title: A549-ACE2_non-infected_48h_rep2_sRNA-seq

city: Paris

first: Germano

last: Cecere

address: Institut Pasteur, 28 Rue Du Docteur Roux, Batiment Monod, 4eme Etage

department: Development and stem cell biology

country: France

institute: Institut Pasteur

description: A549 cells stably expressing ACE2 via lentiviral transduction

data_processing:
The 3' adaptor (TGGAATTCTCGGGTGCCAAGG) was trimmed from the raw reads using cutadapt (version 1.18)
The trimmed reads were sorted by sequence using fastq-sort (from fastq-tools version 0.8) with option -s and deduplicated using a custom haskell program, keeping the highest quality among duplicates, at any given position
The 5' and 3' 4 nt UMIs were removed from the deduplicated reads using cutadapt (version 1.18) with options -u 4 and -u -4
After removing UMIs, the reads from 18 to 26 nt were selected using bioawk version 20110810 (git commit fd40150b7c557da45e781a999d372abbc634cc21)
The size-selected reads were aligned on the Homo sapiens genome (Build version GRCh38) using bowtie2 (version 2.4.2) with options -L 6 -i S,1,0.8 -N 0
Mapped reads were divided in sense and antisense reads in respect to the reference genome using samtools (version 1.3.1) while 22-nucleotide reads were extracted from mapped reads using bioawk
Reads mapping to CoV-2-microRNAs were counted using a custom script. First, a bed file with the coordinates of all the putative 22-nucleotide RNAs encoded by the SARS-CoV-2 genome was created and used to extract their sequences using bedtools. Second, the occurrence of each putative 22-nucleotide RNA among aligned reads was counted.
The resulting alignment was used to generate the normalized bigwig file using the sum of the total number of reads mapping on SARS-Cov-2 genome or Homo sapiens genome as a normalization factor. This was done using deeptools (version 3.5.0).
Genome_build: H. sapiens (GRCh38)
Supplementary_files_format_and_content: bigwig files allowing to display the normalized abundance of 22-nucleotides RNAs along the genome

growth_protocol: Human lung A549-ACE2 cells, which have been modified to stably express ACE2 via lentiviral transduction, were generated in the laboratory of Pr. Olivier Schwartz, (Institut Pasteur, Paris, France). Human colorectal adenocarcinoma Caco-2 and Vero E6 cells were purchased from ATCC. A549-ACE2 and Caco-2  cells were cultured in high-glucose DMEM media (Gibco) supplemented with 10% Fetal Bovine Serum (FBS; Sigma) and 1% penicillin-streptomycin (P/S; Gibco). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2.

extract_protocol:
cells were lysed using TRIzol LS reagent (Thermo Fischer Scientific) and RNA was extracted following manufacturer’s instructions. Agilent 2200 TapeStation System was used to evaluate the RIN indexes of all the RNA preps, and only samples with RIN > 8 was used for downstream applications. 2-4 µg of total RNAs with RIN > 8 was size selected from 15% UREA PAGE gel and used to generate small RNA libraries.
Total RNA was resolved on a 15 % TBE-Urea gel (Invitrogen EC6885BOX). RNA of size between 17-25 nt was excised from the gel and extracted in 0.3 M NaCl overnight at 25°C. Size selected RNA was used to prepare libraries following previously described methodology (Barucci et al 2020), with the exception that size selected monophosphate sRNA were ligated at 3’ with a preadenylated adaptor. Libraries were multiplexed and their quality was assessed on TapeStation (Agilent), and were quantified using the Qubit Fluorometer High Sensitivity dsDNA assay kit (Thermo Fisher Scientific, Q32851) and sequenced on a NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).

treatment_protocol: The strain BetaCoV/France/IDF0372/2020 was supplied by the Pr. S. van der Werf, head of the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France). Viral stocks were produced by amplification on Vero E6 cells, for 72 h in DMEM 2% FBS. The cleared supernatant was stored at -80°C and titrated on Vero E6 cells by using standard plaque assays to measure plaque-forming units per mL (PFU/mL). A549-ACE2 and Caco-2 cells were infected at MOI of 3 and 0,3 respectively, in DMEM without FBS. After 2 h, DMEM with 5% FBS was added to the cells.

tag: cell line

value: no infection

tag: molecule subtype

value: total sRNA


title: N1692= 6w TAC in NRON KO

status: Public on Nov 20 2021

type: SRA

city: Hannover

first: Julia

last: Leonardy

address: Carl-Neuberg-Straße 1

department: IMTTS

country: Germany

institute: Medical School Hannover

description: 6w TAC in NRON KO

library_source: transcriptomic

data_processing:
BCL files were converted to FASTQ files using bcl2fastq Conversion Software version v2.20.0.422
nfcore/rnaseq (v1.3), pipeline: Nextflow (v18.10.1), FastQC (v0.11.8), TrimGalore (v0.5.0), STAR (vSTAR_2.6.1d), RSeQC (v3.0.0), dupRadar, Preseq (v2.0.3), featureCounts (v1.6.4), StringTie (v1.3.5), Sample_correlation, MultiQC (v1.7)
Normalization and differential expression analysis was performed with DESeq2 (Galaxy Tool Version 2.11.40.2) with default settings except for “Output normalized counts table” which was set to “Yes”.
Genome_build: GRCm38; release M18
Supplementary_files_format_and_content: tab-delimited text files of raw read counts from each sample.

molecule: total RNA

organism: Mus musculus

source_name: murine cardiac RNA sample

extract_protocol:
Total RNA was isolated from mouse hearts after 6 weeks of TAC or Sham surgery with miRNeasy mini kit (Qiagen). 250ng of total RNA was used for mRNA enrichment procedure with NEBNext PolyA mRNA magnetic Isolation Module.
Stranded cDNA library was generated using using NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina with an additional purification step using Agencourt® AMPure® XP Beads. cDNA libraries were barcoded by dual indexing approach with NEBNext Multiplex Oligos for Illumina – 96 Unique Dual Index Primer Pairs.

tag: treatment

value: 6w TAC in NRON KO


title: siNC-3#

city: Chengdu

first: Xiaoqiang

last: Tang

address: Renminnan Road

country: China

institute: Sichuan University

data_processing:
Illumina Casava1.7 software used for basecalling.
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 Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: Rnor_6.0
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...

organism: Rattus norvegicus

source_name: Heart

extract_protocol:
RNA was harvested from cardiomyocytes using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) 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: cell type

value: Cardiomyocytes

tag: strain

value: SD

tag: tissue

value: Heart

tag: genotype/variation

value: siNC


title: siNC-4#


title: A549-ACE2_non-infected_24h_rep2_sRNA-seq

organism: Homo sapiens


title: A549-ACE2_non-infected_24h_rep1_sRNA-seq


title: A549-ACE2_non-infected_48h_rep1_sRNA-seq


title: A549-ACE2_infected_48h_rep2_sRNA-seq

description: A549 cells stably expressing ACE2 via lentiviral transduction infected with SARS-CoV2

value: SARS-CoV-2 infection (48 hpi)


title: A549-ACE2_infected_48h_rep1_sRNA-seq


title: siNC-1#


title: siECHS1-4#


title: siECHS1-1#


title: A549-ACE2_infected_24h_rep2_sRNA-seq

value: SARS-CoV-2 infection (24 hpi)


title: D3_WTvsSPTLungs_G02

city: New York

first: Simone

last: Caielli

state: NY

address: 413 E 69th Street

department: Department of Pediatrics

country: USA

institute: Weill Cornell Medicine

data_processing:
Sequencing reads were aligned with STAR_2.4.0f1  against the mm10 mouse genome
SAMTOOLS v0.1.19 was used for sorting and indexing reads
Gene counts were generated using htseq-count
Genome_build: mm10
Supplementary_files_format_and_content: csv file with raw gene counts for every gene and every sample

source_name: Epithelial cells (EpCAM)

growth_protocol: All experiments included sex- and age-matched SPT and WT-littermates between 8 and 12 weeks of age

extract_protocol:
RNA was extracted using Promega Maxwell 16 MDx instrument, (Maxwell 16 LEV simplyRNA Tissue Kit). RNA integrity was verified using the Agilent Bioanalyzer 2100 (Agilent Technologies). cDNA was synthesized from total RNA using Superscript III (Invitrogen)
Specimens were prepared for RNA sequencing using TruSeq RNA Library Preparation Kit v2 or riboZero

treatment_protocol: Mice were intransally inoculated with 5x10E6 TCID50 Human rhinovirus strain A-1B or equal volumes of PBS

tag: genotype

value: SPT

value: Lung

value: Epithelial cells (EpCAM)

tag: infection

value: HRV


title: sample_98

first: Alison

last: Goate

state: New York

address: 1 Gustave L. Levy Place

department: Department of Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

data_processing:
Reads were aligned to the hg19 genome using STAR aligner (v2.5.3.a), and RNA sequence quality was evaluated using RSeQC and Picard.
Samples were imputed to 1000 Genomes using cosmopolitan reference panel (Phase 3, version 5, NCBI GRCh37) using SHAPEIT then Impute2.
Gene expression was corrected for batch, age, sex, rin PMI and alcohol status using the "removeBatchEffect" option from limma package.
Summary data based conditional analysis was performed using Genome-wide Complex Trait Analysis (GCTA) software package.
log10 normalized alcohol consumption was used for differential expression analysis using the DeSeq2 program.
EnrichR and Ingenuity Pathway Analysis (IPA) was used to perform gene ontology and pathway enrichment analyses.
Genome_build: hg19
Supplementary_files_format_and_content: Raw count matrix with counts per gene as obtained by featureCounts

source_name: dorsolateral prefrontal cortex

extract_protocol:
Total RNA was extracted from human PFC brain tissue using the Qiagen RNeasy and Lipid Tissue kit (Qiagen, Valencia, VA, USA) and treated with DNase.
Libraries were prepared in a single batch by one operator using the KAPA Stranded RNA-Seq Kit with RiboErase (v4.16), and paired end sequencing was prformed on the Illumina HiSeq 2500.

value: dorsolateral prefrontal cortex

tag: post-mortem interval (hrs)

tag: rin


title: caco-2_non-infected_48h_rep2_sRNA-seq

description: human colorectal adenocarcinoma Caco-2


title: sample_94


title: sample_97


title: caco-2_non-infected_48h_rep1_sRNA-seq

description: human colorectal adenocarcinoma Caco-2


title: sample_90-92


title: sample_89-610


title: sample_89-577


title: siNC-2#


title: sample_89-221


title: caco-2_non-infected_24h_rep1_sRNA-seq

description: human colorectal adenocarcinoma Caco-2


title: caco-2_non-infected_24h_rep2_sRNA-seq

description: human colorectal adenocarcinoma Caco-2


title: sample_88-274


title: caco-2_infected_24h_rep1_sRNA-seq

description: human colorectal adenocarcinoma Caco-2 infected with SARS-Cov-2


title: A549-ACE2_CoV-2-miR-7a.2_mimic_non-induced_24h_rep1_RNA-seq

data_processing:
Reads were aligned on the Homo sapiens genome (Build version GRCh38) using hisat2 (version 2.2.1) with default parameters
Mapped reads were used to estimate the abundance of protein coding genes using featureCounts (version 2.0.1) with options -O -M --primary and annotations corresponding to protein coding genes (as annotated in the Ensemble release 100.
The alignment was used to generate the normalized bigwig file using normalization CPM. This was done with using bedtools (version 3.5.0).
Genome_build: H. sapiens (GRCh38)
Supplementary_files_format_and_content: bigwig files allowing to display the normalized abundance of RNAs along the genome

growth_protocol: Human lung A549-ACE2 cells, which have been modified to stably express ACE2 via lentiviral transduction, were generated in the laboratory of Pr. Olivier Schwartz, (Institut Pasteur, Paris, France). A549-ACE2 2  cells were cultured in high-glucose DMEM media (Gibco) supplemented with 10% Fetal Bovine Serum (FBS; Sigma) and 1% penicillin-streptomycin (P/S; Gibco). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2.

extract_protocol:
Cells were then lysed using TRIzol LS reagent (Thermo Fischer Scientific) and RNA was extracted following manufacturer’s instructions
DNase-treated total RNA with RIN > 8 was used to prepare strand-specific RNA libraries. DNAse treated RNA with high RIN value was used to deplete ribosomal RNA using NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6350) as per manufacture’s instructions. 100 ng of Ribosomal-depleted RNAs were then used to generate strand-specific RNA libraries using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760S). Multiplexed RNA libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).

