title: BIOCHIP_rep3

status: Public on Dec 01 2020

city: Tokyo

last: Poulain

address: Fw-203, 4-6-1, Komaba, Meguro-ku

department: Institute of Industrial Science

institute: The University of Tokyo

data_processing:
Basecalls performed using CASAVA.
Demultiplexing, filtering, mapping and molecule assembly were performed with the MOIRAI pipeline OP-WORKFLOW-CAGEscan-short-reads-v2.1 (Hasegawa et al., BMC Bioinformatics, 2014. doi: 10.1186/1471-2105-15-144).
Conversion from single pairs in BAM format to single pairs in BED12 format was done with https://github.com/Population-Transcriptomics/pairedBamToBed12
Assembly of single pairs in BED12 format to single molecules in BED12 format was done with https://github.com/mmendez12/umicount
CAGE peaks were called and gene expression tables generated using the CAGEr package (Haberle et al., Nucleic Acids Research, 2015. doi: 10.1093/nar/gkv054).
Genome_build: hg38
Supplementary_files_format_and_content: BED12 files, one RNA molecule per line; the score indicates how many read pairs were used to assemble the molecule.

source_name: induced Pluripotent Stem Cells (iPSC)

growth_protocol: Human iPSCs were cultured in biochips according to the protocol described by Danoy et al., Biotechnol Bioeng., 2019 (doi: 10.1002/bit.26970) using a new biochip design and applying an extended oncostatin stimulation period. Petri dishes cultures were performed following the protocol described by Si-Tayeb et al., Hepatology, 2010 (doi: 10.1002/hep.23354).

extract_protocol:
Total RNA was harvested using Trizol reagent. 250ng of total RNA from each sample were used for the construction of the sequencing libraries.
NanoCAGE sequencing libraries were prepared according to the protocol described by Poulain et al., Methods in Molecular Biology, 2017 (doi: 10.1007/978-1-4939-6716-2_4).

tag: cell type

value: induced pluripotent stem cells (hiPSCs) differentiated into hepatocyte-like cells (HLCs)

tag: cell culture type

value: Biochip

tag: cell differentation stage

value: Hepato-Like Cells (HLC)

tag: nanocage template-switching oligo barcode sequence

value: GCTCTC

tag: nanocage template-switching oligo barcode number


title: BIOCHIP_rep2

type: SRA

first: Stéphane

country: Japan

library_source: transcriptomic

molecule: total RNA

organism: Homo sapiens

value: GAGATA


title: BIOCHIP_rep4

value: GTATGA


title: P128T-E

status: Public on Oct 29 2020

city: Beijing

first: Xiannian

last: Zhang

address: 10 YouAnMen Xitoutiao, Fengtai District

department: Department of neurology

country: China

institute: Captital Medical University

description: Raw data is being made available at https://bigd.big.ac.cn/gsa-human/ under the accession ID of HRA000195

data_processing:
We processed the scRNA-seq data using Cell Ranger Single-Cell Software Suite (10x Genomics, version 2.1.0) with default parameters, aligned to the GRCh38 reference genome and the raw gene expression matrices were generated for each sample.
The expression matrixes of all samples were combined into CD45+ and CD45- tables. The combined tables were imported into the Seurat (version 2.3.4) toolkit for quality filtering and downstream analysis. For quality filtering, we first removed genes that were detected in < 0.1% of all cells and then filtered out cells with less than 500 detected genes or > 20% mitochondrial RNA content.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include UMI counts  for each gene and each cell barcode (CD45xxx_UMIs.txt.gz)
Supplementary_files_format_and_content: tab-delimited text files including cell name, sample and annotated cell type for each cell (cellinfo.CD45xxx.txt)

source_name: Tumor tissue of esophageal carcinoma

extract_protocol: Fresh ESCC tumors and adjacent normal tissues were rinsed with PBS, gently cut into small pieces on ice and digested in RPMI-1640 medium (Invitrogen) containing 2 mg/ml collagenase IV (Gibco) and 0.5 mg/ml hyaluronidase (Sigma Aldrich) for 1 hour at 37oC. The digested cell suspension was subsequently filtered through a 70-μm cell strainer (BD Biosciences) and incubated in 1x red blood cell lysis buffer (BD Biosciences) on ice for 5 min. The remaining cells were suspended in 50 μl of PBS containing 1% FBS after washing once with the same medium. Single-cell suspension was stained with CD45-FITC (BD Biosciences) and sorted into immune (CD45+) or non-immune (CD45-) cells using a FACSAria flow cytometer (BD Biosciences). Sorted cells were examined and counted before subject to 10x Genomics Chips. We targeted for 7,000 cells recovered from each channel and used Chromium Single Cell 5’ Reagent Kits (10x Genomics) to prepared whole transcriptome RNA-sequencing libraries.

treatment_protocol: Fresh ESCC tumors and their adjacent normal tissue samples were placed in RPMI-1640 medium (Invitrogen) with 20% fetal bovine serum (FBS; GE Healthcare Life Sciences) on ice immediately after surgical resection. Tissue sample processing was completed in 10 hours after collection. A portion of sample was cryosectioned, hematoxylin-eosin (H&E) stained and microscopically examined to assure that tumor sample contains > 40% cancer cells and normal tissue contains no cancer cells.

tag: patient

tag: tissue

value: esophageal carcinoma

tag: cell population

tag: info

value: 10X 5' scRNA-Seq for CD45- cells from P128 TUMOR


title: P127T-E

value: 10X 5' scRNA-Seq for CD45- cells from P127 TUMOR


title: P126N-E

source_name: Tumor Normal Adjacent tissue removed from esophageal carcinoma

value: adjacent normal

value: 10X 5' scRNA-Seq for CD45- cells from P126 Normal Tissue


title: P127N-E

value: 10X 5' scRNA-Seq for CD45- cells from P127 Normal Tissue


title: P91T-E

value: 10X 5' scRNA-Seq for CD45- cells from P91 TUMOR


title: RF_pCF10_0

status: Public on Dec 24 2020

city: Minneapolis

first: Julia

last: Willett

state: MN

address: 689 23rd Avenue SE, 4-149 Micro Research Facility

country: USA

institute: University of Minnesota

data_processing:
Sequenced reads were trimmed to remove adapter sequences and low-quality bases using the sliding-window option in Trimmomatic
RPKM and Expression values were calculated using Rockhopper
Genome_build: NC_006827
Supplementary_files_format_and_content: wig

organism: Enterococcus faecalis

source_name: total bacterial cellular RNA

growth_protocol: Overnight bacterial cultures of OG1RF(pCF10) and OG1RF(pCF10ΔprgU) were diluted 1:20 in BHI media and grown for 1 h at 37 °C.

extract_protocol:
Total RNA was extracted using a Qiagen RNeasy kit.  Ribosomal RNA was depleted using a MicrobeExpress kit.  All RNA samples were treated with Turbo DNase (ThermoFisher, rigorous method described by the manufacturer) and submitted to the University of Minnesota Genomics Center for Illumina library prep.
Whole-cell RNA samples were prepared using a non-stranded TruSeq RNAv2 kit, and libraries were size selected to generate inserts of 200 bp. All samples were sequenced as paired-end reads on an Illumina HiSeq 2500 in high output mode (125 bp read length for whole-cell samples).

treatment_protocol: Cells were then induced with 10 ng/mL cCF10 followed by incubation for 1 h at 37 °C.

