverage using SAMtools (H.Li, Bioinformatics,2009) and bedtools (Quinlan, A.R. and Hall, I.M. Bioinformatics 2010) respectively.
Assembly: NCBI:txid451515 (USA300), NCBI:txid546343 (JKD6009)

growth_protocol: S. aureus cells were grown in TSB to OD600 = 3.0 and harvested by centrifugation.

extract_protocol:
RNA extraction was performed using the guanidium thiocyanate (GTC) method. Briefly, 5 ml of cell culture was harvested at exponential phase by centrifugation. Cell pellets were resuspended in 100 µl GTC-Phenol (pH 7; 1:1 ratio) and lysed by vortexing the cells for 5 min with 100 µl of Zirconia beads (0.1 mm; Biospec products 11079101z). Subsequently, 550 µl of GTC was added and the mixture was vortexed for several minutes and incubated at 65ºC for 10 minutes and then 10 minutes on ice. Phases were separated by adding 300 µl chloroform isoamylalcohol (24:1) and 1/10th of a volume of 3M NaAc (pH 5.2) followed by rigorous vortexing. After 5 minutes of centrifugation in an Eppendorf centrifuge (14k rpm), the RNA was purified from the upper phase via two additional rounds of phenol-chloroform extraction and precipitated with 3 volumes of 96% cold ethanol.
Nanopore cDNA libraries were prepared using total RNA isolated from wild type USA300 grown in TSB or human serum. Total RNA was DNase I (TURBO DNase; Thermo Fisher) treated for 1 hour at 37°C in the presence of 2 U of SUPERasin. RNA was subsequently poly(A) tailed using E. coli poly(A) polymerase (NEB) following the manufacturer’s instructions. Afterwards, rRNA was removed using the NEBNext rRNA Depletion Kit (Bacteria) according to the manufacturer’s instructions. After each enzymatic step pior to the use of Nanopore cDNA kit, RNA was purified using RNAClean XP beads (Beckmann Coulter). cDNA barcoded libraries were prepared with Nanopore cDNA-PCR Sequencing kit (SQK-PCS109).  The cDNA was purified without size selection in order not to exclude sRNAs. The pooled barcoded libraries were sequenced on MinION using an R9 flow cell.

tag: media

value: TSB

value: Sample harvested at OD600 = 3 after growth in TSB


title: USA300_human_serum_medium_Nanopore

description: Ribosome-depleted total RNA

value: Human serum


title: OERipAB_0h_RNAseq_OERipAB_control_rep3

value: with 500 μM INA for 0 h


title: 0096_Input_3_S8

description: Input KO

value: 6 months


title: 0096_Input_1_S6

description: Input WT

value: wildtype


title: nSi and ion dissolution products hMSCs Mg 1

description:
hMSCs Mg 1
Mg treatment


title: OERipAB_1h_RNAseq_OERipAB_control_rep2


title: A4T_S10

description: Tumor.Adenoma

value: Non-small cell lung cancer (NSCLC)

value: Adenoma


title: nSi and ion dissolution products hMSCs All Ions 1

description:
hMSCs All Ions 1
All ions treatment


title: nSi and ion dissolution products hMSCs Mg 2

description:
hMSCs Mg 2
Mg treatment


title: 0096_IP_1_S3

description: Total chimeric IP WT


title: A3N_S7

description: Normal.Adenoma

value: lung


title: OERipAB_0h_RNAseq_OERipAB_control_rep2


title: A2T_S6

description: Tumor.Adenoma


title: nSi and ion dissolution products hMSCs Li 2

description:
hMSCs Li 2
Li treatment


title: sTREM-1-3

city: Nanjing

first: Li

last: Lu

address: The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University

institute: Nanjing university

data_processing:
We applied the reads filtering towards the raw reads after sequencing to achieve the clean data following the criteria: a)10% base quality <15 b) 13% base quality <20.
Hisat2 2.0.4 mapping to reference genome
count counting
Genome_build:  hg19_GRCh38
Supplementary_files_format_and_content: TXT file contain counts for each sample

growth_protocol: BV2 cells were seeded into a six-well plate at a density of 2×105 cells/well and cultured overnight (n = 3/group).

extract_protocol:
Total RNA was extracted from cell by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Bioanalyzer 2200 (Aligent) and kept at -80℃.The RNA with RIN >8.0 is right for cDNA library construction.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: BV2 cells were incubated with sTREM-1 recombinant protein (1ug/mL) for 6 h.