treatment_protocol: Mimics RNAs of CoV2-miR-7a.1 and CoV2-miR-7a.2, or control mimic RNA, were synthesized in vitro (Eurofins Genomics). 1 nM of mimic RNAs were transfected in A549-ACE2 or Caco-2 cells using Lipofectamine RNAiMax (Thermo Fischer Scientific). 24 h post-transfection, cells were treated with or without 100U of human Interferon Alpha 2 (PBL Assay Science) for 8, 16 or 24 h.

value: stably expressing ACE2 via lentiviral transduction

value: Cells tranfected with 1nM CoV-2-miR-7a.2 mimic RNA. Post 24 hours transfection, cells without IFN treatment for 24 h

value: total RNA, rRNA-depleted


title: A549-ACE2_CoV-2-miR-7a.2_mimic_non-induced_24h_rep2_RNA-seq


title: caco-2_infected_24h_rep2_sRNA-seq


title: sample_88-225


title: sample_88-304


title: A549-ACE2_CoV-2-miR-7a.2_mimic_IFN-induced_24h_rep2_RNA-seq

value: Cells tranfected with 1nM CoV-2-miR-7a.2 mimic RNA. Post 24 hours transfection, cells were treated with 100U of human Interferon Alpha 2 (IFN-α2) for 24 h


title: caco-2_infected_48h_rep1_sRNA-seq

value: total sRNA


title: sample_650


title: sample_649


title: caco-2_infected_48h_rep2_sRNA-seq


title: A549-ACE2_CoV-2-miR-7a.2_mimic_IFN-induced_24h_rep1_RNA-seq


title: A549-ACE2_control_mimic_not_induced_24h_rep2_RNA-seq

value: Cells tranfected with 1nM control mimic RNA. Post 24 hours transfection, cells without IFN treatment for 24 h


title: sample_642


title: A549-ACE2_control_mimic_not_induced_24h_rep1_RNA-seq


title: sample_591


title: sample_693


title: sample_59


title: sample_510


title: HCM fibrosis 9

type: RNA

city: Hamburg

first: Tilo

last: Thottakara

address: Martinistr 52

department: Kardiologie

institute: Universität Hamburg

description: total miRNA isolated from patient plasma was analyzed by the human v3 miRNA expression assay kit code set (Nanostring Tech.)

data_processing: Background subtraction and quality control for 800 miRNAs tested in each sample were performed using the nSolver Software (Nanostring Tech., USA).  Low abundant miRNAs with expression levels equivalent to the expression  rates of negative controls were excluded from downstream analysis. All  samples were normalized using the geometric mean of the two spike-in miRNA  Oligos cel-miR-254 and osa-miR-414, and three positive ligation controls  provided in the Code set.

label: fluorescence

source_name: Plasma

label_protocol: 1000 attomol of spike-in-miRNA-Oligos osa-miR414  and cel-miR-254

extract_protocol: Isolation using Plasma/Serum Circulating and Exosomal RNA Purification Kit (Slurry Format, #51000, Norgen Biotek, Canada)

tag: disease state

value: hypertrophic cardiomyopathy

tag: patient


title: A549-ACE2_control_mimic_IFN-induced_24h_rep2_RNA-seq

value: Cells tranfected with 1nM control mimic RNA. Post 24 hours transfection, cells were treated with 100U of human Interferon Alpha 2 (IFN-α2) for 24 h


title: HCM fibrosis 12

hyb_protocol: total miRNA isolated from patient plasma was analyzed by the human v3 miRNA expression assay kit code set (Nanostring Tech.)


title: HCM fibrosis 10


title: HCM fibrosis 11


title: HCM 12


title: sample_507


title: HCM 10


title: HCM 11


title: HCM fibrosis 2


title: HCM fibrosis 7


title: sample_476


title: HCM fibrosis 1


title: HCM fibrosis 6


title: HCM fibrosis 4


title: HCM fibrosis 5


title: sample_474


title: A549-ACE2_CoV-2-miR-7a.2_mimic_IFN-induced_16h_rep2_RNA-seq

value: Cells tranfected with 1nM CoV-2-miR-7a.2 mimic RNA. Post 24 hours transfection, cells were treated with 100U of human Interferon Alpha 2 (IFN-α2) for 16 h


title: A549-ACE2_CoV-2-miR-7a.2_mimic_non-induced_16h_rep2_RNA-seq

value: Cells tranfected with 1nM CoV-2-miR-7a.2 mimic RNA. Post 24 hours transfection, cells without IFN treatment for 16 h


title: A549-ACE2_control_mimic_IFN-induced_24h_rep1_RNA-seq


title: A549-ACE2_CoV-2-miR-7a.2_mimic_non-induced_16h_rep1_RNA-seq


title: sample_513


title: HCM 6


title: HCM 9


title: HCM 8


title: A549-ACE2_CoV-2-miR-7a.2_mimic_IFN-induced_16h_rep1_RNA-seq


title: sample_466


title: HCM 7


title: A549-ACE2_control_mimic_not_induced_16h_rep2_RNA-seq

value: Cells tranfected with 1nM control mimic RNA. Post 24 hours transfection, cells without IFN treatment for 16 h


title: HCM fibrosis 3


title: HCM 5


title: sample_459


title: HCM 4


title: HCM 3


title: HCM fibrosis 8


title: A549-ACE2_control_mimic_not_induced_16h_rep1_RNA-seq


title: HCM 2


title: HCM 1


title: A549-ACE2_control_mimic_IFN-induced_16h_rep2_RNA-seq

value: Cells tranfected with 1nM control mimic RNA. Post 24 hours transfection, cells were treated with 100U of human Interferon Alpha 2 (IFN-α2) for 16 h


title: sample_417


title: A549-ACE2_CoV-2-miR-7a.2_mimic_non-induced_8h_rep2_RNA-seq

value: Cells tranfected with 1nM CoV-2-miR-7a.2 mimic RNA. Post 24 hours transfection, cells without IFN treatment for 8 h


title: Control 11

value: healthy


title: sample_410


title: A549-ACE2_control_mimic_IFN-induced_16h_rep1_RNA-seq


title: Control 9


title: Control 10


title: sample_402


title: sample_401


title: A549-ACE2_CoV-2-miR-7a.2_mimic_non-induced_8h_rep1_RNA-seq


title: sample_398


title: A549-ACE2_CoV-2-miR-7a.1_mimic_non-induced_8h_rep2_RNA-seq

value: Cells tranfected with 1nM CoV-2-miR-7a.1 mimic RNA. Post 24 hours transfection, cells without IFN treatment for 8 h


title: Control 2


title: sample_395


title: sample_367


title: Control 5


title: Control 7


title: Control 4


title: sample_442


title: A549-ACE2_CoV-2-miR-7a.2_mimic_IFN-induced_8h_rep2_RNA-seq

value: Cells tranfected with 1nM CoV-2-miR-7a.2 mimic RNA. Post 24 hours transfection, cells were treated with 100U of human Interferon Alpha 2 (IFN-α2) for 8 h


title: A549-ACE2_CoV-2-miR-7a.1_mimic_non-induced_8h_rep1_RNA-seq


title: sample_353

value: dorsolateral prefrontal cortex


title: Control 6


title: Control 8


title: A549-ACE2_CoV-2-miR-7a.1_mimic_IFN-induced_8h_rep1_RNA-seq

value: stably expressing ACE2 via lentiviral transduction

value: Cells tranfected with 1nM CoV-2-miR-7a.1 mimic RNA. Post 24 hours transfection, cells were treated with 100U of human Interferon Alpha 2 (IFN-α2) for 8 h


title: A549-ACE2_control_mimic_not_induced_8h_rep1_RNA-seq

value: Cells tranfected with 1nM control mimic RNA. Post 24 hours transfection, cells without IFN treatment for 8 h

value: total RNA, rRNA-depleted


title: A549-ACE2_control_mimic_not_induced_8h_rep2_RNA-seq


title: A549-ACE2_control_mimic_IFN-induced_8h_rep1_RNA-seq

value: Cells tranfected with 1nM control mimic RNA. Post 24 hours transfection, cells were treated with 100U of human Interferon Alpha 2 (IFN-α2) for 8 h


title: sample_366


title: A549-ACE2_control_mimic_IFN-induced_8h_rep2_RNA-seq


title: sample_337


title: sample_349


title: sample_332


title: sample_335


title: sample_330


title: SEQ12172, 5hmC_Seal

city: Chicago

first: Issam

last: Awad

state: IL

address: 5841 S. Maryland Avenue

department: Department of Neurology

institute: The University of Chicago

library_source: genomic

data_processing:
Library strategy: 5hmC-Seal
Raw sequencing reads were trimmed and filtered for low-quality bases using fastx_toolkit (v 0.0.14), followed by alignment to the human genome reference (hg19) using Bowtie2 (v 2.2.6) with the end-to-end alignment mode
Read pairs were concordantly aligned with fragment length ≤ 500 bp and with average ≤ 1 ambiguous base and up to four mismatched bases per 100 bp length
Aligned read pairs were then sorted, indexed and deduplicated using Picard (v 2.6.0). Alignments with Mapping Quality Score ≥ 10 were counted for gene bodies, according to the gene start and gene end annotations by the GENCODE Project (release 19), using featureCounts from R package Rsubread (v 1.30.9) without strand information
The 5hmC-Seal libraries were sequenced to produce a median number of ~20 million reads in each sample, and a median number of ~7 million unique reads were mapped to the ~20,000 gene body regions
Genome_build: hg19
Supplementary_files_format_and_content: 5hmC_NormCount.csv

molecule: genomic DNA

extract_protocol:
For each study subject, approximately 2-3 mL of frozen plasma was processed by centrifuging twice at 1,350 × g for 12 min, and then centrifuging at 13,500 × g for 12 min, followed by cfDNA extraction using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany)
Each cfDNA sample was first repaired and ligated with adaptors. Next, the T4 bacteriophage enzyme β-glucosyltransferase (β-GT) was used to transfer an engineered glucose moiety containing an azide-group to 5hmC in duplex DNA. A biotin tag was then installed onto the azide group using Huisgen cycloaddition (“click”) chemistry, followed by capturing of 5hmC-containing DNA fragments using avidin beads. The 5hmC-Seal library for each cfDNA sample was then constructed through PCR amplification, followed by paired-end sequencing (PE38) on the Illumina NextSeq500 platform at Shanghai Epican Genetech Co. Ltd. (Shanghai, China), according to the manufacturer’s protocol. We randomized cfDNA samples when constructing the 5hmC-Seal libraries and sequencing. The 5hmC-Seal was shown to be technically robust and stable in our previous studies using duplicate samples and spike-in controls

tag: population

value: Chinese

tag: diagnosis

value: Non-DR

value: Plasma

value: circulating cell-free DNA (cfDNA)


title: A549-ACE2_infected_24h_rep1_sRNA-seq

value: SARS-CoV-2 infection (24 hpi)

value: total sRNA


title: A549-ACE2_CoV-2-miR-7a.2_mimic_IFN-induced_8h_rep1_RNA-seq


title: Control 3


title: SEQ12151, 5hmC_Seal

value: DR


title: A549-ACE2_CoV-2-miR-7a.1_mimic_IFN-induced_8h_rep2_RNA-seq


title: sample_329


title: SEQ12203, 5hmC_Seal


title: Control 1


title: sample_326


title: SEQ12152, 5hmC_Seal


title: Sample 13_266 RNA-seq (RLI)