tag: strain

value: OG1RF pCF10

tag: pheromone


title: P130T-E

value: 10X 5' scRNA-Seq for CD45- cells from P130 TUMOR


title: P107T-E

value: 10X 5' scRNA-Seq for CD45- cells from P107 TUMOR


title: P104T-E

value: 10X 5' scRNA-Seq for CD45- cells from P104 TUMOR


title: P94T-E

value: 10X 5' scRNA-Seq for CD45- cells from P94 TUMOR


title: P126T-E

value: 10X 5' scRNA-Seq for CD45- cells from P126 TUMOR


title: P87T-E

value: 10X 5' scRNA-Seq for CD45- cells from P87 TUMOR


title: P89T-E

value: 10X 5' scRNA-Seq for CD45- cells from P89 TUMOR


title: IIH41540: patient with idiopathic intracranial hypertension (IIH), patient#IIH41540

status: Public on Dec 11 2020

city: Muenster

first: I-Na

last: Lu

state: NRW

address: Domagkstraße 20

department: Department of Pediatric Hematology and Oncology

country: Germany

institute: University Children's Hospital Münster

description: single-cell RNA seq data of a patient with idiopathic intracranial hypertension (IIH), patient#IIH41540 (previously uploaded in GSE138266)

data_processing:
Raw bcl files were de-multiplexed using the cellranger mkfastq pipeline (cellranger v3.1.0)
Subsequent reads alignments and transcript counting was done individually for each sample using the cellranger count pipeline with standard parameters
 The cellranger aggr pipeline was employed to aggregate samples with mapping normalization to leave each sample with similar sequencing depth per cell.
Genome_build: GRCh38
Supplementary_files_format_and_content: Standard cellranger output format

source_name: CSF

extract_protocol:
CSF samples were collected into round bottom polypropylene tubes and then centrifuged for 10 min at 300 x g. The CSF supernatant was removed and CSF cells were resuspended in 5 ml of X-Vivo15 media (Lonza) and stored at 4°C. The samples were then transported to the laboratory of the Department of Neurology with Institute of Translational Neurology at the University Clinic Münster, Germany for scRNA-seq. Transport time of samples collected in Münster was 10-30 minutes. Transport time of samples collected in Essen was < 60 minutes. Cell suspensions were then centrifuged again for 5 min at 400 x g and then resuspended in 40 μl of media. Out of this volume, 6 μl were used for cell counting and the remaining volume was entirely used for scRNA-seq.
Four Neuro-COVID samples were processed with the Chromium Single Cell 5’ Library & Gel Bead Kit and barcoded, full-length VDJ segments from T cells were enriched from first-strand cDNA via PCR amplification with the Chromium Single Cell VDJ Enrichment Kit, Human T Cell (from 10X Genomics) following the manufacturer’s instructions. Further library preparation was performed using the Chromium Single Cell 5' Library Construction Kit (10X Genomics). Three Neuro-COVID samples were processed with the Chromium Single Cell 3’ Library & Gel Bead Kit and we developed a novel method to sequence antigen receptor information  from 3’ scRNA-seq libraries. The method allows removing the constant region of antigen receptors during enrichment, while maintaining their cell barcode and unique molecular identifier (UMI) information attached to the 3' of the cDNA molecules. In summary, the method involves self-circulating the cDNA library, enriching the VDJ region and re-linearizing. Each step maintains the VDJ region together with the cell barcode and UMI in the same molecule. Sample processing and library preparation was performed according to manufacturer instructions using AMPure XP beads (Beckman Coulter). Sequencing was either carried out on a local Illumina Nextseq 500 using the High-Out 75 cycle kit with a 26-8-0-57 read setup or commercially (Microanaly, China) on a NovaSeq 6000 using the 300 cycle kit with paired end 150 read setup. All the samples were sequenced with a sequencing depth >50,000 reads per cell.

value: CSF-derived leukocytes

tag: diagnosis

value: IIH

tag: type of library

value: RNA expression

tag: subject id


title: P84T-E

value: esophageal carcinoma

value: 10X 5' scRNA-Seq for CD45- cells from P84 TUMOR


title: P83T-E

value: 10X 5' scRNA-Seq for CD45- cells from P83 TUMOR


title: P82T-E

value: 10X 5' scRNA-Seq for CD45- cells from P82 TUMOR


title: P130N-E

value: adjacent normal

value: 10X 5' scRNA-Seq for CD45- cells from P130 Normal Tissue


title: P79T-E

value: 10X 5' scRNA-Seq for CD45- cells from P79 TUMOR


title: BIOCHIP_rep1

value: induced pluripotent stem cells (hiPSCs) differentiated into hepatocyte-like cells (HLCs)

value: Biochip

value: Hepato-Like Cells (HLC)