value: stimulated with sTREM-1 recombinant protein


title: OERipAB_0h_RNAseq_OERipAB_control_rep1


title: nSi and ion dissolution products hMSCs Li 1

description:
hMSCs Li 1
Li treatment


title: 0096_IP_3_S5

description: Total chimeric IP KO

value: FVB N/J


title: sTREM-1-2


title: sTREM-1-1


title: A2N_S5

description: Normal.Adenoma


title: WT_1h_RNAseq_OERipAB_control_rep3

value: wild type


title: CON 1-2

value: control


title: CON 1-3


title: nSi and ion dissolution products hMSCs Si 2

description:
hMSCs Si 2
Si treatment

value: Bone marrow derived mesenchymal stem cells


title: A18T-24_S2

description: Tumor.Adenoma

value: Tumor


title: WT_1h_RNAseq_OERipAB_control_rep2


title: nSi and ion dissolution products hMSCs 2

description:
hMSCs 2
Control


title: A1N_S3

description: Normal.Adenoma

value: Normal


title: WT_1h_RNAseq_OERipAB_control_rep1

value: with 500 μM INA for 1 h


title: A15T_S59

description: Tumor.Adenoma


title: nSi and ion dissolution products hMSCs 1

description:
hMSCs 1
Control


title: Mouse oocyte KO-4 weeks-MII-Rep3

city: Hangzhou

first: Yunwen

last: Wu

state: Zhejiang

address: Yu Hang Tang Road

department: Life Sciences Institute

institute: Zhejiang University

description:
Aging-Mouse-FPKM.txt
KO-4 weeks-MII-Rep3

data_processing:
RNA-Seq was performed with biological replicates for all samples. Raw reads were trimmed to 50 bp and mapped to the human genome (hg19), mouse genome (mm9) and ERCC spike-in sequences with Tophat v2.1.1 respectively. Only uniquely mapped reads were subsequently assembled into transcripts guided by the reference annotation (UCSC gene models) with Cufflinks v2.2.1. Expression level of each gene was quantified with normalized FPKM (fragments per kilobase of exon per million mapped fragments) and was further normalized with the ERCC spike-in.
Genome_build: hg19, mm9
Supplementary_files_format_and_content: Tab-delimited text files include FPKM values of all RNA-seq samples

source_name: Mouse KO MII

extract_protocol:
Oocytes were collected from indicated species (1 oocyte per sample for human and 10 oocytes per sample for mouse). Each sample was directly lysed with 4.2 ul lysis buffer (0.2% Triton X-100, RNase inhibitor, dNTPs, oligo-dT primers, and 1:1000 ERCC spike-in) and immediately used for cDNA synthesis using the Smart-seq2 method as described previously (Picelli et al, 2014).
Sequencing libraries were constructed from 500 pg of amplified cDNA using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD503) according to manufacturer’s instructions. Barcoded libraries were pooled and sequenced on the Illumina HiSeq X Ten platform with 150 bp paired-end reads

value: oocyte


title: A1T_S4

description: Tumor.Adenoma

data_processing:
Reads were mapped with Hisat2. Only uniquely mapping reads were allowed.
The mapped reads were then assembled into transcripts with Stringtie and the GENECODE gene model.
Gene expression (FPKM) was quantile normalized using the R statistical system.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include quantile normalized FPKM values for each Sample


title: WT_0h_RNAseq_OERipAB_control_rep2


title: A15N_S58

description: Normal.Adenoma


title: A18N-24_S60

description: Tumor.Adenoma


title: A3T_S8

description: Tumor.Adenoma


title: CON 1-1


title: WT_0h_RNAseq_OERipAB_control_rep3


title: nSi and ion dissolution products hMSCs Si 1

description:
hMSCs Si 1
Si treatment


title: WT_0h_RNAseq_OERipAB_control_rep1


title: Metabolic_syndrome (KC1-G36-H1297), total RNA

city: Daejeon

first: Su-Jin

last: Baek

address: 1672, Yuseong-daero

country: South Korea

institute: KOREA INSTITUTE of ORIENTAL MEDICINE

data_processing:
read trimming: skewer
mapping: STAR
quantification: Cuffquant
Genome_build: hg38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: PBMC

extract_protocol:
Total RNA was isolated from   PBMC cell using TRIzol® RNA Isolation Reagents (Life technologies, Carlsbad, CA) based method. The quantity and quality of the RNA were evaluated using Agilent Nono Kit and assessing the RNA integrity number (RIN >7). One mg of total RNA was processed for preparing mRNA sequencing library using MGIEasy RNA Directional Library Prep Kit (MGI) according to manufacturer’s instruction.
RNA libraries were prepared for sequencing using standard MGI protocols