city: Rochester

first: Joseph

middle: Benjamin

last: Lillegard

state: MN

address: 200 First St. SW

department: Surgery

institute: The Mayo Clinic

data_processing:
RNA-seq:
The mRNA-seq data were aligned with STAR version 2.5.2b to the Sscrofa11.1 assembly of the pig genome.  Raw gene expression was quantified with the featureCounts software.  Normalization and differential expression analyses were performed using R version 3.5.3 with the edgeR version 3.24.3 and limma version 3.38.3 packages.  Network and functional term enrichment analyses were processed through Qiagen's Ingenuity application on the identified differentially expressed genes.  Heat maps visualizing the z-scores of the relevant genes' normalized expression values were created using the clustvis web tool.
After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate p-values and Log2 fold changes. Genes with adjusted p-values < 0.05 and absolute log2 fold changes > 1 were called as differentially expressed genes for each comparison. A gene ontology analysis was performed on the statistically significant set of genes by implementing the software GeneSCF. The mgi GO list was used to cluster the set of genes based on their biological process and determine their statistical significance. A PCA analysis was performed using the "plotPCA" function within the DESeq2 R package. The plot shows the samples in a 2D plane spanned by their first two principal components. The top 500 genes, selected by highest row variance, were used to generate the plot.
NGS DNA:
Quality control of the sequenced read pairs was performed using the FASTQC software. The average base quality of the sequenced reads was >Q30 on the Phred scale. Uniform distribution of A,G,T and C nucleotides was seen across the length of the reads without bias for any specific bases. The number of unknown “N” bases was less than 1% across the length of the reads. High sequence duplication (> 80%) was observed, however, this was anticipated due to the nature of the experiment and the amplification of specific library fragments.
Reads were trimmed to remove the viral sequence in two separate steps. In step 1, the viral sequence was trimmed from the R1 reads using cutadapt84 with a mismatch rate (e=0.3) from the 5’ end of each read. In step 2, if the linker sequence was present, it was similarly trimmed from the 3’end of the read. Trimmed reads with a length less than 15bps were removed from the rest of the analysis to reduce ambiguous alignments. Untrimmed reads were also removed from the rest of the analysis because they did not contain a viral segment that could be used as evidence of a viral mediated integration.
The remaining R1 reads were aligned to the susScr11 build of the pig reference genome using BWA-MEM in single-end mode. Default BWA-MEM parameters were used. The reads used to identify genomic points of viral integrations had to be uniquely mapped to the genome with a BWA-MEM mapping quality score greater than zero.
An integration point was defined by the position of R1’s first aligned base on the susScr1 genome. Unique integration points were identified across the genome without any constraints on coverage. However, for downstream annotation and analysis, only those integration points with 5 or more supporting reads were used to minimize calling false positive integration points.
Locations of integration points were categorized into: exons, introns, 3 prime UTRs, 5 prime UTRs and intergenic regions using information extracted from the susScr11 refflat file maintained by UCSC’s Genomics Institute. To avoid conflict of feature categorization arising from multiple overlapping gene definitions, only the definition of the longest gene was used to annotate integration points. Additional features where computed including: the distance to the nearest transcription start site (TSS), makeup of CpG-rich regions of the genome (“CpG islands”), and enrichment to tumor-associated genes. This tumor gene list was comprised of 745 clinically validated tumor-associated human genes (from Mayo Clinic internal data). Those genes were related to their pig homologs based on GenBank’s gene names.
In order to look for selective enrichment of any specific biological pathways, the genes identified as integration sites were entered into the WEB-based GEne SeT AnaLysis Toolkit. Results were identified using the over-representation analysis (ORA) method with identified KEGG pathways. A false discovery rate (FDR) of <0.05 was considered significant.
Genome_build: NGS DNA: susScr11; RNA-seq: Sscrofa11.1
Supplementary_files_format_and_content: "bed" (.txt); Rawcounts

organism: Sus scrofa

source_name: liver

growth_protocol: All animal procedures were reviewed and approved by Mayo Clinic’s  Institutional Animal Care and Use Committee, and all animals received humane care and husbandry for the  duration of the study.

extract_protocol:
(RNA-Seq samples 1-13) Total RNA was extracted from fresh frozen tissue using Qiagen RNeasy Plus Universal mini kit followed by Manufacturer’s instructions (Qiagen, Hilden, Germany). RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA).
(NGS DNA samples 14-22) Genomic DNA was isolated from snap-frozen tissue fragments using a Gentra Puregene Tissue Kit (Qiagen, Hilden, Germany).  Ligation-mediated PCR (LM-PCR) was used for efficient isolation of integration sites.  Restriction enzyme digestions with MseI were performed on genomic DNA samples; the digested DNA samples were then ligated to linkers and treated with ApoI to limit amplification of the internal vector fragment downstream of the 5' LTR.  Samples were amplified by nested PCR and and sequenced using the Illumina HiSeq 2500 Next-Generation Sequencing System (San Diego, CA).
Samples 1-13 contain paired-end reads of extracted mRNA.  Samples 14-22 contain paired end reads of DNA amplicons for mapping lentiviral integration sites.
(RNA-seq samples 1-13) RNA libraries were prepared using 200 ng of total RNA according to the manufacturer's instructions for the truSeq Stranded mRNA Sample Prep Kit (Illumina, Sna Diego, CA). The concentration and size distribution of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA).  Library was sequenced at 600 million fragment reads per sample following Illumina's standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100x2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HiSeq Control Software HD 3.4.0.38 collection softward. Base-calling was performed using Illumina's RTA version 2.7.7.
(NGS DNA samples 14-22)We used Picard software’s (http://broadinstitute.github.io/picard/) insert size metrics function to calculate the average fragment length per sample. This metric averaged across all the samples was 158 base pairs (bps) long with an average standard deviation of 29bps. Since 150bp long reads were sequenced, most of the paired read one (R1) and read two (R2) reads overlapped, providing redundant information. R2 reads were therefore not considered in the analysis.

treatment_protocol: Animals with the genotype "Fah +/+" were positive controls which did not exhibit the diseased phenotype of HT1 received no treatment. Pig number 266 was used as a negative control and received no consitant treatment, however, was treated on cycles of the drug NTBC. Animals with the genotpye "Fah -/-" which recieved the NTBC treatment were used as controls for the current standard of care of HT1 patients.  Animals with the genotype "Fah -/-" which recieved the LV-FAH treatment were the subjects of the study for the lentiviral gene therapy.

value: parenchyma

value: Fah -/-


title: sample_339


title: Sample 11_266 RNA-seq (LL)


title: Sample 10_283 NTBC RNA-Seq

value: NTBC


title: Sample 12_266 RNA-seq (LM)


title: SEQ12171, 5hmC_Seal


title: SEQ12153, 5hmC_Seal


title: SEQ12159, 5hmC_Seal


title: SEQ12173, 5hmC_Seal


title: Sample 9_282 NTBC RNA-Seq


title: Sample 7 _12 mo WT Sow

value: Fah +/+


title: Sample 5_WT 6 mo


title: SEQ12197, 5hmC_Seal


title: SEQ12175, 5hmC_Seal


title: SEQ12158, 5hmC_Seal


title: sample_299

value: dorsolateral prefrontal cortex


title: sample_303


title: sample_312


title: sample_301


title: sample_315


title: sample_313


title: sample_295


title: Sample 3_p162 RNA-seq (treated 6 mo)

value: LV-FAH


title: Sample 4_Fah -/- 6 mo untreated


title: Sample 2_p167 RNA-seq (12 mo)


title: Sample 6_Fah -/- 12 mo sick (p266 #65)


title: sample_305


title: W89A30

city: Chapel Hill

first: Brian

middle: David

last: Strahl

state: NC

address: 120 Mason Farm Road 3060 Genetic Medicine

department: Biochemistry

institute: UNC-Chapel Hill

description: YEATS-domain pocket mutant yaf9W89A

data_processing:
Library preparation for transcriptome sequencing
Clustering and sequencing
Data analysis including quality control, mapping to reference genome, novel gene prediction, quantification, differential expression analysis, enrichment analysis, alternative splicing analysis, and mutation analysis.
Genome_build: mm9
Supplementary_files_format_and_content: excel files of all the sample readcounts as well as the comparisons between wt and yaf9W89A samples

organism: Saccharomyces cerevisiae

source_name: Saccharomyces Cerevisiae yeast

growth_protocol: the chemostat protocal was listed in the method section of the manuscript. Both strains grow initially under high nutrient environment with constant O2 supply until they starve. 6 hours after starvation, low glucose media is slowly added into the vessels. The YMC is recored under nutrient-limiting conditions.

extract_protocol:
5 ml cells were extracted from the YMC cycles according to their time points
RNA libraries were referred to Graeme et al., 2019

treatment_protocol: 5 time points of YMC samples were collected across three consecutive cycles for both wt and yaf9W89A strains. Samples WT1-WT5 cover one wt YMC cycle. WT6-WT10 and WT11-WT15 are replicates; Samples W89A16-W89A20 cover one yaf9W89A YMC cycle. W89A21-W89A25 and W89A26-W89A30 are replicates.

tag: strain background

value: YEATS-domain pocket mutant yaf9W89A

value: low glucose environment


title: Sample 1_p166 RNA-seq (12 mo)


title: W89A28

description: YEATS-domain pocket mutant yaf9W89A


title: W89A29

description: YEATS-domain pocket mutant yaf9W89A


title: Sample 8_279 NTBC RNA-Seq


title: W89A27

description: YEATS-domain pocket mutant yaf9W89A


title: sample_286


title: SEQ12157, 5hmC_Seal


title: piwi_n4357_COPAS_lateL4_rep2_GRO-seq

data_processing:
library strategy: GRO-seq
The 3' adaptor (TGGAATTCTCGGGTGCCAAGG) was trimmed from the raw reads using cutadapt (version 1.18). The reads where the adaptor was not found were nonetheless kept ("untrimmed")
The 5' and 3' 4 nt UMIs were removed from the trimmed reads using cutadapt (version 1.18) with options -u 4 and -u -4
The 5' 4 nt UMIs as well as 7 nt in 3' (to be sure to eliminate possibly unrecognized first adaptor bases, as well as preceding 3' 4 nt UMIs) were removed from the "untrimmed" reads using cutadapt (version 1.18) options -u 4 and -u -7
Both types of reads were mapped on the C. elegans genome (WBcel235) using bowtie2 (version 2.3.4.3) with default parameters, and the resulting alignment files were merged using samtools (version 1.9)
Mapped reads were used to estimate the abundance of protein coding genes using featureCounts (version 1.6.3) with options -O -M --primary -s 1 --fracOverlap 0 and annotations corresponding to protein coding genes (as annotated in the iGenome distribution of WBcel235 obtained at ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Caenorhabditis_elegans/Ensembl/WBcel235/Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz)
The merged alignment was used to generate the normalized bigwig file using millions of summed forward reads per kilobase in protein coding genes as normalizer. This was done with a custom bash script using bedtools (version 2.27.1), bedops (version 2.4.26) and bedGraphToBigWig (version 4)
Genome_build: C. elegans ce11 (WBcel235)
Supplementary_files_format_and_content: bigwig files allowing to display the normalized abundance of nascent RNAs along the genome

organism: Caenorhabditis elegans

source_name: nematodes

growth_protocol: Strains were maintained at 20°C, using standard methods. (Stiernagle, T. Maintenance of C. elegans. WormBook : the online review of C. elegans biology 1–11 (2006))

extract_protocol:
Synchronous populations of L1 worms were grown at 20°C on NGM plates seeded with OP50 E. coli concentrated food at a density of maximum 40,000 animals per 15 cm Petri dish. Worms were collected at the indicated developmental stage: early L4 ( 38 hours post hatching (hph)), late L4 (44hph) or Young Adult ( 48hph). The harvested worms were washed three times with M9 buffer and nuclei were extracted in Nuclei extraction buffer using a metal dounce on ice. GRO-seq experiments were performed as described by Barucci et al., 2014 with minor modifications.
Biotinylated nascent RNAs were fragmented and immunoprecipitated using streptavidin conjugated beads (Invitrogen, 65001). 5’-OH of fragmented RNAs was repaired using Polynucleotide kinase (New England biolabs, M0201S). RNA was ligated to 3’end adapter using T4 RNA ligase 2 Truncated KQ and subjected to a second round of immunoprecipitation with streptavidin beads. RNA was ligated at 5’ end using T4 RNA ligase 1 (New England biolabs, M0204S) and biotinylated RNA was enriched for a third time. Purified RNA was reverse transcribed using SuperScript IV Reverse Transcriptase (Invitrogen, 18091050). cDNA was PCR amplified with specific primers using Q5 High-Fidelity 2X Master Mix (New England biolabs, M0492S). The purified libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced either on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).

treatment_protocol: A COPAS Biosorter was used to obtain homogeneously staged worm populations at the indicated developmentals stages.