value: CGATGA


title: P128N-E

value: 10X 5' scRNA-Seq for CD45- cells from P128 Normal Tissue


title: P76T-E

value: 10X 5' scRNA-Seq for CD45- cells from P76 TUMOR


title: P80T-E

value: 10X 5' scRNA-Seq for CD45- cells from P80 TUMOR


title: P75T-E

value: 10X 5' scRNA-Seq for CD45- cells from P75 TUMOR


title: P65T-E

value: 10X 5' scRNA-Seq for CD45- cells from P65 TUMOR


title: P63T-E

value: 10X 5' scRNA-Seq for CD45- cells from P63 TUMOR


title: P62T-E

value: esophageal carcinoma

value: 10X 5' scRNA-Seq for CD45- cells from P62 TUMOR


title: P52T-E

value: 10X 5' scRNA-Seq for CD45- cells from P52 TUMOR


title: P56T-E

value: 10X 5' scRNA-Seq for CD45- cells from P56 TUMOR


title: P61T-E

value: 10X 5' scRNA-Seq for CD45- cells from P61 TUMOR


title: P47T-E

value: 10X 5' scRNA-Seq for CD45- cells from P47 TUMOR


title: P49T-E

value: 10X 5' scRNA-Seq for CD45- cells from P49 TUMOR


title: P36T-E

value: 10X 5' scRNA-Seq for CD45- cells from P36 TUMOR


title: P39T-E

value: 10X 5' scRNA-Seq for CD45- cells from P39 TUMOR


title: P54T-E

value: 10X 5' scRNA-Seq for CD45- cells from P54 TUMOR


title: P42T-E

value: 10X 5' scRNA-Seq for CD45- cells from P42 TUMOR


title: P31T-E

value: 10X 5' scRNA-Seq for CD45- cells from P31 TUMOR


title: P48T-E

value: 10X 5' scRNA-Seq for CD45- cells from P48 TUMOR


title: P38T-E

value: 10X 5' scRNA-Seq for CD45- cells from P38 TUMOR


title: P74T-E

value: 10X 5' scRNA-Seq for CD45- cells from P74 TUMOR


title: P32T-E

value: 10X 5' scRNA-Seq for CD45- cells from P32 TUMOR


title: P23T-E

value: 10X 5' scRNA-Seq for CD45- cells from P23 TUMOR


title: P37T-E

value: esophageal carcinoma

value: 10X 5' scRNA-Seq for CD45- cells from P37 TUMOR


title: P19T-E

value: 10X 5' scRNA-Seq for CD45- cells from P19 TUMOR


title: P20T-E

value: 10X 5' scRNA-Seq for CD45- cells from P20 TUMOR


title: P24T-E

value: 10X 5' scRNA-Seq for CD45- cells from P24 TUMOR


title: P22T-E

value: 10X 5' scRNA-Seq for CD45- cells from P22 TUMOR


title: P16T-E

value: 10X 5' scRNA-Seq for CD45- cells from P16 TUMOR


title: P28T-E

value: 10X 5' scRNA-Seq for CD45- cells from P28 TUMOR


title: P30T-E

value: 10X 5' scRNA-Seq for CD45- cells from P30 TUMOR


title: P26T-E

value: 10X 5' scRNA-Seq for CD45- cells from P26 TUMOR


title: P57T-E

value: 10X 5' scRNA-Seq for CD45- cells from P57 TUMOR


title: P27T-E

value: 10X 5' scRNA-Seq for CD45- cells from P27 TUMOR


title: P17T-E

value: 10X 5' scRNA-Seq for CD45- cells from P17 TUMOR


title: P21T-E

value: 10X 5' scRNA-Seq for CD45- cells from P21 TUMOR


title: P12T-E

value: 10X 5' scRNA-Seq for CD45- cells from P12 TUMOR


title: P15T-E

value: 10X 5' scRNA-Seq for CD45- cells from P15 TUMOR


title: P40T-E

value: 10X 5' scRNA-Seq for CD45- cells from P40 TUMOR


title: P9T-E

value: esophageal carcinoma

value: 10X 5' scRNA-Seq for CD45- cells from P9 TUMOR


title: P4T-E

value: 10X 5' scRNA-Seq for CD45- cells from P4 TUMOR


title: P10T-E

value: 10X 5' scRNA-Seq for CD45- cells from P10 TUMOR


title: P5T-E

value: 10X 5' scRNA-Seq for CD45- cells from P5 TUMOR


title: P1T-E

value: 10X 5' scRNA-Seq for CD45- cells from P1 TUMOR


title: P2T-E

value: 10X 5' scRNA-Seq for CD45- cells from P2 TUMOR


title: P128N-I

value: adjacent normal

value: 10X 5' scRNA-Seq for CD45+ cells from P128 Normal Tissue


title: P130T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P130 TUMOR


title: P126N-I

value: 10X 5' scRNA-Seq for CD45+ cells from P126 Normal Tissue


title: P127N-I

value: 10X 5' scRNA-Seq for CD45+ cells from P127 Normal Tissue


title: P104T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P104 TUMOR


title: P84T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P84 TUMOR


title: P127T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P127 TUMOR


title: P83T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P83 TUMOR


title: P8T-E

value: 10X 5' scRNA-Seq for CD45- cells from P8 TUMOR


title: P87T-I

value: esophageal carcinoma

value: 10X 5' scRNA-Seq for CD45+ cells from P87 TUMOR


title: P126T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P126 TUMOR


title: P76T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P76 TUMOR


title: P94T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P94 TUMOR


title: P128T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P128 TUMOR


title: P107T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P107 TUMOR


title: P79T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P79 TUMOR


title: P82T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P82 TUMOR


title: P11T-E

value: 10X 5' scRNA-Seq for CD45- cells from P11 TUMOR


title: P63T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P63 TUMOR


title: P91T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P91 TUMOR


title: P74T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P74 TUMOR


title: P89T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P89 TUMOR


title: P130N-I

value: adjacent normal

value: 10X 5' scRNA-Seq for CD45+ cells from P130 Normal Tissue


title: P80T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P80 TUMOR


title: P61T-I

value: esophageal carcinoma

value: 10X 5' scRNA-Seq for CD45+ cells from P61 TUMOR


title: P62T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P62 TUMOR


title: P57T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P57 TUMOR


title: P56T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P56 TUMOR


title: P54T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P54 TUMOR


title: P47T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P47 TUMOR


title: P75T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P75 TUMOR


title: P40T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P40 TUMOR


title: P65T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P65 TUMOR


title: P39T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P39 TUMOR


title: P52T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P52 TUMOR


title: P48T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P48 TUMOR


title: P42T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P42 TUMOR


title: P44T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P44 TUMOR


title: P26T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P26 TUMOR


title: P27T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P27 TUMOR


title: P20T-I

value: esophageal carcinoma

value: 10X 5' scRNA-Seq for CD45+ cells from P20 TUMOR


title: P28T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P28 TUMOR


title: P23T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P23 TUMOR


title: P36T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P36 TUMOR


title: P31T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P31 TUMOR


title: P32T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P32 TUMOR


title: P38T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P38 TUMOR


title: P16T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P16 TUMOR


title: P17T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P17 TUMOR


title: P21T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P21 TUMOR


title: P44T-E

value: 10X 5' scRNA-Seq for CD45- cells from P44 TUMOR


title: P24T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P24 TUMOR


title: P37T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P37 TUMOR


title: P49T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P49 TUMOR


title: P22T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P22 TUMOR


title: P19T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P19 TUMOR


title: P30T-I

value: esophageal carcinoma

value: 10X 5' scRNA-Seq for CD45+ cells from P30 TUMOR


title: P11T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P11 TUMOR


title: P10T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P10 TUMOR


title: P12T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P12 TUMOR


title: P8T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P8 TUMOR


title: P4T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P4 TUMOR


title: P9T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P9 TUMOR


title: P15T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P15 TUMOR


title: P1T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P1 TUMOR


title: P2T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P2 TUMOR


title: Plate_396_Q7_SS62862 (Single-cell RNA-Seq)

status: Public on Sep 14 2020

city: Ashburn

first: Vivek

last: Jayaraman

state: VA

address: 19700 Helix Drive

institute: Janelia Research Campus, HHMI

description: Single-cell RNA-Seq

data_processing:
Sequencing adapters were trimmed and aligned as done for the bulk RNA-seq data. Gene counts were generated with the STARsolo algorithm using the following additional parameters: "--soloType CB_UMI_Simple --soloCBwhitelist smartscrb_whitelist.txt --soloCBstart 2 --soloCBlen 8 --soloUMIstart 10 --soloUMIlen 10 -soloBarcodeReadLength 0 --soloCBmatchWLtype 1MM_multi_pseudocounts --soloStrand Forward --soloFeatures Gene --soloUMIdedup 1MM_Directional --soloUMIfiltering MultiGeneUMI --soloCellFilter None".  The full set of 384 barcodes designed for this assay was used as the whitelist. Gene counts for the subset of barcodes used in this experiment were extracted using custom R scripts.
For each sample, raw read counts were normalized by the total number of reads per million mapped reads per sample to yield counts per million (CPM) measurements.
The single-cell data was filtered in Python by: 1) Selecting the single cells where more genes were detected than in the ERCC-alone wells, 2) selecting the single cells that were within 2 S.D.s of the mean for number of ERCC genes detected and 3) selecting the single cells that had a correlation of >0.75 between the ERCC genes counts and the expected # of molecules.
The counts for each gene were averaged across the filtered population.
For the statistical analysis of differential gene expression, the edgeR package (Robinson et al., 2010) in R was used on the raw count data, with the standard workflow; this consists of estimating the negative binomial fit of the mean-dispersion relationship through a weighted-likelihood empirical Bayesian approach (estimateDisp function), and then calculating p-values through a gene-wise negative binomal generalized linear model with quasi-likelihood tests (glmQLFit function).
Genome_build: Drosophila melanogaster r6.17 genome assembly on Flybase (Thurmond et al., 2019).
Supplementary_files_format_and_content: 3 Excel files with raw count values for each sample; 1 Excel file with statistical data from DGE analyses.

organism: Drosophila melanogaster

growth_protocol: Drosophila adults were collected daily as they eclosed, and aged 3-5 days prior to dissection.