value: PBMC

tag: Sex

value: Female


title: Mouse oocyte KO-4 weeks-MII-Rep2

description:
Aging-Mouse-FPKM.txt
KO-4 weeks-MII-Rep2


title: Mouse oocyte KO-4 weeks-GV-Rep3

description:
Aging-Mouse-FPKM.txt
KO-4 weeks-GV-Rep3

source_name: Mouse KO GV


title: SC_NS-6

city: Pittsburgh

first: Chandan

middle: K

last: Sen

state: Pennsylvania

address: 450 Technology Drive

department: Surgery

institute: University of Pittsburgh

data_processing:
BAM files were generated directly from raw Illumina BCL files using 10X Genomics Cellranger with default paramaters (cellranger)
Gene-barcode matrices were determined using 10X Genomics Cellranger  with default parameters (cellranger run)
Quality control, clustering and differential gene expression analysis were performed using Seurat v.3.1.0
Genome_build: hg:38
Supplementary_files_format_and_content: barcode files
Supplementary_files_format_and_content: gene names file
Supplementary_files_format_and_content: count matrix file

source_name: human unwounded skin tissue

growth_protocol: Human tissue samples

extract_protocol:
The tissue was chopped into small pieces and subjected to enzymatic dissociation using whole skin dissociation kit (cat. no. 130-101-540) in MACS C Tubes (cat. no. 130-093-237) (Miltenyi Biotec Inc., San Diego, CA). The samples were incubated in a water bath at 37°C for approximately 3 hours. After incubation, the samples were diluted by adding 0.5 mL of cold cell culture medium in order to stop the enzymatic reaction. The C Tubes were then tightly closed and attached upside down onto the sleeve of the gentleMACS™ Octo dissociator and UW human skin dissociation program was performed. The cell suspension was subjected to a pre-Separation Filter, 70 µm, placed on a 15 mL tube in order to remove the debris. RBC were lysed using 10X RBC lysis buffer (cat. no. 420301, BioLegend, San Diego, CA).
The resulting cell suspension was utilized for single cell RNA sequencing using the 10x Genomics platform using Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1(10x Genomics, Inc.) and sequenced on an Illumina NovaSeq 6000 (Illumina, Inc).

treatment_protocol: Single-cell suspensions were generated from theunwounded human skin biopsies.

tag: gender

value: M

tag: age (years)


title: A14N_S45

description: Normal.Adenoma

extract_protocol:
Total RNA was purified using the RNeasy plus MiniKit (Qiagen) and the integrity of RNA was assessed using the RNA pico chip on Bioanalyzer 2100 (Agilent) and quantified with Qubit Fluorometer (Thermo Fisher). RNA Integrity Numbers (RINs) were used to evaluate the integrity of the RNA samples with > 7.0 considered intact and selected these samples for RNA library prep.
The RNA libraries were prepared and sequenced at the University of Houston Seq-N-Edit Core per standard protocols. RNA libraries were prepared with SMARTer Stranded Total RNA-Seq Kit v1 - Pico Input Mammalian Kit (Takara) using 10ng input RNA. RNA was fragmented, reverse transcribed into cDNA with random primers for the first strand and LNA-TSO switching oligo for the second stranded to give stranded information. Illumina adaptors were ligated to the cDNA at the 5’ and 3’ ends and rRNA was cleaved with ZapR enzyme. The uncleaved cDNA was amplified and size selection for libraries was performed using SPRIselect beads (Beckman Coulter). The purity of the libraries was analyzed using the DNA high sensitivity chip on Bioanalyzer 2100 (Agilent) and sequences were ~400bp in size. The indexed libraries were pooled and sequenced using NextSeq 500 (Illumina); generating ~20million 2×76 bp paired-end reads per samples.

tag: patient diagnosis


title: Mouse oocyte KO-4 weeks-GV-Rep2

description:
Aging-Mouse-FPKM.txt
KO-4 weeks-GV-Rep2


title: SC_NS-5


title: A14T_S56

description: Tumor.Adenoma


title: control (KC1-G36-H1549), total RNA


title: SC_NS-3


title: control (KC1-G36-H1806), total RNA


title: Mouse oocyte WT-14 months-MII (no PB)-Rep2

description:
Aging-Mouse-FPKM.txt
WT-14 months-MII (no PB)-Rep2

source_name: Mouse WT old MII (no PB)