value: whole worm

tag: developmental stage

value: late L4

value: sorted population


title: sample_285


title: SEQ12156, 5hmC_Seal

value: Chinese

value: Non-DR


title: W89A25

description: YEATS-domain pocket mutant yaf9W89A


title: W89A26

description: YEATS-domain pocket mutant yaf9W89A


title: piwi_n4357_COPAS_lateL4_rep1_GRO-seq


title: sample_282


title: W89A24

description: YEATS-domain pocket mutant yaf9W89A


title: CSR1_FLAG_YA_IP_rep2_sRNA-seq

data_processing:
The 3' adaptor (TGGAATTCTCGGGTGCCAAGG) was trimmed from the raw reads using cutadapt (version 1.18)
The trimmed reads were sorted by sequence using fastq-sort (from fastq-tools version 0.8) with option -s and deduplicated using a custom haskell program, keeping the highest quality among duplicates, at any given position
The 5' and 3' 4 nt UMIs were removed from the deduplicated reads using cutadapt (version 1.18) with options -u 4 and -u -4
After removing UMIs, the reads from 18 to 24 nt were selected using bioawk version 20110810 (git commit fd40150b7c557da45e781a999d372abbc634cc21)
The size-selected reads were mapped on the C. elegans genome (WBcel235) using bowtie2 (version 2.3.4.3) with options -L 6 -i S,1,0.8 -N 0
The reads that failed to map were inspected using grep -E -B 1 -A 2 "^G[ACGTN]{20,25}T+$" to detect possible reads starting with G with 20 to 25 nt followed by a poly-U tail that might have prevented the mapping, and this tail was removed from such reads using a custom haskell program before re-mapping them.
Mapped and remapped reads were used to estimate the abundance of small RNAs derived from structural RNAs using featureCounts (version 1.6.3) with options -O -s 1 --fracOverlap 1 and annotations corresponding to tRNA, snRNA, snoRNA, rRNA or RNA (as annotated in the iGenome distribution of WBcel235 obtained at ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Caenorhabditis_elegans/Ensembl/WBcel235/Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz)
The abundance of non-structural RNAs was estimated by subtracting the above counts from the number of mapped and remapped reads.
Initially mapped reads were classified using a custom python program according to their length, composition and on the annotations on which they mapped. Reads that didn't match miRNA and piRNA annotations were considered as potential endo-siRNAs.
The potential endo-siRNAs of size 21 to 23 nt that started with G were classified as “si_22G” if they mapped antisense to annotation belonging to the following categories: DNA transposons, RNA transposons, satellites, simple repeats (as annotated in http://hgdownload.cse.ucsc.edu/goldenPath/ce11/database/rmsk.txt.gz) or pseudogene or protein-coding genes (as annotated in the iGenome distribution of WBcel235 obtained at ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Caenorhabditis_elegans/Ensembl/WBcel235/Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz)
The “si_22G” reads were re-mapped on the C. elegans genome (WBcel235) using bowtie2 (version 2.3.4.3) with options -L 6 -i S,1,0.8 -N 0
The resulting alignment was used to generate the normalized bigwig file using millions of non-structural RNAs as normalizer. This was done with a custom bash script using bedtools (version 2.27.1), bedops (version 2.4.35) and bedGraphToBigWig (version 4)
Genome_build: C. elegans ce11 (WBcel235)
Supplementary_files_format_and_content: bigwig files allowing to display the normalized abundance of “si_22G” RNAs antisense to protein coding genes along the genome

extract_protocol:
Synchronous populations of L1 worms were grown at 20°C on NGM plates seeded with OP50 E. coli concentrated food at a density of maximum 40,000 animals per 15 cm Petri dish. 80.000 worms were collected at the indicated developmental stage: early L4 ( 38 hours post hatching (hph)), late L4 (44hph) or Young Adult ( 48hph). Worm pellets were washed three times with M9 buffer and suspended in the extraction buffer (50 mM Tris, 500 mM NaCl, 1 mM EDTA, 1% NP40, protease inhibitor cocktails (Thermo Scientific, 1860932)) followed by 30 strokes using a metal dounce on ice. Crude protein extracts were spun at 12,000 rpm at 4°C for 10 minutes. For CSR-1 IPs, Protein concentration was quantified by the Bradford assay, and 1 mg of protein extract was incubated with 15 μl of packed Anti-FLAG M2 Magnetic Agarose Beads (Sigma M8823). For HRDE-1 IPs, 2 mg of protein extract was incubated with 25uL GFP-trap Agarose beads (ChromoTek) for 1 hour at 4°C. After four washes with IP washing buffer (50 mM Tris, 500 mM NaCl, 1 mM EDTA, 1% NP40, protease inhibitor cocktails (Thermo Scientific, 1860932)), the beads were resuspended in 750 µL of TRIzol LS reagent (Ambion) to extract the immunoprecipitated RNAs. 750 µL of TRIzol LS reagent (Ambion) was also added to 10% of protein extract before the immunoprecipitation (Input). RNA extracted from IPs and Inputs were size selected from 15% UREA PAGE gel.
The library preparation was performed as essentially described by Jayaprakash et al., 2011, except that a 5’ polyphosphatase (lucigen RP8092H) treatment was performed to be able to clone tri-phosphate small RNAs, and that the PAGE gel extraction after each ligation was substitute with purification by 1.8 volumes of Agencourt RNAClean XP Beads (Beckman Coulter, NC0068576) and 3 volumes of isopropanol. The multiplexed amplified libraries were further purified using PippinPrep DNA size selection with 3% gel cassettes and the following parameters for the selection: BP start (115) and the BP end (165). The purified libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced either on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).

value: Young Adult

tag: rna population

value: immunoprecipitated sRNA

tag: rip antibody


title: SEQ12170, 5hmC_Seal

value: Plasma

tag: molecule subtype

value: circulating cell-free DNA (cfDNA)


title: W89A22

description: YEATS-domain pocket mutant yaf9W89A

tag: treatment


title: sample_278


title: sample_270


title: sample_277-1


title: SEQ12142, 5hmC_Seal

value: DR


title: W89A20

description: YEATS-domain pocket mutant yaf9W89A


title: CSR1_FLAG_earlyL4_IP_rep2_sRNA-seq

value: early L4

value: Anti-FLAG M2 Magnetic Agarose Beads (Sigma M8823)


title: sample_259


title: W89A23

description: YEATS-domain pocket mutant yaf9W89A


title: sample_257


title: CSR1_FLAG_earlyL4_IP_rep1_sRNA-seq


title: SEQ12136, 5hmC_Seal


title: CSR1_FLAG_YA_input_rep2_sRNA-seq

value: total sRNA

value: none (input)


title: SEQ12135, 5hmC_Seal


title: CSR1_FLAG_YA_input_rep1_sRNA-seq


title: sample_256

value: dorsolateral prefrontal cortex


title: W89A18

description: YEATS-domain pocket mutant yaf9W89A

value: YEATS-domain pocket mutant yaf9W89A


title: SEQ12168, 5hmC_Seal


title: W89A17

description: YEATS-domain pocket mutant yaf9W89A

value: low glucose environment


title: CSR1_FLAG_earlyL4_input_rep2_sRNA-seq

value: whole worm


title: sample_246


title: SEQ12174, 5hmC_Seal


title: SEQ12125, 5hmC_Seal


title: W89A21

description: YEATS-domain pocket mutant yaf9W89A


title: SEQ12126, 5hmC_Seal


title: SEQ12195, 5hmC_Seal


title: W89A16

description: YEATS-domain pocket mutant yaf9W89A


title: sample_245


title: CSR1_FLAG_earlyL4_input_rep1_sRNA-seq


title: WT15

description: wt

value: wt


title: SEQ12140, 5hmC_Seal


title: piwi_HRDE-1_GFP_lateL4_IP_rep2_sRNA-seq

value: piwi(gc035[STOP]) I ; ggSi1[hrde-1p::3xflag::gfp::hrde-1] II

value: late L4

value: GFP-trap Agarose beads (ChromoTek)


title: W89A19

description: YEATS-domain pocket mutant yaf9W89A


title: sample_236


title: SEQ12145, 5hmC_Seal

value: Chinese


title: CSR1_FLAG_YA_IP_rep1_sRNA-seq

value: Young Adult

value: sorted population

value: immunoprecipitated sRNA


title: sample_239


title: SEQ12133, 5hmC_Seal

value: DR

value: Plasma

value: circulating cell-free DNA (cfDNA)


title: piwi_HRDE-1_GFP_lateL4_input_rep2_sRNA-seq

value: total sRNA


title: piwi_HRDE-1_GFP_lateL4_IP_rep1_sRNA-seq

value: piwi(gc034[STOP]) I ; ggSi1[hrde-1p::3xflag::gfp::hrde-1] II


title: sample_234

value: dorsolateral prefrontal cortex


title: WT13

description: wt


title: sample_232


title: piwi_HRDE-1_GFP_lateL4_input_rep1_sRNA-seq

value: none (input)


title: SEQ12132, 5hmC_Seal

value: Non-DR


title: SEQ12165, 5hmC_Seal


title: HRDE-1_GFP_YA_IP_rep2_sRNA-seq

value: ggSi1[hrde-1p::3xflag::gfp::hrde-1] II

value: whole worm


title: WT10

description: wt

value: wt

value: low glucose environment


title: WT11

description: wt


title: HRDE-1_GFP_YA_IP_rep1_sRNA-seq

value: GFP-trap Agarose beads (ChromoTek)


title: HRDE-1_GFP_YA_input_rep2_sRNA-seq


title: SEQ12150, 5hmC_Seal

value: Chinese


title: WT14

description: wt


title: sample_227


title: SEQ12128, 5hmC_Seal


title: sample_225


title: sample_218


title: HRDE-1_GFP_YA_input_rep1_sRNA-seq

value: Young Adult


title: sample_217


title: WT7

description: wt


title: SEQ12149, 5hmC_Seal


title: HRDE-1_GFP_lateL4_IP_rep2_sRNA-seq

value: late L4

value: sorted population


title: SEQ12134, 5hmC_Seal


title: WT8

description: wt


title: sample_212


title: SEQ12167, 5hmC_Seal


title: WT6

description: wt


title: HRDE-1_GFP_lateL4_IP_rep1_sRNA-seq

value: immunoprecipitated sRNA


title: sample_228


title: WT12

description: wt


title: HRDE-1_GFP_lateL4_input_rep2_sRNA-seq


title: WT5

description: wt


title: SEQ12143, 5hmC_Seal

value: DR

value: Plasma


title: sample_209


title: sample_204


title: WT3

description: wt


title: SEQ12181, 5hmC_Seal

value: circulating cell-free DNA (cfDNA)


title: HRDE-1_GFP_lateL4_input_rep1_sRNA-seq

value: total sRNA


title: WT9

description: wt


title: WT4

description: wt


title: SEQ12177, 5hmC_Seal


title: SEQ12129, 5hmC_Seal


title: HRDE-1_GFP_earlyL4_IP_rep1_sRNA-seq

value: early L4


title: HRDE-1_GFP_earlyL4_input_rep2_sRNA-seq

value: none (input)


title: SEQ12166, 5hmC_Seal


title: WT1

description: wt


title: SEQ12139, 5hmC_Seal

value: Non-DR


title: HRDE-1_GFP_earlyL4_input_rep1_sRNA-seq

value: ggSi1[hrde-1p::3xflag::gfp::hrde-1] II


title: SEQ12120, 5hmC_Seal


title: N2_COPAS_earlyL4_rep4_RNA-seq

description: total RNA, rRNA-depleted

data_processing:
Reads were mapped on the C. elegans genome (WBcel235) using hisat2 (version 2.1.0) with default parameters
Mapped reads were used to estimate the abundance of protein coding genes using featureCounts (version 1.6.3) with options -O -M --primary -s 2 --fracOverlap 0 and annotations corresponding to protein coding genes (as annotated in the iGenome distribution of WBcel235 obtained at ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Caenorhabditis_elegans/Ensembl/WBcel235/Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz)
The alignment was used to generate the normalized bigwig file using millions of summed forward reads per kilobase in protein coding genes as normalizer. This was done with a custom bash script using bedtools (version 2.27.1), bedops (version 2.4.35) and bedGraphToBigWig (version 4)
Genome_build: C. elegans ce11 (WBcel235)
Supplementary_files_format_and_content: bigwig files allowing to display the normalized abundance of RNAs along the genome

extract_protocol:
Synchronous populations of L1 worms were grown at 20°C on NGM plates seeded with OP50 E. coli concentrated food at a density of maximum 40,000 animals per 15 cm Petri dish. Worms were collected at the indicated developmental stage: early L4 ( 38 hours post hatching (hph)), late L4 (44hph) or Young Adult ( 48hph). The harvested worms were washed three times with M9 buffer and the pellet was frozen in dry ice with Trizol Reagent (Ambion). After five repetitions of freeze and thaw, total RNA was isolated according to the Trizol Reagent protocol. Ten micrograms of RNA were treated with 2 U of Turbo DNase (Ambion) at 37°C for 30 minutes followed by phenol-extraction and isopropanol precipitation. Agilent 2200 TapeStation System was used to evaluate the RIN indexes of all the RNA preps, and only samples with RIN > 8 were used for downstream applications.
DNase-treated total RNA with RIN > 8 was used to prepare strand-specific RNA libraries. We developed an RNase H based method to degrade C. elegans and mitochondrial ribosomal RNAs (rRNAs) using 50nt oligos complementary to rRNA and mtRNA C. elegans sequences. 100 ng of DNase-treated total RNA was mixed with 1.5 µg of oligos at equimolar concentration and 1X probe hybridization buffer (200 mM NaCl, 10 mM Tris pH 7.5) and incubated in a thermocycler using the following parameters: 2 minutes at 95°C followed by 0.1°C/sec at 95-45°C, and 2 minutes hold at 45°C. Next 2 µl of Thermostable RNase H (epicentre) was added to the reactions together with 1X RNase H reaction buffer 50 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2) and incubated for 30 minutes at 45°C. Next, digested RNAs were treated with 2 U of Turbo DNase (Ambion) at 37°C for 30 minutes followed by purification using 2.2 volumes of Agencourt RNAClean XP Beads (Beckman Coulter, NC0068576) following the manufacturer’s instructions. 100 ng of Ribosomal-depleted RNAs were then used to generate strand-specific RNA libraries using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760S). Multiplexed RNA libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).