extract_protocol:
For each sample, 60-100 brains were dissected in freshly prepared, ice cold Adult Hemolymph Solution (AHS, 108 mM NaCl, 5 mM KCl, 2 mM CaCl2, 8.2 mM MgCl2, 4 mM NaHCO3, 1 mM NaH2PO4, 5 mM HEPES, 6 mM Trehalose, 10 mM Sucrose), and the major tracheal branches removed. The brains were transferred to a 1.5 ml Eppendorf tube containing 500 μl 1 mg/ml Liberase DH (Roche, prepared according to the manufacturer’s recommendation) in AHS, and digested for 1 h at room temperature. The Liberase solution was removed and the brains washed twice with 800 microliters ice cold AHS. The final wash was removed completely and 400 μl of AHS+2% Fetal Bovine Serum (FBS, Sigma) were added. The brain samples were gently triturated with a series of fire-polished, FBS-coated Pasteur pipettes of descending pore sizes until the tissue was homogenized, after which the tube was allowed to stand for 2-3 m so that the larger debris could settle. For hand sorting, the cell suspension was transferred to a Sylgard-lined Glass Bottom Dish (Willco Wells), leaving the debris at the bottom of the Eppendorf tube, and distributed evenly in a rectangular area in the center of the plate with the pipet tip. The cells were allowed to settle for 10-30 m prior to picking. Fluorescent cells were picked with a mouth aspirator consisting of a 0.8 mm Nalgene Syringe Filter (Thermo), a short stretch of tubing, a plastic needle holder, and a pulled Kwik-Fil Borosilicate Glass capillary (Fisher). Cells picked off the primary plate were transferred to a Sylgard-lined 35 mm Mat Tek Glass Bottom Microwell Dishes (Mat Tek) filled with 170 μl AHS+2%FBS, allowed to settle, and then re-picked. Three washes were performed in this way before the purified cells were picked and transferred into 50 μl buffer XB from the PicoPure RNA Isolation Kit (Life Technologies), lysed for 5 m at 42˚C, and stored at -80˚C. For FACS, the samples were triturated in AHS+2%FBS that was run through a 0.2 μm filter, and the cell suspension was passed through a Falcon 5 ml round-bottom tube fitted with a 35 μm cell strainer cap (Fisher). Samples were sorted on a SONY SH800 cell sorter gated for single cells with a fluorescence intensity exceeding that of a non-fluorescent control. For bulk samples, GFP+tdTom+ events were purity sorted directly into 50 microliters PicoPure XB buffer, the sample lysed for 5 m at 42˚C, and stored at -80˚C. For low-cell samples, replicates of 40 cells were either purity sorted directly or yield sorted to enrich for GFP+tdTom+ cells followed by a purity sort into 3 μL Lysis Buffer (0.2% Triton X-100, 0.1 U/μL RNAsin) and flash frozen on dry ice. For single-cell (scRNA-Seq), individual cells were purity sorted directly into a 96-well plate containing 3 μL Lysis Buffer per well.
For bulk samples, total RNA was extracted from 100-500 pooled cells using the PicoPure kit (Life Technologies) according to the manufacturer’s recommendation, including the on-column DNAse1 step. The extracted RNA was converted to cDNA and amplified with the Ovation RNA-Seq System V2 (NuGEN), and the yield quantified by NanoDrop (Thermo). The cDNA was fragmented and the sequencing adaptors ligated onto the fragments using the Ovation Rapid Library System (NuGEN). Library quality and concentration was determined with the Kapa Illumina Library Quantification Kit (KK4854, Kapa Biosystems), and the libraries were pooled and sequenced on an Illumina NextSeq 550 with 75 base pair reads. Sequencing adapters were trimmed from the reads with Cutadapt (Martin, 2011) prior to alignment with STAR (Dobin et al., 2013) to the Drosophila r6.17 genome assembly (Flybase). The resulting transcript alignments were passed to RSEM (Li and Dewey, 2011) to generate gene expression counts. For low-cell and single-cell samples, one microliter of harsh lysis buffer (50 mM Tris pH 8.0, 5 mM EDTA pH 8.0, 10 mM DTT, 1% Tween-20, 1% Triton X-100, 0.1 g/L Proteinase K (Roche), 2.5 mM dNTPs (Takara), and ERCC Mix 1 (Thermo-Fisher) diluted to 1e-7) and 1 μl10 μM barcoded RT primer were added, and the samples were incubated for 5 m at 50˚C to lyse the cells, followed by 20 m at 80˚C to inactivate the Proteinase K. Reverse transcription master mix (2 μL 5X RT Buffer (Thermo-Fisher), 2 μL 5M Betaine (Sigma-Aldrich), 0.2 μL 50 μM E5V6NEXT template-switch oligo (Integrated DNA Technologies), 0.1 μL 200 U/μL Maxima H-RT (Thermo-Fisher), 0.1 μL 40U/μL RNAsin (Lucigen), and 0.6 μL nuclease-free water (Thermo-Fisher) was added to the lysis reaction and incubated at 42˚C for 1.5 h, followed by 10 m at 75˚C to inactivate the reverse transcriptase. PCR was performed by adding 10 μL 2X HiFi PCR Mix (Kapa Biosystems) and 0.5 μL 60 μM SINGV6 primer and incubating at 98˚C for 3 m, followed by 20 cycles of 98˚C for 20 s, 64˚C for 15 s, 72˚C for 4 m, and a final extension of 5 m at 72˚C. Groups of 8 reactions were pooled to yield ~250 μL and purified with Ampure XP Beads (0.6x ratio; Beckman Coulter), washed twice with 75% Ethanol, and eluted in 40 μL nuclease-free water. The DNA concentration of each sample was determined using Qubit High-Sensitivity DNA kit (Thermo-Fisher). To prepare the Illumina sequencing library, 600 pg cDNA from each pooled sample was used in a modified Nextera XT library preparation (Illumina) (Soumillon et al., 2014) using the P5NEXTPT5 primer and extending the tagmentation time to 15 m. The resulting libraries were purified according to the Nextera XT protocol (0.6x ratio) and quantified by qPCR using Kapa Library Quantification (Kapa Biosystems). Six to eight sequencing libraries were loaded on a NextSeq High Output flow cell reading 26 bases in Read 1, including the spacer, sample barcode and UMI, 8 bases in the i7 index read, and 50 bases in Read 2 representing the cDNA fragment from the 3' end of the transcript.

tag: neuronal cell type

tag: genetic driver

tag: reporter

value: nlstdTomato

tag: Sex

value: Female

tag: sorting

value: FACS

tag: cell number

value: single-cell


title: P5T-I

value: 10X 5' scRNA-Seq for CD45+ cells from P5 TUMOR


title: Plate_389_Q8_SS62862 (Single-cell RNA-Seq)

description: Single-cell RNA-Seq


title: Plate_395_Q6_SS62862 (Single-cell RNA-Seq)

description: Single-cell RNA-Seq


title: Plate_394_Q5_SS62862 (Single-cell RNA-Seq)

description: Single-cell RNA-Seq


title: CITE-seq Met v Naïve ADT

city: Dallas

first: Siyuan

address: 6001 Forest Park Rd

department: Department of Pathology

institute: UT Southwestern Medical Center

data_processing:
library strategy: CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing)
Demultiplexing: cellranger mkfastq
10x genomics Cellranger V2.1 was used for alignment, filtering, barcoding Counting and UMI Counting.
CITE-seq-Count v1.3.4 (https://github.com/Hoohm/CITE-seq-Count) was used for ADT and HTO counts.
HTO1-4 were used for sample level demultiplexing and group assignment. HTO1: naive brain, replicate 1; HTO2: naive brain, replicate 2; HTO3: brain metastasese-bearing brain, replicate 1; HTO4: brain metastasese-bearing brain, replicate 2
After generating the digital gene expression data matrices, the Seurat R package V2.3.4 (Satija, 2015) was used to perform data normalization, dimension reduction, clustering and differential expression analysis.
Genome_build: mm10-1.2.0
Supplementary_files_format_and_content: tab-delimited text files that include UMI values for gene UMI, HTO and ADT counts

molecule: protein

organism: Mus musculus

source_name: brain cells and metastatic tumor cells

extract_protocol:
Mice were perfused with PBS and brains extracted. Brains digested with papain-based neural tissue dissocation kit (Miltenyi). Mononuclear cells enriched by 70%/37%/30% percoll gradient centrifugation.
For RNA expression (cDNA), we followed 10x genomics single cell 3’ v2 assay standard protocol. For ADT and HTO, we followed CITE-seq and Cell hashing protocol as provided here:https://citeseq.files.wordpress.com/2019/02/cite-seq_and_hashing_protocol_190213.pdf
10x genomics single cell 3’ v2 assay

tag: mouse strain

tag: cell type

value: brain cells and metastatic tumor cells

tag: molecule type

value: ADT


title: CITE-seq Met v Naïve HTO


title: CITE-seq Met v Naïve mRNA/cDNA (GEX)

value: mRNA/cDNA


title: Plate_393_Q8_SS02232 (Single-cell RNA-Seq)

description: Single-cell RNA-Seq


title: Plate_388_Q7_SS02232 (Single-cell RNA-Seq)

description: Single-cell RNA-Seq


title: Plate_391_Q6_SS62862 (Single-cell RNA-Seq)

description: Single-cell RNA-Seq


title: Plate_387_Q6_SS02232 (Single-cell RNA-Seq)

description: Single-cell RNA-Seq


title: Plate_390_Q5_SS02232 (Single-cell RNA-Seq)

description: Single-cell RNA-Seq


title: Plate_386_Q5_SS02232 (Single-cell RNA-Seq)

description: Single-cell RNA-Seq


title: Sample_SS00238 (Low-cell RNA-Seq)

description: Low-cell RNA-Seq

value: 40 cells per replicate


title: Sample_SS02232_2 (Low-cell RNA-Seq)

description: Low-cell RNA-Seq

value: 35 cells per replicate


title: Sample_19_7_6_whole_brain_qRNA_r2 (bulk RNA-Seq)

description: bulk RNA-Seq

value: whole brain


title: Sample_20_8_1_SS02232_r2 (bulk RNA-Seq)

description: bulk RNA-Seq

value: 1095 cells


title: Sample_SS02232_1 (Low-cell RNA-Seq)

description: Low-cell RNA-Seq


title: Sample_20_7_2_SS02191_r2 (bulk RNA-Seq)

description: bulk RNA-Seq

source_name: PEG

value: PEG

value: 1247 cells


title: Plate_392_Q7_SS62862 (Single-cell RNA-Seq)

description: Single-cell RNA-Seq


title: Sample_20_8_5_SS00131_r2 (bulk RNA-Seq)

description: bulk RNA-Seq

source_name: EPG

value: EPG

value: 865 cells


title: Sample_20_7_1_SS02191_r1 (bulk RNA-Seq)