title: Mouse oocyte WT-14 months-MII (no PB)-Rep1

description:
Aging-Mouse-FPKM.txt
WT-14 months-MII (no PB)-Rep1


title: Mouse oocyte KO-4 weeks-GV-Rep1

description:
Aging-Mouse-FPKM.txt
KO-4 weeks-GV-Rep1


title: A12N_S23

description: Normal.Adenoma


title: SC_NS-1

value: F


title: Mouse oocyte KO-4 weeks-MII-Rep1

description:
Aging-Mouse-FPKM.txt
KO-4 weeks-MII-Rep1


title: control (KC1-G36-H0826), total RNA


title: Mouse oocyte WT-14 months-MII-Rep3

description:
Aging-Mouse-FPKM.txt
WT-14 months-MII-Rep3

source_name: Mouse WT old MII


title: Metabolic_syndrome (KC1-G36-H1614), total RNA


title: A10N_S1

description: Normal.Adenoma


title: Mouse oocyte WT-14 months-MII-Rep2

description:
Aging-Mouse-FPKM.txt
WT-14 months-MII-Rep2


title: Mouse oocyte WT-14 months-MII-Rep1

description:
Aging-Mouse-FPKM.txt
WT-14 months-MII-Rep1


title: SC_NS-4


title: control (KC1-G36-H1176), total RNA


title: 7-CCA1_BRCO

first: Haoyu

address: Kai Yuan Avenue 190

institute: GIBH

data_processing:
Bulk RNA-seq was performed by Novogene (Beijing, China) resulting in 20-30 million, 150 nucleotides long, paired-end reads.
Mapped reads were translated to counts using FeatureCounts (version 2.0.1) applying the built-in hg38 gernome annotation file.
CCA primary tissue bulk RNA-seq data from patient CCA1 was retrieved from a previously published manuscript [Broutier et al., Nat Med, 2017] (accession number GSE84073) and incorporated into our dataset. Raw counts were batch corrected using ComBat-seq (available via sva package version 3.36.0) using a negative binomial regression model.
Analysis of differential expression was performed using edgeR (version 3.32.1) and Limma (version 3.46.0) R packages. Genes were considered differentially expressed when the adjusted p-value was <0.05 (Benjamini Hochberg FDR corrected).
Subsequent GSEA on the log2FC values was done using fgsea (version 1.16.0) and gage (version 2.40.1) R packages. To increase specificity, fgsea results were filtered using gage results so that only pathways which are significant and in the same direction in both methods are retained.
Genome_build: Hg38
Supplementary_files_format_and_content: Dataframe counts and genes
Supplementary_files_format_and_content: Metadata information samples

source_name: Cholangiocarcinoma tumor organoids

growth_protocol: Broutier et al. Nat Med 2017. For BRCCAOs, branching organoids were created by switching culture conditions to non-canonical WNT stimulating conditions, consisting of William's E medium (WE, ThermoFisher Scientific) supplemented with 10 mM nicotinamide (Sigma), 17 mM sodium bicarbonate (Sigma), 0.2 mM 2-phospho-L-ascorbic acid trisodium salt (Sigma), 6.3 mM sodium pyruvate (Gibco), 14 mM glucose (Sigma), 20 mM HEPES (Fisher Scientific), ITS + premix (Life Technologies), 0.1 μM dexamethasone (Sigma), 2 mM Ultraglutamine (Fisher Scientific), 100 µg/mL penicillin/streptomycin (Fisher Scientific), 20 ng/mL EGF (Peprotech), 10% RSPO1 conditioned media (homemade) and 100 ng/mL DKK-1 (Abcam Ltd), after 3 passages.

extract_protocol:
RNA from CCA primary tissue and CCAOs cultured in EM and branching medium conditions was collected in lysis buffer (QIAzol lysing reagent, Qiagen) and the RNA was isolated using the miRNeasy mini kit (Qiagen) according to the manufacturer’s protocol
Sequencing data were uploaded to the Galaxy Web platform public server usegalaxy.org (Afgan E., Baker D., van den Beek M., Blankenberg D., Bouvier D., et al., 2016 The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 44: W3–W10). Sequences were trimmed for adapters using Trim Galore! (version 0.4.3.1), and mapped using RNA STAR (version 2.6.8a) against the human reference genome GRCh38).

value: human CCAOs

tag: medium

value: cultured in branching medium


title: Metabolic_syndrome (KC1-G36-H0844), total RNA


title: 10-CCA27_BRCO


title: 6-CCA27_EM

value: cultured in expansion medium


title: control (KC1-G36-H1099), total RNA

value: Male


title: 9-CCA14_BRCO


title: Mouse oocyte WT-14 months-GV-Rep3

description:
Aging-Mouse-FPKM.txt
WT-14 months-GV-Rep3

source_name: Mouse WT old GV


title: Metabolic_syndrome (KC1-G36-H0623), total RNA


title: A10T_S12

description: Tumor.Adenoma

value: Non-small cell lung cancer (NSCLC)

tag: cancer subtype

value: Adenoma

value: lung


title: Metabolic_syndrome (KC1-G36-H1080), total RNA