value: N2 - C. elegans wild isolate (Bristol)

value: whole worm


title: sample_193

value: dorsolateral prefrontal cortex


title: sample_190


title: sample_211


title: sample_185


title: SEQ12107, 5hmC_Seal


title: sample_182


title: N2_COPAS_earlyL4_rep3_RNA-seq

description: total RNA, rRNA-depleted


title: HRDE-1_GFP_earlyL4_IP_rep2_sRNA-seq

value: GFP-trap Agarose beads (ChromoTek)


title: sample_153


title: SEQ12122, 5hmC_Seal

value: Chinese


title: sample_150


title: hrde1_tm1200_YA_rep1_RNA-seq

description: total RNA, rRNA-depleted

value: Young Adult


title: SEQ12099, 5hmC_Seal


title: sample_146


title: SEQ12113, 5hmC_Seal


title: N2_YA_rep2_RNA-seq

description: total RNA, rRNA-depleted


title: N2_YA_rep1_RNA-seq

description: total RNA, rRNA-depleted


title: WT2

description: wt

value: wt

value: low glucose environment


title: SEQ12178, 5hmC_Seal


title: sample_192


title: SEQ12180, 5hmC_Seal


title: N2_COPAS_earlyL4_rep1_RNA-seq

description: total RNA, rRNA-depleted

value: sorted population


title: H2AK119ub KO-1 Day_40

city: Stanford

first: Andrey

last: Krokhotin

state: CA

address: 279 Campus Drive W

institute: HHMI

data_processing:
Library strategy: CUT&RUN
The adapters were trimmed from the raw reads using trim_galore and cutadapt. The trimmed reads were aligned to GRCh38 human genome assembly using bowtie2 with --very-sensitive -X2000 parameters. Not aligned reads and paired reads with at least one read in a pair falling below quality score threshold 10 were removed using samtools and awk text editor. Additionally, paired reads were removed if each read in a pair have multiple alignment sites. PCR and optical duplicates were removed using Picard. Macs2 (v2.1.1) was used to reconstruct peaks from fragments with the following parameters --format BEDPE -B --call-summits --nolambda -p 0.01.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include raw counts and TPM values for each RNA-seq data set
Supplementary_files_format_and_content: bigWig files generated for each ATAC-seq, H3K27me3 and H2AK119ub data sets

source_name: neural progenitor cells

growth_protocol: Human iPSC cultures were maintained in mTeSR1 medium (StemCell Technologies). To start NPC differentiation, cells were dissociated to single cells using Accutase (Life Technologies) and re-aggregated for 48h in Ultra-Low Cluster 96-well round bottom plates (CoStar) at a density of 7500 cells per well in mTeST1 supplemented with 1 uM Thiazovivin. On day 1 of differentiation, an equal volume of Neural Induction Medium (NIM) was added: 10% Knock-Out Serum Replacement, Penicillin/Streptomycin, and GlutaMax in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies) supplemented with 10 uM RepSox (Tocris), 1 uM LDN-193189 (StemCell Technologies), and 1 uM Thiazovivin. From day 2 to day 5, half of the spent medium was removed daily and replaced with fresh NIM without Thiazovivin. Starting on day 6, Neural Progenitor Medium (NPM) was used, containing 2% B27 Supplement without vitamin A, Penicillin/Steptomycin, and GlutaMax in DMEM/F12 (Life Technologies) supplemented with 20 ng/ml EGF and 20 ng/ml FGF (PeproTech); 10 uM RepSox was only added until day 10. On day 7, cell aggregates were transferred to Geltrex (Life-Technologies)-coated 6-well plates. Neural rosettes were carefully dissected from the adherent culture on days 12-14 and maintained as neurospheres in suspension, with medium change every 1-2 days and gentle dissociation by trituration every 4-5 days until the cells were used in experiments. EGF and FGF were omitted from the NPM medium starting on day 40.

extract_protocol:
Cultures were washed 1X with ice-cold PBS and dissociated to single cells using Accutase (Life Technologies). CUT&RUN was performed with an anti-H2AK119ub antibody (Ubiquityl-Histone H2A (Lys119) (D27C4) rabbit mAb Cell Signaling Technologies #8240) as described in (PMID: 31232687). Briefly, exactly 250,000 cells were counted with an automated cell counter (Countess, Life Technologies), bound to Concanavalin A beads (BioMagPlus, Bangs Laboratories #BP531). The anti-H3K27me3 antibody was added (1:50 dilution) and incubated with cells under 800rpm shaking at 4OC for 2 hrs. After wash steps, a 1:200 dilution of proteinA-MNase was added and incubated for 1hr at 4OC. After washing, 100mM CaCl2 was added at 0OC and incubated for 15 mins to activate the MNase. The reaction was stopped by addition of a 10mM EDTA/EGTA Stop Buffer, incubated at 37OC for 10mins, and centrifuged at 4OC, 5000xg for 15 mins. The supernatant containing DNA was taken and purified with the DNA Clean and Concentrator Kit (Zymo). Samples were quantified on a HS DNA Qubit Fluorometer (Life Technologies) and fragment size distribution checked with a Tapestation (Agilent).
Dual-index libraries were prepared from CUT&RUN eluted DNA using the NEBNext Ultra II DNA Library Prep (E7103S) with NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, E7600S) following manufacturer instructions. Libraries were quantified by qPCR using a PhiX (Illumina) standard curve, and checked for appropriate fragment size distribution by Tapestation (Agilent). Libraries were pooled and multiplexed 1:1 and run on a HiSeq3000 (Illumina) for 2x150 paired-end sequencing (Novogene).

tag: cell type

value: neural progenitor cells (NPCs) differentiated from iPSC line derived from a patient with Coffin-Siris syndrome

value: day 40 after start of iPSC differentiation to NPCs

tag: chip antibody


title: H2AK119ub KO-2 Day_40

value: rabbit mAb Cell Signaling Technologies #8240


title: S-patient-exo2

city: Daejeon

first: Ho Yeop

last: Lee

address: 266, Munhwa-ro, Jung-gu

country: South Korea

institute: Chungnam national university

data_processing:
FastQC v0.11.7 software used for quality check
Find and remove adapter sequences, primers, poly-A tails and other types of unwanted sequence from high-throughtput sequencing reads using Cutadapt 1.16
miRDeep2 used to discover novel microRNAs with high accuracy
The index alinged through ultrafast, memory-efficient short read aligner Bowtie. And use fast and sensitive ailgnment software HISAT2 for mapping next-generation sequencing reads to genomes.
gene and isoform expression levels from RNA-Seq data estimated using RSEM software.
Hairpin portion of a miRNA transcript are represented using miRBase database.
Genome_build: hg19

source_name: Salivary exosome

growth_protocol: In all, 16 randomized Korean patients with diabetes (mean ± s.d. age, 57.25 ± 11.08 years) who visited the Department of Oral Medicine, Kyungpook National University Hospital were enrolled in the study after obtaining their informed consent. All the patients fulfilled the clinicopathological criteria of erosive/atrophic OLP (van der Meij and van der Waal, 2003), including visual validation of whitish, atrophic, or erosive erythematous mucosal lesions on both the buccal mucosae and positive results of incisional biopsy. In addition, eight age-matched Korean volunteers (mean ± s.d. age, 54.8 ± 9.18 years) without OLP or any other oral mucosal disease were recruited as controls after their dental and medical examination. This study was approved by the Institutional Review Board of Kyungpook National University Hospital (IRB number: KNUH 2014-07-049-001), and all participants signed an informed consent. All saliva samples were collected between 10:00 and 12:00 a.m. to minimize the variability in salivary components. To reduce contamination with oral debris, all the subjects underwent standardized mechanical tooth and tongue cleansing without bleeding, performed by professionals. After the mouth was rinsed with distilled water for 1 min and waiting for additional 5 min, 5–10 ml of unstimulated whole saliva was collected in chilled 50-ml conical tubes from both the patients with OLP and healthy controls.

extract_protocol:
Salivary exosomes were isolated using ExoQuick precipitation solution (SBI, Mountain View, CA, USA). In all, 500 μl of cell-free saliva was thoroughly mixed with an equal volume of ExoQuick solution, incubated overnight at 4°C, and centrifuged at 1500 × g and 4°C for 15 min. The pellet was resuspended in PBS
RNA libraries were preparaed for sequencing using SMARTer smRNA-Seq Kit for Illumina User Manual

tag: age (y)

tag: tissue

value: salivary gland

tag: disease group

value: Type2 Diabetes


title: H2AK119ub HET-2 Day_40

tag: genotype


title: H2AK119ub HET-1 Day_40


title: SEQ12198, 5hmC_Seal

value: Plasma

value: circulating cell-free DNA (cfDNA)


title: sample_105

tag: post-mortem interval (hrs)

tag: rin


title: N2_COPAS_earlyL4_rep1_GRO-seq

data_processing:
Library strategy: GRO-seq
The 3' adaptor (TGGAATTCTCGGGTGCCAAGG) was trimmed from the raw reads using cutadapt (version 1.18). The reads where the adaptor was not found were nonetheless kept ("untrimmed")
The 5' and 3' 4 nt UMIs were removed from the trimmed reads using cutadapt (version 1.18) with options -u 4 and -u -4
The 5' 4 nt UMIs as well as 7 nt in 3' (to be sure to eliminate possibly unrecognized first adaptor bases, as well as preceding 3' 4 nt UMIs) were removed from the "untrimmed" reads using cutadapt (version 1.18) options -u 4 and -u -7
Both types of reads were mapped on the C. elegans genome (WBcel235) using bowtie2 (version 2.3.4.3) with default parameters, and the resulting alignment files were merged using samtools (version 1.9)
Mapped reads were used to estimate the abundance of protein coding genes using featureCounts (version 1.6.3) with options -O -M --primary -s 1 --fracOverlap 0 and annotations corresponding to protein coding genes (as annotated in the iGenome distribution of WBcel235 obtained at ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Caenorhabditis_elegans/Ensembl/WBcel235/Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz)
The merged alignment was used to generate the normalized bigwig file using millions of summed forward reads per kilobase in protein coding genes as normalizer. This was done with a custom bash script using bedtools (version 2.27.1), bedops (version 2.4.26) and bedGraphToBigWig (version 4)
Genome_build: C. elegans ce11 (WBcel235)
Supplementary_files_format_and_content: bigwig files allowing to display the normalized abundance of nascent RNAs along the genome

value: early L4

value: biotinylated RNA


title: N2_COPAS_earlyL4_rep2_GRO-seq

value: N2 - C. elegans wild isolate (Bristol)

value: whole worm


title: hrde1_tm1200_YA_rep2_RNA-seq

description: total RNA, rRNA-depleted


title: SEQ12176, 5hmC_Seal

value: DR


title: S-con-exo2

value: healthy


title: SEQ12123, 5hmC_Seal

value: Chinese


title: hrde1_tm1200_COPAS_lateL4_rep2_GRO-seq

value: late L4


title: hrde1_tm1200_COPAS_lateL4_rep1_GRO-seq


title: N2_COPAS_lateL4_rep2_GRO-seq


title: S-con-exo1


title: SEQ12109, 5hmC_Seal

value: Non-DR


title: SEQ12106, 5hmC_Seal


title: H2AK119ub WT-2 Day_40

value: wild type


title: H2AK119ub WT-1 Day_40

value: neural progenitor cells (NPCs) differentiated from iPSC line derived from a patient with Coffin-Siris syndrome