description: bulk RNA-Seq

value: 1170 cells


title: Sample_20_7_5_SS02232_r1 (bulk RNA-Seq)

description: bulk RNA-Seq

value: nlstdTomato

value: 993 cells


title: hRO-D120

status: Public on Sep 05 2020

city: Wenzhou

first: Zibing

last: Jin

state: Zhejiang

address: No.270, Xueyuan road

department: Division of Ophthalmic Genetics, The Eye Hospital

institute: Laboratory for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research

library_source: genomic

data_processing:
The raw reads of WGBS data were trimmed by trim_galore (Version 0.5.0) and then mapped to hg38 (human) using Bismark (Version 0.18.2) with default parameters.
We extracted the methylated CpGs with bismark methylation extractor script.
The number of converted and unconverted reads, total coverage and the fractional methylation at each individual CpG were reported.
Bedgraph format data were convert to bigwig data using bedGrapthToBigWig
Genome_build: GRCh38
Supplementary_files_format_and_content: bigwig

molecule: genomic DNA

source_name: human retinal organoids

growth_protocol: Human retinal and Rb organoids differentiation were performed as previously described with minor modifications. On day 0, RB1Wt/Wt and RB1Mut/Mut hESCs maintained on Matrigel-coated plates were pretreated with 10 μM Y-27632 for 2h, and then carefully dissociated into single-cell suspensions using TrypLE Select (containing 0.05 mg/ml DNase I and 20 μM Y-27632), plated at a density of 12,000 live cells per well in 96-well low-attachment V-bottom plates in retinal differentiation medium I containing 44% Iscove’s modified Dulbecco’s medium, 44% Hams F12, 10% KnockOut Serum Replacement, 1% GlutaMAX-I, 1% Penicillin-Streptomycin (10,000 U/ml), and 20 μM Y-27632. 1.5nM (55 ng/ml) recombinant human BMP4 was added to the culture on day 6, and its concentration was semi-reduced with half medium change every 3 days. Retinal differentiation media Ⅱ [DMEM/F12-Glutamax medium containing 10% Fetal Bovine Serum, 1% N2 supplement, 1% Penicillin-Streptomycin (10,000 U/ml), 0.5 μM retinoic acid and 0.1 mM taurine] was exchanged until day 18 when the aggregate bodies were transferred to 9 cm nonadherent dishes (30 bodies per dish) for further long-term culture in a 37℃, 5% CO2/40% O2 incubator, and half of the volume changed with fresh retinal differentiation media Ⅱ every 5 days. The state and morphology of organoids was monitored in real time using the inverted fluorescence microscope.

extract_protocol:
Genomic DNA was isolated from each sample using the DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s protocol. DNA was quantified using NanoDrop 2000 and Qubit 2.0 Fluorometer.
1µg genomic DNA was used to construct WGBS library. Bisulfite conversion of genomic DNA was performed using the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer’s recommendation. Bisulfite-converted DNA was subjected to process as described, and then sequencing (PE125/PE150) on an Illumina HiSeq platform.

tag: source cell type

value: normal hESCs

tag: organoids

value: human ES-derived retinal organoids (hROs)

tag: developmental/tumor stage

value: 120-day old


title: Sample_19_7_5_whole_brain_qRNA_r1 (bulk RNA-Seq)

description: bulk RNA-Seq

value: Female


title: HEK293T cells - TMEM119 silencing rep2

status: Public on Aug 28 2020

city: Chicago

first: Hoang Van

last: Phan

state: Illinois

address: 5640 S Ellis Ave

department: Priztker School of Molecular Engineering

institute: The University of Chicago

description:
Bulk RNA-seq of fresh live cells with TMEM119 silencing
S20-589-siTMEM-2_S20
processed data file: counts_KSHV.txt.gz

data_processing:
Base calling was done automatically in Illumina's BaseSpace platform
Single-cell data was processed using Drop-seq tools v1.13 and STAR aligner according to the online manual
Bulk data was aligned using STAR aligner, and counted using featureCounts.
Single-cell count data was analyzed with monocle 2 and Seurat 3
Bulk data count data was analyzed with DESeq2
Genome_build: GRCh38, hg19 and mm10 mixed, OC43 genome NC_006213.1
Supplementary_files_format_and_content: Single-cell data: tab-delimited text file of UMI counts per single cell
Supplementary_files_format_and_content: Bulk data: tab-delimited text file of read counts per sample

molecule: polyA RNA

growth_protocol: BC3 cells were grown in RPMI media with 20% FBS, 2 mM GlutaMax and 1% P/S. HEK293T cells were grown in DMEM with 10% FBS, 2 mM GlutaMAX and 1% P/S. A549 cells were grown in DMEM with 10% FBS. 3T3 cells were grown in DMEM with 10% FBS, 1% GlutaMAX and 1X P/S

extract_protocol:
Single cells were captured and lysed in droplets in a microfluidic chip as per Drop-seq. Bulk RNA samples were extracted using HP Total RNA Kit.
The single-cell cDNA was tagmented using Nextera XT kit as per Drop-seq protocol. Bulk library was prepared using NEBNext Ultra II RNA Library Prep Kit for Illumina.

value: Human embryonic kidney cells

tag: virus type

tag: treatment

value: TMEM119 siRNA


title: Sample_20_6_1_SS00096_r1 (bulk RNA-Seq)

description: bulk RNA-Seq

value: FACS

value: 998 cells


title: Sample_20_7_3_SS30295_r1 (bulk RNA-Seq)

description: bulk RNA-Seq

value: 1250 cells


title: Sample_20_6_2_SS00096_r2 (bulk RNA-Seq)

description: bulk RNA-Seq

value: 809 cells


title: Sample_20_5_8_SS02238_r2 (bulk RNA-Seq)

description: bulk RNA-Seq

value: 754 cells


title: HEK293T cells - TMEM119 overexpression rep3

description:
Bulk RNA-seq of fresh live cells with TMEM119 overexpression
S15-589-TMEM-3_S15
processed data file: counts_KSHV.txt.gz

value: TMEM119 overexpression


title: HEK293T cells - GFP overexpression rep3

description:
Bulk RNA-seq of fresh live cells with GFP overexpression control
S12-589-GFP-3_S12
processed data file: counts_KSHV.txt.gz

value: GFP overexpression


title: hRBO-D120

source_name: human Rb organoids

value: RB1 mutation knock-in hESCs

value: human ES-derived retinoblastoma organoids (hRBOs)


title: HEK293T cells - TMEM119 overexpression rep1

description:
Bulk RNA-seq of fresh live cells with TMEM119 overexpression
S13-589-TMEM-1_S13
processed data file: counts_KSHV.txt.gz


title: HEK293T cells - TMEM119 silencing rep3

description:
Bulk RNA-seq of fresh live cells with TMEM119 silencing
S21-589-siTMEM-3_S21
processed data file: counts_KSHV.txt.gz


title: HEK293T cells - TMEM119 overexpression rep2

description:
Bulk RNA-seq of fresh live cells with TMEM119 overexpression
S14-589-TMEM-2_S14
processed data file: counts_KSHV.txt.gz


title: Sample_central_brain_qRNA_50pg (Low-cell RNA-Seq)

description: Low-cell RNA-Seq

value: central brain

value: 50 pg per replicate


title: Sample_20_5_7_SS02238_r1 (bulk RNA-Seq)

description: bulk RNA-Seq

value: nlstdTomato

value: 885 cells


title: HEK293T cells - GFP overexpression rep2

description:
Bulk RNA-seq of fresh live cells with GFP overexpression control
S11-589-GFP-2_S11
processed data file: counts_KSHV.txt.gz


title: Sample_20_8_3_SS00131_r1 (bulk RNA-Seq)

description: bulk RNA-Seq

value: EPG

value: 762 cells


title: HEK293T cells - GFP overexpression rep1

description:
Bulk RNA-seq of fresh live cells with GFP overexpression control
S10-589-GFP-1_S10
processed data file: counts_KSHV.txt.gz

value: Human embryonic kidney cells


title: HEK293T cells - siRNA control rep1

description:
Bulk RNA-seq of fresh live cells with control siRNA
S16-589-siCtrl-1_S16
processed data file: counts_KSHV.txt.gz

value: siRNA control


title: HEK293T.rKSHV219 cells - TMEM119 overexpression rep2

description:
Bulk RNA-seq of fresh live cells with TMEM119 overexpression
S08-570-TMEM-2_S8
processed data file: counts_KSHV.txt.gz

value: Recombinant KSHV.219


title: Sample_20_7_4_SS30195_r2 (bulk RNA-Seq)

description: bulk RNA-Seq

value: Female

value: FACS

value: 1270 cells


title: HEK293T.rKSHV219 cells - TMEM119 overexpression rep1

description:
Bulk RNA-seq of fresh live cells with TMEM119 overexpression
S07-570-TMEM-1_S7
processed data file: counts_KSHV.txt.gz


title: HEK293T.rKSHV219 cells - GFP overexpression rep3

description:
Bulk RNA-seq of fresh live cells with GFP overexpression control
S06-570-GFP-3_S6
processed data file: counts_KSHV.txt.gz


title: HEK293T.rKSHV219 cells - TMEM119 overexpression rep3

description:
Bulk RNA-seq of fresh live cells with TMEM119 overexpression
S09-570-TMEM-3_S9
processed data file: counts_KSHV.txt.gz


title: HEK293T.rKSHV219 cells - GFP overexpression rep1

description:
Bulk RNA-seq of fresh live cells with GFP overexpression control
S04-570-GFP-1_S4
processed data file: counts_KSHV.txt.gz


title: HEK293T.rKSHV219 cells - GFP overexpression rep2

description:
Bulk RNA-seq of fresh live cells with GFP overexpression control
S05-570-GFP-2_S5
processed data file: counts_KSHV.txt.gz


title: HEK293T cells - siRNA control rep3

description:
Bulk RNA-seq of fresh live cells with control siRNA
S18-589-siCtrl-3_S18
processed data file: counts_KSHV.txt.gz


title: HEK293T.rKSHV219 cells - CDH1 overexpression rep2

description:
Bulk RNA-seq of fresh live cells with CDH1 overexpression
S02-570-CDH1-2_S2
processed data file: counts_KSHV.txt.gz

value: CDH1 overexpression


title: HEK293T.rKSHV219 cells - CDH1 overexpression rep3

description:
Bulk RNA-seq of fresh live cells with CDH1 overexpression
S03-570-CDH1-3_S3
processed data file: counts_KSHV.txt.gz


title: Primary fibroblasts 2

status: Public on Jun 16 2020

city: Buffalo

first: Katerina

last: Gurova

state: NY

address: Elm and Carlton Str

department: Cell Stress Biology

institute: Roswell Park Cancer Institute

data_processing:
MNase-seq reads were aligned to the mm10 genome assembly using bwa
PCR duplicates were marked using picard MarkDuplicates
The big wiggle files were generated using deeptools and normalized by RPKM
Genome_build: mm10
Supplementary_files_format_and_content: big wiggle files

source_name: Tail

growth_protocol: DMEM, 10% FBS, 1% pen/strep

extract_protocol:
Nucleosomal DNA was  collected by MNase treatment of chromatin extracts as described by Jason M. Rizzo and Satrajit Sinha (2014, Analyzing the global chromatin structure of keratinocytes by MNase-Seq. Methods in Molecular Biology, 1195:49-59).
DNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: untreated/treated with 2 ug/ml 4OH-tamoxifen for 96 hours

value: Primary

value: control

tag: replicate


title: Primary fibroblasts 1


title: Transformed fibroblasts clone 2 2

value: Transformed


title: Immortalized fibroblasts clone 2 treated with 4OH-tamoxifen for 96 hours 2

value: Immortalized

value: 4OH-tamoxifen for 96 hours


title: HEK293T.rKSHV219 cells - CDH1 overexpression rep1

description:
Bulk RNA-seq of fresh live cells with CDH1 overexpression
S01-570-CDH1-1_S1
processed data file: counts_KSHV.txt.gz


title: Primary fibroblasts treated with 4OH-tamoxifen for 96 hours 2


title: Transformed fibroblasts clone 2 1


title: Immortalized fibroblasts clone 2 1


title: Immortalized fibroblasts clone 2 2


title: n3_A_CDDPplusplus

status: Public on Apr 30 2020

city: Paris Cedex 05

first: Nicolas

last: Servant

address: 26 rue d'ulm

country: France

institute: Institut Curie

description:
replicate 3
3'-seq

data_processing:
Raw reads were trimmed in their 5’ and 3’ ends to remove uninformative nucleotides due to primer sequences, nucleotides added by SMART Scribe RT enzyme and polyA tail of mRNAs.
Trimmed reads of 25 bp or more were aligned on the Mouse reference genome (mm9) using Bowtie2 (version 2.2.5) (Langmead and Salzberg 2012). Only reads with a mapping quality score (MAPQ) of 20 or more were retained (Samtools version 1.1) for downstream analysis.
Reads were then clustered along the genome using Bedtools (version 2.17.0) (Quinlan and Hall 2010), allowing a maximum distance of 170 bp (max library fragment length (300bp) – min fragment length (60bp) – read length (50bp) – oligodT length (25bp)) and a minimum number of 5 reads per peak.
Peaks with a stretch of 6 consecutive As (or 8 As out of 9 nucleotides) within 150 bp downstream were filtered out, as they are likely due to internal priming of oligo-dT.
Genome_build: mm9
Supplementary_files_format_and_content: .bed

source_name: Non-small cell lung cancer cell line

growth_protocol: Cells were cultured in RPMI-1640-Glutamax medium (GibcoBRL, Life Technologies, Cergy Pontoise, France), supplemented with 10% (v/v) heat-inactivated fetal calf serum (GibcoBRL), in 5% CO2 at 37°C.

extract_protocol:
RNA was extracted from total cells, cytosol fraction or heavy polysomes.
5 μg of total RNA were used for poly(A)+ RNA purification with Dynabeads mRNA DIRECT Micro kit (ThermoFisher Scientific). Poly(A)+ RNA was fragmented at 70°C for 5 min in RNA Fragmentation Reagents (ThermoFisher Scientific). After ethanol precipitation, RNA was reverse transcribed using anchored oligo-dT and SMARTScribe RT enzyme (Clontech) allowing to add adapter sequences to the 5’ and 3’ ends of RNA fragments. Libraries were amplified by 16 cycles of PCR with GoTaq Flexi DNA Polymerase (Promega), purified using Agencourt AMPure XP (Beckman Coulter) magnetic beads, and quantified with Quant-iT Picogreen dsDNA kit (ThermoFisher Scientific). A size selection step was done using SPRIselect (Beckman Coulter) magnetic beads to obtain fragments of 150–300 bp. Purified libraries were controlled by capillary electrophoresis (LabChip - Perkin) and quantitated by qPCR (KAPA Library Quantification Kits Illumina Platforms - Roche). Pooled libraries (12 pM) with 30% of phiX were subjected to single-end, 50 bp sequencing using the HiSeq 2500 machine (Illumina). Read 1 was read with primer HP6 (Illumina) with 3 dark cycles (first 3 bases of read 1 were not read). Index i7 (6 pb barcode) was read with primer HP8 (Illumina).

treatment_protocol: Treatments with cisplatin and oxaliplatin (Selleckchem, Euromedex, Souffelweyersheim, France) were done at 100 µM. Treatment with vehicle (DMSO) was always done in parallel.