title: SEQ12111, 5hmC_Seal


title: SEQ12163, 5hmC_Seal


title: S-patient-exo1


title: UAS-Jeb, Alk activation with twi2xPE-GAL4 promoter replicate 3

city: Gothenburg

first: Erik

last: Larsson

address: Medicinaregatan 9A

country: Sweden

institute: University of Gothenburg

description:
GATC_counts.txt
tj_peak.txt
Sample tj3

data_processing:
Library strategy: DAMID-seq
Basecalls performed using CASAVA version 1.4
Reads were aligned to the dmel r6.21 genome assembly using Bowtie2 (–very-sensitive-local)
Counts of reads mapped on edge to GATC fragments were generated using a script (GATC_mapper.pl) from DamID-Seq pipeline (Maksimov et al.,2016).
GATC sites were merged into peaks following the methods prescribed in a previous study (Tosti et al.,2018). In brief, logFC for individual GATCs were generated using Limma (Jeb versus Dam and DN versus Dam) (P<1e-5) and the GATC sites were merged into peaks based on median GATC fragment distance in the Drosophila genome using mergeWindows and combineTests function from the csaw package (Lun and Smyth, 2016).
Genome_build: dmel r6.21
Supplementary_files_format_and_content: GATC raw counts text files
Supplementary_files_format_and_content: peak text files

organism: Drosophila melanogaster

source_name: Drosophila embryonic DNA

growth_protocol: Embryos were collected over a 4 h period and aged at 25°C to stage 10-13, followed by dechorionation in 2% sodium hypochlorite solution for 2 min and subsequent washing steps in PBS. A total of 50 µl embryos per sample was used as starting material.

extract_protocol:
Genomic DNA was extracted (QIAGEN Blood and Tissue DNA extraction kit) and methylated DNA was processed and amplified as previously described (Choksi et al., 2006; Sun et al., 2003), with the following modifications.
Purified PCR products were used for PCR-free library preparation, followed by pair-end sequencing on Illumina HiSeq X Ten platform (BGI Tech Solutions, Hong Kong).

tag: expression

value: UAS-Jeb

tag: promoter

tag: replicate


title: UAS-Jeb, Alk activation with twi2xPE-GAL4 promoter replicate 2

description:
GATC_counts.txt
tj_peak.txt
Sample tj2


title: H3K27me3 KO-2 Day_60

data_processing:
Library strategy: CUT&RUN
The adapters were trimmed from the raw reads using trim_galore and cutadapt. The trimmed reads were aligned to GRCh38 human genome assembly using bowtie2 with  --very-sensitive -X2000 parameters. Not aligned reads and paired reads with at least one read in a pair falling below quality score threshold 10 were removed using samtools and awk text editor. Additionally, paired reads were removed if each read in a pair have multiple alignment sites. PCR and optical duplicates were removed using Picard. Macs2 (v2.1.1) was used to reconstruct peaks from fragments with the following parameters --format BEDPE -B --call-summits --nolambda -p 0.01. To derive consensus peak set we (1) merged individual biological replica data sets across each condition (WT, HET, KO), (2) applied macs2 on the merged data sets and filtered out all peaks falling below the random quality threshold 200, (3) merged resulting peaks using bedtools. The raw counts in the peak regions were calculated using bedtools. Peak differential analysis was performed using DESeq2. GO-term enrichment analysis was performed using GREAT web server on a set of peaks with significant change in H3K27me3 level across conditions. A significant change was defined as |log2FC| > 0.5 and FDR < 0.05. A set of all peaks was used as a background.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include raw counts and TPM values for each RNA-seq data set
Supplementary_files_format_and_content: bigWig files generated for each ATAC-seq, H3K27me3 and H2AK119ub data sets

extract_protocol:
Cultures were washed 1X with ice-cold PBS and dissociated to single cells using Accutase (Life Technologies). CUT&RUN was performed with an anti-H3K27me3 antibody (ActiveMotif rabbit polyclonal #39155) as described in (PMID: 31232687). Briefly, exactly 250,000 cells were counted with an automated cell counter (Countess, Life Technologies), bound to Concanavalin A beads (BioMagPlus, Bangs Laboratories #BP531). The anti-H3K27me3 antibody was added (1:50 dilution) and incubated with cells under 800rpm shaking at 4OC for 2 hrs. After wash steps, a 1:200 dilution of proteinA-MNase was added and incubated for 1hr at 4OC. After washing, 100mM CaCl2 was added at 0OC and incubated for 15 mins to activate the MNase. The reaction was stopped by addition of a 10mM EDTA/EGTA Stop Buffer, incubated at 37OC for 10mins, and centrifuged at 4OC, 5000xg for 15 mins. The supernatant containing DNA was taken and purified with the DNA Clean and Concentrator Kit (Zymo). Samples were quantified on a HS DNA Qubit Fluorometer (Life Technologies) and fragment size distribution checked with a Tapestation (Agilent).
Dual-index libraries were prepared from CUT&RUN eluted DNA using the NEBNext Ultra II DNA Library Prep (E7103S) with NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, E7600S) following manufacturer instructions. Libraries were quantified by qPCR using a PhiX (Illumina) standard curve, and checked for appropriate fragment size distribution by Tapestation (Agilent). Libraries were pooled and multiplexed 1:1 and run on a HiSeq3000 (Illumina) for 2x150 paired-end sequencing (Novogene).

value: day 60 after start of iPSC differentiation to NPCs

value: ActiveMotif rabbit polyclonal #39155


title: H3K27me3 KO-1 Day_60


title: N2_COPAS_lateL4_rep1_GRO-seq

value: sorted population


title: H3K27me3 HET-2 Day_60


title: UAS-Alk.DN, Alk deactivation with twi2xPE-GAL4 promoter replicate 3

description:
GATC_counts.txt
tn_peak.txt
Sample tn3


title: UAS-Jeb, Alk activation with twi2xPE-GAL4 promoter replicate 1

description:
GATC_counts.txt
tj_peak.txt
Sample tj1


title: H3K27me3 HET-1 Day_60


title: SEQ12101, 5hmC_Seal

tag: population

tag: diagnosis

value: Plasma


title: SEQ12162, 5hmC_Seal

value: circulating cell-free DNA (cfDNA)


title: UAS-Alk.DN, Alk deactivation with twi2xPE-GAL4 promoter replicate 2

description:
GATC_counts.txt
tn_peak.txt
Sample tn2


title: H3K27me3 WT-2 Day_60

tag: developmental stage


title: H3K27me3 WT-1 Day_60


title: UAS-Alk.DN, Alk deactivation with twi2xPE-GAL4 promoter replicate 1

description:
GATC_counts.txt
tn_peak.txt
Sample tn1


title: SEQ12119, 5hmC_Seal


title: SEQ12115, 5hmC_Seal


title: Dam-PolII expression with twi2xPE-GAL4 promoter replicate 2

description:
GATC_counts.txt
Sample td2

value: Dam-PolII


title: Dam-PolII expression with twi2xPE-GAL4 promoter replicate 3

description:
GATC_counts.txt
Sample td3


title: SEQ12103, 5hmC_Seal


title: H3K27me3 KO-2 Day_40

value: day 40 after start of iPSC differentiation to NPCs


title: H3K27me3 KO-1 Day_40


title: sample_180

value: dorsolateral prefrontal cortex


title: SEQ12104, 5hmC_Seal


title: Dam-PolII expression with twi2xPE-GAL4 promoter replicate 1

description:
GATC_counts.txt
Sample td1


title: SEQ12137, 5hmC_Seal


title: H3K27me3 HET-1 Day_40


title: H3K27me3 HET-2 Day_40


title: UAS-Jeb, Alk activation with bap-GAL4 promoter replicate 2

description:
GATC_counts.txt
bj_peak.txt
Sample bj2


title: H3K27me3 WT-2 Day_40


title: UAS-Jeb, Alk activation with bap-GAL4 promoter replicate 3

description:
GATC_counts.txt
bj_peak.txt
Sample bj3


title: SEQ12118, 5hmC_Seal


title: UAS-Jeb, Alk activation with bap-GAL4 promoter replicate 1

description:
GATC_counts.txt
bj_peak.txt
Sample bj1


title: SEQ12102, 5hmC_Seal


title: H3K27me3 WT-1 Day_40


title: MDA-MB 231 Cells :Set 2 GNE-140 B

city: Tallahassee

first: Elizabeth

middle: A

last: Mazzio

state: Florida

address: 1515 MLK Blvd

department: College of Pharmacy & Pharmaceutical Science

institute: Florida Agricultural & Mechanical University

description: Gene expression data from MDA-MB-231 cells grown under variable experimental treatment

hyb_protocol: Hybrization was conducted according to manufacturer's instructions as per the GeneChipTM WT PLUS Reagent Manual for Whole Transcript (WT) Expression Arrays. (Affymetrix, ThermoFisher Scientific).

scan_protocol: Arrays were scanned with GeneAtlas Imaging station scanner  (Affymetrix, ThermoFisher Scientific)

data_processing: The data was processed using Affymetrix (Applied biosystems transcriptome analysis console software) (Affymetrix, ThermoFisher Scientific)

label: Biotin

source_name: MDA-MB-231 ATCC® HTB-26™ cells - 3-O-Acetyl-β-boswellic acid 46ug/ml

label_protocol: cDNA was labeled using according to manufacturer's instructions as per the GeneChipTM WT PLUS Reagent Manual for Whole Transcript (WT) Expression Arrays. (Affymetrix, ThermoFisher Scientific).

growth_protocol: MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle’s Medium - high glucose with 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, supplemented with 7% FBS and 100 U/ml penicillin G sodium /100 μg/ml streptomycin sulfate. Cells were grown at 37°C with 95% air and 5% CO2 and sub-cultured every three to five days.

extract_protocol: After treatment, cells were washed three times in HBSS, rapidly frozen and stored at -80 C. Total RNA was isolated/ purified using the TRIzol/chloroform method, quality was assessed and concentration was equalized to 82ng/ul in nuclease free water

treatment_protocol: GNE-140 was purchased from Millipore, Sigma (St. Louis, MO, USA). A stock solution of GNE-140 was prepared in DMSO (10mg/ml) and microarray experiments were conducted in accordance with preliminary  comprehensive data to establish the most appropriate concentration. Both controls and treatments (MDA-MB-231 cells) were grown in 75cm2 flasks , where treatments were added and at 24 hours, the cells were QC checked as having retained morphological shape, flask attachment and had no obvious signs of cell death.  At this point, cells were rapidly washed in HBSS 3x, spun and frozen at -80C.

value: mammary gland/breast; derived from metastatic site:pleural effusion

tag: cell line


title: MDA-MB 231 Cells :Set 2 GNE-140 A


title: TADA

description:
GATC_counts.txt
bn_peak.txt
Sample bn3


title: ATAC-seq KO-2 Day_60

data_processing:
The adapters were trimmed from the raw reads using trim_galore and cutadapt. The trimmed reads were aligned to GRCh38 human genome assembly using bowtie2 with  --very-sensitive -X2000 parameters. Not aligned reads and paired reads with at least one read in a pair falling below quality score threshold 10 were removed using samtools and awk text editor. Additionally, paired reads were removed if each read in a pair have multiple alignment sites. PCR and optical duplicates were removed using Picard. Tn5 insertion sites were defined as the start read position offset by +4 bp for the reads aligned to + strand and as the end read position offset by -5 bp for the reads aligned to - strand. Macs2 (v2.1.1) was used to reconstruct accessibility peaks around Tn5 insertion sites with the following parameters --shift -75 --extsize 150 --nomodel --call-summits --nolambda --keep-dup all -p 0.01. To derive consensus peak set we (1) merged individual biological replica data sets across each condition (WT, HET, KO), (2) applied macs2 on the merged data sets and filtered out all peaks falling below the random quality threshold 100, (3) merged resulting peaks using bedtools. The raw counts in the peak regions were calculated using bedtools. Peak differential analysis was performed using DESeq2. GO-term enrichment analysis was performed using GREAT web server on a set of peaks with differential accessibility, with a set of all accessibility peaks used as a background. HOMER was used to perform TF enrichment analysis in the differentially accessible peaks, defined as peaks with |log2FC| > 0.5 and FDR < 0.05. A heatmap was produced using deeptools (version 3.3.0)
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include raw counts and TPM values for each RNA-seq data set
Supplementary_files_format_and_content: bigWig files generated for each ATAC-seq, H3K27me3 and H2AK119ub data sets

extract_protocol:
Cells were washed and collected in ice-cold PBS using a cell lifter (Corning #3008), and then counted using a Countess II FL cell counter (Applied Biosystems).
Accessible chromatin from 75,000 cells per biological sample were transposed and tagged using Tn5 (Nextera DNA Sample Prep Kit from Illumina, cat# FC-121-1030), then barcoded and amplified following a published protocol (PMID: 24097267). DNA fragment sizes were determined using a TapeStation (Agilent) with High Sensitivity D1000 Screentapes (Agilent cat# 5067-5584) and showed the expected pattern for non-nucleosomal and nucleosomal sizes. Libraries were quantified by PCR using a PhiX library (Illumina cat# TG-110-3001) standard curve and then multiplexed at a 1:1 ratio. Pooled libraries were run on a HiSeq3000 (Illumina) for 2x75 paired-end sequencing at the Stanford Functional Genomics Facility.


title: MDA-MB 231 Cells :Set 2 Control B

value: control


title: SEQ12164, 5hmC_Seal

tag: molecule subtype


title: DIPG007_TP53KO_RG7388_3

city: Durham

first: Matthew

last: Waitkus

address: 203 Research Drive, Medical Science Research Building 1, Room 181A

institute: Duke University

data_processing:
mRNA seqeuncing data was aligned to Human hg38 by HISAT2
Bam files was counted by featureCounts packages to generate gene expression data.
DESeq2 was used to analyze differentially expressed features and normalized counts.
Genome_build: Human hg38
Supplementary_files_format_and_content: tabular, data matrix

source_name: Diffuse intrinsic pontine glioma

growth_protocol: DIPG007 cell line was grown in stem cell media containing half quantity of DMED/F12 and half of Neurobasal medium supplemented with Glutamax, Pytuvate, Non-essential amino acid, Hepes buffer, antibiotic/antimycotic, heparin, human EGF, human basic FDF, PDGF-AA, PDGF-BB

extract_protocol:
Three samples for each condition were harvested and RNA extractions were done using Maxwell RSC simplyRNA Cells Kit (Promega #AS1390).
mRNA library preparation (poly A enrichment) performed by novogene
NovaSeq PE150 (6 G raw data per sample ) performed by novogene

treatment_protocol: HSJD-DIPG-007-NTC and HSJD-DIPG-TP53 KO were treated with 100 nM RG7388 or DMSO for 24 h.

value: cancer cell line

tag: treatment

value: RG7388 treated

tag: edit

value: TP53 wildtype/PPM1D mutant


title: MDA-MB 231 Cells :Set 1 GNE-140 B

source_name: MDA-MB-231 ATCC® HTB-26™ cells - Boswellia Serrata Extract 128 ug/ml


title: MDA-MB 231 Cells :Set 1 Control B

source_name: MDA-MB-231 ATCC® HTB-26™ cells - No treatment


title: MDA-MB 231 Cells :Set 2 Control A


title: MDA-MB 231 Cells :Set 1 Control A


title: MDA-MB 231 Cells :Set 1 GNE-140 A


title: MDA-MB 231 Cells :Set 1 Control C


title: ATAC-seq KO-1 Day_60

tag: cell type


title: UAS-Alk.DN, Alk deactivation with bap-GAL4 promoter replicate 2

description:
GATC_counts.txt
bn_peak.txt
Sample bn2


title: DIPG007_TP53KO_RG7388_1


title: SEQ12121, 5hmC_Seal

value: Chinese

value: DR


title: ATAC-seq HET-2 Day_60

value: neural progenitor cells (NPCs) differentiated from iPSC line derived from a patient with Coffin-Siris syndrome


title: DIPG007_TP53KO_DMSO_3

value: DMSO control


title: SEQ12155, 5hmC_Seal

value: Non-DR


title: UAS-Alk.DN, Alk deactivation with bap-GAL4 promoter replicate 1

description:
GATC_counts.txt
bn_peak.txt
Sample bn1


title: Dam-PolII expression with bap-GAL4 promoter replicate 3

description:
GATC_counts.txt
Sample bd3


title: ATAC-seq HET-1 Day_60

value: day 60 after start of iPSC differentiation to NPCs


title: SEQ12116, 5hmC_Seal


title: SEQ12201, 5hmC_Seal


title: DIPG007_TP53KO_DMSO_2


title: ATAC-seq WT-2 Day_60

value: wild type


title: DIPG007_TP53KO_RG7388_2


title: DIPG007_TP53KO_DMSO_1


title: Dam-PolII expression with bap-GAL4 promoter replicate 2

description:
GATC_counts.txt
Sample bd2


title: Dam-PolII expression with bap-GAL4 promoter replicate 1

description:
GATC_counts.txt
Sample bd1


title: ATAC-seq WT-1 Day_60


title: DIPG007_NTC_RG7388_3


title: SEQ12196, 5hmC_Seal

value: Plasma


title: SEQ12199, 5hmC_Seal

value: circulating cell-free DNA (cfDNA)


title: SEQ12182, 5hmC_Seal


title: SEQ12179, 5hmC_Seal


title: DIPG007_NTC_RG7388_2


title: CT26-scr 3

city: Taichung

first: Yu-Ting

last: Yen

address: No.91, Hsueh-Shih Road

department: Graduate Institute of Biomedical Sciences

country: Taiwan

institute: China Medical University

data_processing:
Library strategy: TCR-seq
Libraries were pooled to a final pool concentration of 4 nM. Pooled libraries were then diluted to a final concentration of 13.5 pM, including a 10% PhiX Control v3 (Illumina, Cat. No. FC-110-3001) spike-in.
Libraries were sequenced on an Illumina MiSeq® sequencer using the 600-cycle MiSeq Reagent Kit v3 (Illumina, Cat. No. MS-102-3003) with paired-end, 2 x 300 base pair reads.
Data analysis was performed using MiXCR.

source_name: peripheral blood sample

extract_protocol:
SMARTer Mouse TCR a/b Profiling Kit-obtain full-length sequences of TCR-alpha and TCR-beta V(D)J variable regions (TaKaRa)
Library preparation and PCR strategy for TCR profiling using the SMARTer based on protocol of TaKaRa

treatment_protocol: TCR rearrangements identified in peripheral blood from mice injected with the indicated CT26 clones

tag: strain

value: BALB/c

value: ctrl

value: peripheral blood


title: ATAC-seq HET-2 Day_40

value: day 40 after start of iPSC differentiation to NPCs


title: ATAC-seq KO-1 Day_40


title: ATAC-seq KO-2 Day_40


title: DIPG007_NTC_DMSO_3


title: CT26-scr 2

growth_protocol: mice injected with the indicated CT26 clones

tag: tissue


title: DIPG007_NTC_DMSO_2


title: ATAC-seq HET-1 Day_40


title: ATAC-seq WT-2 Day_40


title: AEG2

city: Parkville

first: Michael

middle: James

last: Haydon

state: VIC

address: BioSciences 4, Royal Parade

country: Australia

institute: University of Melbourne

data_processing:
Image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument.
SOAPnuke v2.1.0 (Chen et al., 2018) was used to remove adapter sequences and low-quality reads from the sequencing data.
HiSAT2 v2.2.1 (Kim et al., 2015) to align the sequencing reads to the Arabidopsis thaliana Col-0 genome (TAIR10).
RSEM v1.2.22 (Li & Dewey, 2011) to quantify the gene expression level of each replicate.
Genome_build: TAIR10
Supplementary_files_format_and_content: TAIR_gene_expression.txt: Abundance measurements matrix.

organism: Arabidopsis thaliana

source_name: 14 d old seedlings

growth_protocol: 12 h light/12 h dark, 14 d on 1/2MS media.

extract_protocol:
Total RNA extracted with ISOLATE II RNA Plant Kit (Meridian Bioscience).
NEBNext® UltraTM RNA Library Prep Kit for Illumina® and NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~360 bp (with the approximate insert size of 300 bp) were recovered. Each sample was then amplified by PCR for 11 cycles using P5 and P7 primers. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer, and quantified by Qubit 2.0 Fluorometer (Invitrogen). Libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument and sequenced using a 2x150bp paired-end (PE) configuration.

treatment_protocol: 1/2MS media containing DMSO, 10 µM DPI, 25 µM pentamidine isethionate, 12.5 µM AEG3482 or 2 µM Tyrphostin AG879.

tag: ecotype

value: Seedlings

tag: age

value: 14 days

value: AEG

tag: time point


title: CT26-scr 1

tag: genotype


title: TYR3

value: TYR


title: DIPG007_NTC_DMSO_1

value: cancer cell line

value: TP53 wildtype/PPM1D mutant


title: TYR2


title: ATAC-seq WT-1 Day_40

value: neural progenitor cells (NPCs) differentiated from iPSC line derived from a patient with Coffin-Siris syndrome


title: AEG1


title: TYR1


title: AEG3


title: CT26-shppp2r1a 2

value: knockdown ppp2r1a


title: CT26-shppp2r1a 3


title: PI3

value: PI


title: RNA-seq KO-2 Day_60

data_processing:
Raw reads were pseudoaligned to GENCODE v33 human transcriptome assembly and transcript abundance was quantified using kallisto (version 0.44.0). Transcript abundance files from kallisto output were imported to R using tximport. DESeq2 was used for analysis of differential gene expression. For visualization and gene ranking we used effect size shrinkage for Log2FC estimates as encoded by ashr() function. Enrichr was used for GO-term analysis of differentially expressed genes, defined as genes with |log2FC| > 0.5 and FDR < 0.05. PANTHER Pathway analysis was performed using PANTHER.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include raw counts and TPM values for each RNA-seq data set
Supplementary_files_format_and_content: bigWig files generated for each ATAC-seq, H3K27me3 and H2AK119ub data sets

extract_protocol:
Cultures were washed 1X with ice-cold PBS and total RNA was extracted with Trisure (Bioline), purified and DNAse-treated using the Direct-zol RNA kit (Zymo).
Libraries were prepared from 500ng RNA with dual-index unique barcodes using the SMARTer Stranded Total RNA Sample Prep HI Kit (Clontech), quantified by qPCR using a PhiX (Illumina) standard curve, and checked for appropriate fragment size distribution by Tapestation (Agilent). Libraries were pooled and multiplexed 1:1 and run on a HiSeq3000 (Illumina) for 2x150 paired-end sequencing (Novogene) with a 20% PhiX spike-in.

value: day 60 after start of iPSC differentiation to NPCs


title: PI2


title: 1824 library, metastatic rumor

city: Washington

first: Yanxin

last: Pei

state: DC

address: 111 Michigan Ave NW

department: Center for Cancer and Immunology

institute: Children's National Medical Center

data_processing:
 all alignment and quantification was done using cell ranger version v6.2.1
Genome_build: GrCh38 human genome
Supplementary_files_format_and_content: Generic data matrices in csv format with cell

source_name: patient-derived xenograft MB002

growth_protocol: Tumor cells were transplanted into the cerebella of R2G2 mice and monitored for growth weekly by bioluminescent imaging with an IVIS imager.

extract_protocol:
After nanoliter-scale Gel Beads-in-emulsion (GEM) generation and barcoding, GEMs are broken and pooled fractions are recovered. Silane magnetic beads are used to purify the first-strand cDNA from the post GEM-RT reaction mixture (please refer to the USER GUIDE of Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index)).
Libraries were prepared according to 10X genomics' instructions accompanying the Chromium Next GEM Single Cell 3’ Reagent Kits V3.1(Dual Index) (Part# 0801-0303). Briefly, around 10000 cells were partitioned into nanoliter-scale Gel Beads-in-emulsion (GEM) by combining barcoded Single Cell 3’ V3.1 Gel Beads, a Master Mix containing cells, and Partitioning Oil onto Chromium Next GEM Chip G. Immediately following GEM generation, full-length cDNA was reverse-transcripted from poly-adenylated mRNA and barcoded. The barcoded, full-length cDNA was amplified via PCR to generated mass for library construction. After enzymatic fragmentation and size selection, P5, P7, i5 sample index, and TruSeq Read 2 (read 2 primer sequence) are added into cDNA. Finally a Chromium Single Cell 3’ Gene Expression Dual Index library comprises standard Illumina paired-end constructs which begin and end with P5 AND P7.
3' Gene Expression Dual Index Library Construction using 10X Genomics Library Construction Kit (PN-1000196).