tag: cell line

value: Cisplatin

tag: extract

value: Polysomes


title: n2_A_CDDPplusplus

description:
replicate 2
3'-seq


title: n1_A_CDDPplusplus

description:
replicate 1
3'-seq


title: HEK293T cells - TMEM119 silencing rep1

description:
Bulk RNA-seq of fresh live cells with TMEM119 silencing
S19-589-siTMEM-1_S19
processed data file: counts_KSHV.txt.gz

value: TMEM119 siRNA


title: n2_A_DMSOplusplus

description:
replicate 2
3'-seq

value: Vehicle (DMSO)


title: RD-i-3

description:
replicate 3
3'-seq

value: Cytosol


title: n1_A_DMSOplusplus

description:
replicate 1
3'-seq


title: RC-i-3

description:
replicate 3
3'-seq


title: Transformed fibroblasts clone 2 treated with 4OH-tamoxifen for 96 hours 2


title: Primary fibroblasts treated with 4OH-tamoxifen for 96 hours 1


title: Transformed fibroblasts clone 2 treated with 4OH-tamoxifen for 96 hours 1


title: AD-i-3

description:
replicate 3
3'-seq


title: RD-i-2

description:
replicate 2
3'-seq


title: n3_A_DMSOplusplus

description:
replicate 3
3'-seq


title: RC-i-2

description:
replicate 2
3'-seq


title: Immortalized fibroblasts clone 2 treated with 4OH-tamoxifen for 96 hours 1


title: AC-i-2

description:
replicate 2
3'-seq


title: AD-i-2

description:
replicate 2
3'-seq


title: H358-OXPT-T-3

description:
replicate 3
3'-seq

value: Oxaliplatin

value: Total cells


title: H358-CDDP-T-3

description:
replicate 3
3'-seq


title: RC-i-1

description:
replicate 1
3'-seq


title: AD-i-1

description:
replicate 1
3'-seq


title: H358-DMSO-T-3

description:
replicate 3
3'-seq


title: H358-CDDP-T-2

description:
replicate 2
3'-seq


title: H358-OXPT-T-2

description:
replicate 2
3'-seq


title: AC-i-3

description:
replicate 3
3'-seq


title: AC-i-1

description:
replicate 1
3'-seq


title: H358-CDDP-T-1

description:
replicate 1
3'-seq


title: H358-DMSO-T-1

description:
replicate 1
3'-seq


title: H358-OXPT-T-1

description:
replicate 1
3'-seq


title: RD-i-1

description:
replicate 1
3'-seq


title: HD3

status: Public on Apr 21 2020

city: Washington

first: Jerry

last: Xiao

state: DC

address: 3900 Reservoir Rd NW, PCS Bldg, Room GR10C

department: Tumor Biology

institute: Georgetown University

description: RNA sample from plasma + buffy coat of healthy individual

data_processing:
Base-calling was performed by Genewiz.
adapter were trimmed using cutadapt v2.6 -a CTGTCTCTTATA -A CTGTCTCTTATA -j 23 -q 30 -m 40
Reads were aligned to the human genome (Star Genecode v32) using STAR aligner v2.7.3a --runThreadN 23 --outSAMtype BAM Unsorted
reads were counted using feature counts in R annot.inbuilt='hg38' isPairedEnd = TRUE nthreads=23
Differential Expression was calculated using DESeq2 in R
Genome_build: GRCH38.p13
Supplementary_files_format_and_content: raw and normalized gene counts (CSV) created from DESeq2

source_name: Healthy Donor 3

growth_protocol: Samples were cultured at 37C in culture medium consisting of Advanced DMEM/F12 supplemented with B27 supplement (1x; Thermo Fisher, Grand Island, NY, USA, cat. no. 17504044), epidermal growth factor (10 ng/mL; Sigma-Aldrich, Saint Louis, MO, USA, cat. no. E9644), basic fibroblast growth factor (10 ng/mL; R&D systems, Minneapolis, MN, USA, cat. no. 233-FB), heparin (10 ug/mL; Sigma-Aldrich cat. no. H3149), Y-27632 (10 uM; Enzo Life Sciences, Farmingdale, NY, USA, cat. no. ALX-270-333), Adenine, L-Glutamine (2 mM; Thermo Fisher cat. no. 25030149), and Antibiotic-antimycotic (1x; Thermo Fisher cat. no. 15240062). Cultures were washed every 6 days with 1X PBS by centrifugation. After one month in culture, cells were isolated for RNA extraction.

extract_protocol:
RNA was extracted using Invitrogen RNAqueous Micro-RNA kit following manufacturer protocol.
Clonetech SMARTer kit was used for conversion to cDNA from ulta-low input RNA. NEBNextUltra DNA library prep kit was then used to finish the library construction. Library contstructions were done through a third-party company, Genewiz. Libraries were read using an Illumina HiSeq 2x150 PE HO configuration.
Genewiz was consulted for the construction and preparation of all libraries.

value: plasma + buffy coat

tag: gender

value: Male

tag: disease state

value: Healthy Individual


title: HD4

source_name: Healthy Donor 4


title: HD5

source_name: Healthy Donor 5

value: Female


title: HD1

source_name: Healthy Donor 1


title: HD2

source_name: Healthy Donor 2


title: CTC10

description: RNA sample of cultured circulating tumor cell

source_name: MBC Patient 10

value: cultured circulating tumor cell

value: Metastatic Breast Cancer


title: CTC9

source_name: MBC Patient 9


title: CTC3

source_name: MBC Patient 3


title: CTC8

source_name: MBC Patient 8


title: 4950N

status: Public on Mar 01 2020

city: New York

first: Yang

last: Hu

state: New York

address: 1305 York Avenue Y-1322

department: Department of Physiology and Biophysics

institute: Weill Cornell Medical College

description: single cell RNA-seq

data_processing:
Single cell RNA sequencing data was collected on a Nextseq 500 Illumina instrument
Alignment using Cell Ranger (2.1.1) from 10xgenomics
Removed outlier cells which are three median absolute deviations (MADs) below than the median gene per cell, median transcripts per cell, and removed cells MADs above median mitochondrion percentage
Cells with less than 400 genes or 1000 UMIs or more than 15% of mitochondria genes were filtered out from the analysis
Genome_build: mm10 and hg19
Supplementary_files_format_and_content: tab-delimited raw counts for each sample

source_name: Blader cancer

growth_protocol: Bladder cancer

extract_protocol:
After single cell suspension, cells were sorted by CD45 antiboy and split into CD45 negative and position group.
Chromium Single Cell 3’ Library & Gel Bead Kit v2

treatment_protocol: mice were treated with OHBBN (0.1% (TCI, B0938) for 14 weeks followed  by progression over an additional 4 weeks.

tag: tissue

value: Blader cancer

tag: markers

value: CD45 Negative sorted


title: 8524N

description: single cell RNA-seq


title: 357

description: single cell RNA-seq

source_name: OHBBN-induced mouse bladder tumors

value: OHBBN-induced mouse bladder tumors

value: CD45 negative sorted


title: CTC12

source_name: MBC Patient 12


title: CTC4

source_name: MBC Patient 4


title: H358-DMSO-T-2

description:
replicate 2
3'-seq


title: 8525N

description: single cell RNA-seq


title: PROseq_unCond_K562_scr_HS0_rep2

status: Public on Mar 04 2020

city: Stockholm

first: Anniina

last: Vihervaara

address: Tomtebodavägen 23A

department: Gene Technology

country: Sweden

institute: KTH Royal Institute of Technology

description:
nascent RNA
Normalized_PROseq_unCond_K562_scr_HS0_minusStrand.bigWig
Normalized_PROseq_unCond_K562_scr_HS0_plusStrand.bigWig

data_processing:
Adapters (TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC) were removed using cutadapt (Marcel, 2011)
The reads were trimmed and filtered, and complements of reads were generated using fastx (http://hannonlab.cshl.edu/fastx_toolkit/).
The reads were aligned to combined hg19-dm3 genome using Bowtie2 (Langmead et al., 2009).
Reads that uniquely mapped to hg19 were processed to bed, bedgraph and bigWig files. The number of uniquely mapping reads to Drosophila genome (whole-genome invariant spike-in) was used for sample normalization.
Genome_build: hg19
Supplementary_files_format_and_content: Normalized and (where applicable) replicate-combined bigWig files. Since PRO-seq reports positions of transcription complexes strand-specifically, the plus strand and minus strand are given in separate bigWig files.

growth_protocol: Human K562 erythroleukemia cells were maintained at 37°C in a humidified 5% CO2 atmosphere and cultured in RPMI medium (Sigma), containing 10% fetal calf serum, 2 mM L-glutamate, 100 μg/ml streptomycin, and 100 U/ml penicillin. The cells were grown below 5x10^5 cells/ml confluency.