treatment_protocol: Once tumors reached a luminescence signal of 10^7, animals in the treatment group were administered radiotherapy with a RS 2000 Biological System Irradiator.  Animals were treated with 2Gy per dose at a rate of approximately 120 rads/minute every other day for a total cumulative dose of 18Gy.  Residual tumor cells were collected 24 hours after the last time of irradiation. For recurrent tumors, animals were maintained following radiotherapy until they relapsed.

value: PDX MB002 live cells

tag: host

value: Rag2-Il2rg double Knockout mice

value: radiation for 18 Gy, metastatic tumor cells from forebrain and skull


title: 1669 library, metastatic tumor

value: radiation for 18 Gy, metastatic tumor cells from forebrain


title: CT26-shppp2r1a 1

value: BALB/c


title: RNA-seq HET-2 Day_60


title: RNA-seq KO-1 Day_60


title: Sample 22_p167 NGS

description: Pig-Kellyanne.integrations.txt

data_processing:
RNA-seq:
The mRNA-seq data were aligned with STAR version 2.5.2b to the Sscrofa11.1 assembly of the pig genome.  Raw gene expression was quantified with the featureCounts software.  Normalization and differential expression analyses were performed using R version 3.5.3 with the edgeR version 3.24.3 and limma version 3.38.3 packages.  Network and functional term enrichment analyses were processed through Qiagen's Ingenuity application on the identified differentially expressed genes.  Heat maps visualizing the z-scores of the relevant genes' normalized expression values were created using the clustvis web tool.
After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate p-values and Log2 fold changes. Genes with adjusted p-values < 0.05 and absolute log2 fold changes > 1 were called as differentially expressed genes for each comparison. A gene ontology analysis was performed on the statistically significant set of genes by implementing the software GeneSCF. The mgi GO list was used to cluster the set of genes based on their biological process and determine their statistical significance. A PCA analysis was performed using the "plotPCA" function within the DESeq2 R package. The plot shows the samples in a 2D plane spanned by their first two principal components. The top 500 genes, selected by highest row variance, were used to generate the plot.
NGS DNA:
Quality control of the sequenced read pairs was performed using the FASTQC software. The average base quality of the sequenced reads was >Q30 on the Phred scale. Uniform distribution of A,G,T and C nucleotides was seen across the length of the reads without bias for any specific bases. The number of unknown “N” bases was less than 1% across the length of the reads. High sequence duplication (> 80%) was observed, however, this was anticipated due to the nature of the experiment and the amplification of specific library fragments.
Reads were trimmed to remove the viral sequence in two separate steps. In step 1, the viral sequence was trimmed from the R1 reads using cutadapt84 with a mismatch rate (e=0.3) from the 5’ end of each read. In step 2, if the linker sequence was present, it was similarly trimmed from the 3’end of the read. Trimmed reads with a length less than 15bps were removed from the rest of the analysis to reduce ambiguous alignments. Untrimmed reads were also removed from the rest of the analysis because they did not contain a viral segment that could be used as evidence of a viral mediated integration.
The remaining R1 reads were aligned to the susScr11 build of the pig reference genome using BWA-MEM in single-end mode. Default BWA-MEM parameters were used. The reads used to identify genomic points of viral integrations had to be uniquely mapped to the genome with a BWA-MEM mapping quality score greater than zero.
An integration point was defined by the position of R1’s first aligned base on the susScr1 genome. Unique integration points were identified across the genome without any constraints on coverage. However, for downstream annotation and analysis, only those integration points with 5 or more supporting reads were used to minimize calling false positive integration points.
Locations of integration points were categorized into: exons, introns, 3 prime UTRs, 5 prime UTRs and intergenic regions using information extracted from the susScr11 refflat file maintained by UCSC’s Genomics Institute. To avoid conflict of feature categorization arising from multiple overlapping gene definitions, only the definition of the longest gene was used to annotate integration points. Additional features where computed including: the distance to the nearest transcription start site (TSS), makeup of CpG-rich regions of the genome (“CpG islands”), and enrichment to tumor-associated genes. This tumor gene list was comprised of 745 clinically validated tumor-associated human genes (from Mayo Clinic internal data). Those genes were related to their pig homologs based on GenBank’s gene names.
In order to look for selective enrichment of any specific biological pathways, the genes identified as integration sites were entered into the WEB-based GEne SeT AnaLysis Toolkit. Results were identified using the over-representation analysis (ORA) method with identified KEGG pathways. A false discovery rate (FDR) of <0.05 was considered significant.
Genome_build: NGS DNA: susScr11; RNA-seq: Sscrofa11.1
Supplementary_files_format_and_content: "bed" (.txt); Rawcounts

source_name: lung

value: parenchyma

value: Fah -/-

value: LV-FAH


title: PI1

value: Seedlings

value: 14 days


title: DIPG007_NTC_RG7388_1

value: RG7388 treated


title: DPI3

value: DPI


title: 1637 library, residual tumor

value: radiation for 18 Gy


title: Sample 21_p167 NGS

description: Pig-Kellyanne.integrations.txt

source_name: lymph node


title: 1634 library, residual tumor


title: DPI1


title: RNA-seq WT-1 Day_60

value: neural progenitor cells (NPCs) differentiated from iPSC line derived from a patient with Coffin-Siris syndrome

value: wild type


title: 1636 library, recurrent tumor

value: radiation for 18 Gy, then tumor relapsed


title: Sample 20_p166 NGS

description: Pig-Mike.integrations.txt


title: DPI2


title: RNA-seq WT-2 Day_60

value: day 60 after start of iPSC differentiation to NPCs


title: 1668 library, metastatic tumor

value: PDX MB002 live cells


title: RNA-seq KO-2 Day_40

value: day 40 after start of iPSC differentiation to NPCs


title: DMSO3

value: DMSO


title: 1626 library, recurrent tumor

value: Rag2-Il2rg double Knockout mice


title: Sample 19_p162 NGS

description: Pig-Donald.integrations.txt


title: 1554 library, recurrent tumor


title: N2_COPAS_earlyL4_rep3_GRO-seq

first: Germano

last: Cecere

address: Institut Pasteur, 28 Rue Du Docteur Roux, Batiment Monod, 4eme Etage

institute: Institut Pasteur

value: N2 - C. elegans wild isolate (Bristol)

value: whole worm

value: early L4

value: sorted population

tag: rna population

value: biotinylated RNA


title: DMSO1

value: Seedlings

value: 14 days


title: RNA-seq KO-1 Day_40


title: DMSO2


title: 1632 library, primary tumor

value: untreated


title: Sample 16_p166 NGS

description: Pig-Mike.integrations.txt

value: parenchyma

value: Fah -/-

value: LV-FAH


title: RNA-seq HET-1 Day_40

value: neural progenitor cells (NPCs) differentiated from iPSC line derived from a patient with Coffin-Siris syndrome


title: Sample 15_p167 NGS

description: Pig-Kellyanne.integrations.txt


title: RNA-seq HET-1 Day_60

value: day 60 after start of iPSC differentiation to NPCs


title: 1631 library, primary tumor

value: PDX MB002 live cells


title: RNA-seq HET-2 Day_40

value: day 40 after start of iPSC differentiation to NPCs


title: 1635 library, residual tumor

value: radiation for 18 Gy


title: NT3

value: Untreated


title: RNA-seq WT-2 Day_40

value: wild type


title: Sample 14_p162 NGS

description: Pig-Donald.integrations.txt


title: RNA-seq WT-1 Day_40


title: NT1


title: CMK9_MS009_051

status: Public on Nov 19 2021

city: Kyoto

first: Yukihiro

last: Yabuta

state: Kyoto

address: Yoshida-Konoe-cho, Sakyo-ku

department: Anatomy and Cell Biology

country: Japan

institute: Kyoto University, Graduate school of medicine

description: Single cell transcriptome of amplified cDNA from cynomolgus monkey ESC CMK9.

data_processing:
All the reads were surveyed and the adaptor or the poly-A sequences were trimmed by cutadapt-1.3 with options "-e 0.1 -q 20 -n 2 -O 1 -m 30 -a CTCGAGGGCGCGCCGGATCC -g CTCGAGGGCGCGCCGGATCC -a AAAAAAAAAAAAAAAAAAAA -a TTTTTTTTTTTTTTTTTTTT". The trimmed reads with less than 30 bp were discarded.
Untrimmed and trimmed reads of 30 bp or longer were mapped onto the Cynomolgus monkey genome Macfas5.0 and the ERCC spike-in RNA sequences with tophat-1.4.1/bowtie1.0.1 with the “—no-coverage-search” option.
Mapped reads on the genome and the ERCC were separated, and the reads on the genome were converted into the expression levels (RPM) by cufflinks-2.2.0 using the “—compatible-hits-norm”, “—max-mle-iterations 50000", “—no-length-correction” and “—library-type fr-secondstrand” options and macfas5.0 reference gene annotations with extended TTSs.
Mapped reads were also processed with HTSeq v0.11.0  to calculate number of read counts per gene.
For the reference gene annotations used in cufflinks and HTSeq, we extended the TTSs of the reference genes up to 10 kb downstream to correctly estimate the expression levels of genes whose transcripts are longer than the reference toward the 3 prime.
Genome_build: Macaca fascicularis 5.0 (macFas5)
Supplementary_files_format_and_content: *_rpm.txt: tab-delimited text files include RPM or raw count values for each Sample.

organism: Macaca fascicularis

source_name: Cynomolgus monkey ESC CMK9

growth_protocol: For cultivation of CMK6 and CMK9 cyESCs, they were cultured with conventional hESC medium [DMEM/F12 supplemented with 20% (vol/vol) of KSR, 1 mM of sodium pyruvate, 2 mM of GlutaMax, 0.1 mM of non-essential amino acids, 0.1 mM of 2-mercaptoethanol, 1000 U/ml of ESGRO mouse LIF, and 4 ng/ml of recombinant human bFGF] on mouse embryonic feeders (MEFs).

extract_protocol: For SC3-seq analysis, cDNA synthesis / amplification from single cells and library construction from the cDNAs were performed as described previously [Nakamura et al. 2015, NAR, 43, e60; Ishikura et al. 2016, Cell Reports, 17, 2789].

value: Undifferentiated embryonic stem cell

tag: sequencer

value: SOLiD, 50bp, ECC


title: CMK9_MS009_052


title: CMK9_MS009_050


title: CMK9_MS009_048


title: CMK9_MS009_045


title: CMK9_MS009_046


title: E08_MS273_221

description: Single cell transcriptome of amplified cDNA from cynomolgus monkey embryo at E08

data_processing:
All the reads were surveyed and the adaptor or the poly-A sequences were trimmed by cutadapt-1.3 with options "-e 0.1 -q 20 -n 2 -O 1 -m 30 -a CTCGAGGGCGCGCCGGATCC -g CTCGAGGGCGCGCCGGATCC -a AAAAAAAAAAAAAAAAAAAA -a TTTTTTTTTTTTTTTTTTTT". The trimmed reads with less than 30 bp were discarded.
Untrimmed and trimmed reads of 30 bp or longer were mapped onto the Cynomolgus monkey genome Macfac5.0 and the ERCC spike-in RNA sequences with tophat-1.4.1/bowtie1.0.1 with the “—no-coverage-search” option.
Mapped reads on the genome and the ERCC were separated, and the reads on the genome were converted into the expression levels by cufflinks-2.2.0 using the “—compatible-hits-norm”,  “—max-mle-iterations 50000", “—no-length-correction” and “—library-type fr-secondstrand” options and macfas5.0 reference gene annotations with extended TTSs. For the reference gene annotations used in cufflinks, we extended the TTSs of the reference genes up to 10 kb downstream to correctly estimate the expression levels of genes whose transcripts are longer than the reference toward the 3 prime.
Genome_build: Macaca fascicu;aris 5.0
Supplementary_files_format_and_content: tab-delimited text files *rpm.txt include RPM values (cufflinks) or *count.txt include Read counts (HTSeq) for each Sample

source_name: Cynomolgus monkey embryo E08

value: embryo

value: Nextseq550, High, 75 cycle


title: CMK9_MS009_047


title: 1628 library, primary tumor

value: Rag2-Il2rg double Knockout mice


title: E08_MS273_209