extract_protocol:
After treatments, the cells were harvested by centrifugation, and washed twice with PBS. Nuclei were isolated and run-on reactions were performed with biotinylated nucleotides as described by Kwak H, Fuda NJ, Core LJ, Lis JT. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 2013 Feb 22;339(6122):950-3. The run-on reaction was conducted in the presence of nuclei from S2 cells, which provides an invariant whole-genome spike-in control between samples.
The libraries were generated using TruSeq small-RNA adaptors, amplified 11 cycles, gel-purified and size-selected to 125-400 bp fragments. The libraries were sequenced using NextSeq 500 (Illumina).
PRO-seq

treatment_protocol: Preconditioning of K562 cells was conducted by submerging cells in a 175 mL flask into a 42°C heat bath. Nine 1-hour heat treatments were conducted in total, divided to three heat treatments per day for three consecutive days. Recovery from stress was conducted by placing the cells into a 37°C incubator for two hours (between the 1h heat shocks within the day) or over night (between the days). After nine heat shock treatments, the cells were allowed to recover for 48h in 37°C incubator prior to an additional, single heat shock was introduced. The single additional heat shock was conducted by concentrating 1x10^7 cells in a 10 mL media and placing the sample either into 37°C incubator (0 min) or submerging into 42°C water bath for 30 min (HS30) or 60 min (HS60). Transfections of cells (scrambled sequence or short hairpin targeting HSF1 mRNA) was conducted 24 h prior to initiating the preconditioning.

tag: hsf1 depletion

value: no depletion (scrambled sequence transfected)

tag: preconditioning

tag: heat shock (additional to preconditioning)

value: no heat stress applied


title: 4950P

description: single cell RNA-seq

value: CD45 Positive sorted


title: 8524P

description: single cell RNA-seq


title: 8525P

description: single cell RNA-seq


title: 7h-MMS-100

status: Public on Feb 18 2020

city: Ottawa

first: Matthew

last: Meier

state: Ontario

address: 251 Sir Frederick Banting Driveway

department: Health Canada

country: Canada

institute: Government of Canada

data_processing:
Raw sequencing data were analyzed and aligned to the reference genome (Human genome Hg 19) using the Ion Torrent Suite software (v5.04) for the Ion Proton.
The AmpliSeq plugin was employed to get the counts file.
Genome_build: Hg19
Supplementary_files_format_and_content: AmpliSeq output files rpm and raw count files.

source_name: Human Hepatoma HepaRG Cell Line

growth_protocol: Human cryopreserved No-Spin HepaRG™ cells were purchased from Triangle Research Labs (TRL), Durham, North Carolina, USA; which has since been acquired by Lonza Bioscience. Human HepaRG™ cells were cultured according to a method adapted from Jossé et al. (2012) for use in a slide-based in vitro MN assay. Differentiated HepaRG™ cells were seeded into collagen-coated wells at approximately 1.0-1.75 x105 viable cells per well in 12-well plates in TRL’s Thawing and Plating Medium for 24 hours, then switched to TRL’s Pre-Induction/Tox medium (supplemented with MHPIT) for cell maintenance and treatment. The 12-well plate format was chosen in order to provide sufficient numbers of cells per replicate for RNA extraction and the MN assay without the need to pool wells. Cells were incubated for 7 days following seeding to allow the cells to regain peak metabolic function.

extract_protocol:
Total RNA was extracted from exposed and control HepaRG™ cell pellets (n=3) using the Qiagen RNeasy Mini kit (Qiagen, Toronto, Ontario, Canada) with an on-column DNase I digestion, according to the supplier’s protocol. Purified RNA was quantified and assessed for quality with a NanoDrop® ND-1000 spectrophotometer and an Agilent TapeStation 2200. High quality RNA was used for gene expression analysis (A260/280 ≥ 2.0 and RINe ranging from 8.3 to 10).
The human AmpliSeq library kit was used to build the libraries.
RNA-Seq (Human Whole Transcriptome AmpliSeq)

treatment_protocol: Cells were incubated for 7 days following seeding to allow the cells to regain peak metabolic function, then they were treated with six concentrations of each test chemical and refreshed with media and test article daily for 3 days (i.e. 3-day repeat exposures; 0 hr, 24 hr, and 48 hr). A multiple exposure procedure was selected to allow for a more gradual induction of metabolic activity that led to more effective bioactivation of certain chemicals, specifically cyclophosphamide. Furthermore, use of this repeat exposure scenario mimics the protocol used for induction of enzymes in the S9 fractions of rat livers following administration of a mixture of phenobarbital and 5,6-benzoflavone. Seven hours following the third treatment (55 hr total exposure time), a subset of cells were detached using TrypLE (Waltham, MA), washed with 1X phosphate buffered saline, pelleted, flash-frozen and stored at -80°C for RNA extraction and subsequent whole transcriptome profiling. This sampling time was selected as it most aligned with the optimized protocol for use of the TK6 cell line in the presence of rat liver S9, in which a 4 hr chemical exposure was followed by a 3 to 4 hr recovery time for optimal TGx-DDI performance. Chemical treatments continued for MN frequency testing for a full 24 hr following the last treatment (e.g. 3-day repeat exposures for 24 hr each; 72 hr total treatment time). Test articles were then removed, media was re-freshed and the cells were stimulated with human Epidermal Growth Factor-1 (hEGF) for a further 72 hr to induce cell division (i.e., 144 hr total time following the last chemical exposure). hEGF-1 (Cell SignalingTechnology, Danvers, MA)  was applied to the cultures at 200 ng/mL immediately following chemical removal and media refreshment and again 48 hr later. The 3-day mitogen stimulation was found to increase the cell population by approximately 2.3-fold. All experiments were run in duplicate for the MN assay and in triplicate for RNA extraction (RNA-Seq was run as a pooled sample for each condition), with concurrent media and vehicle controls.

value: Methyl methanesulfonate (MMS)

tag: concentration


title: 7h-2DG-10

value: 2-Deoxy-D-Glucose (2DG)

value: 10 mM


title: 7h-VCP6

value: Vehicle Control for ETP (DMSO)

value: 0 uM


title: 7h-2DG-1.25


title: 7h-VCP8

value: Vehicle Control for ASC (media)

value: 0 mM


title: 7h-VCP9

value: Vehicle Control for AMP (media)


title: 7h-COL-0.1

value: Colchicine (COL)


title: 359

description: single cell RNA-seq


title: 7h-VCP7

value: Vehicle Control for AFB1 & COL (DMSO)


title: 7h-2NF-50

value: 2-Nitrofluorene (2NF)

value: 50 uM


title: 7h-VCP4

value: Vehicle Control for MMS (water)


title: PROseq_unCond_K562_scr_HS0_rep1


title: 7h-CISP-2a

value: Cisplatin (CISP)

value: 2 uM


title: 7h-ETP-0.5

value: Etoposide (ETP)


title: 7h-NaCl-10

value: Sodium Chloride (NaCl)


title: 7h-AFB1-1

value: Alflatoxin B1 (AFB1)

value: 1 uM


title: 7h-VCP3

value: Vehicle Control for 2DG (media)


title: 7h-COL-0.05


title: 7h-VCP2

value: Vehicle Control for 2NF (DMSO)


title: 7h-ETP-10

value: 10 uM


title: 7h-AMP-10

value: Ampicillin Trihydrate (AMP)

value: 10 mM


title: 7h-VCP1

value: Vehicle Control for NaCl (media)

value: 0 mM


title: 7h-CISP-5

value: 5 uM


title: 7h-2DG-5

value: 2-Deoxy-D-Glucose (2DG)

value: 5 mM


title: 7h-COL-0.3

value: Colchicine (COL)


title: 7h-2NF-10

value: 2-Nitrofluorene (2NF)


title: 7h-ETP-2.5


title: 7h-AFB1-0.25


title: 7h-ASC-2

value: Sodium Ascorbate (ASC)

value: 2 mM


title: 7h-MMS-200

value: Methyl methanesulfonate (MMS)


title: 7h-AFB1-2.5

value: Alflatoxin B1 (AFB1)


title: 7h-NaCl-1

value: Sodium Chloride (NaCl)

value: 1 mM


title: 7h-NaCl-2.5


title: 7h-AMP-3

value: 3 mM


title: wy_hz_21

status: Public on Dec 15 2019

city: Ramat Gan

first: Tirza

middle: T

last: Doniger

state: Not US or Canada

address: Bar Ilan University

department: The Mina and Everard Faculty of Life Sciences

country: Israel

institute: Bar Ilan University

description: treated wy hz 21

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 mm10 whole genome using was aligned using STAR (v 2.5.0a)  (Dobin et al., 2013) with default parameters.
The UCSC mm10 iGenome build and its corresponding gtf were used to build the STAR reference database.
The number of reads per gene were quantified using HTSeq-Count (v0.6.1p1) (Anders et al., 2015).
The R Bioconductor package, DESeq2 (v1.18.1)  (Love et al.,2014),  was used to classify genes as differentially expressed (Benjamini-Hochberg adjusted p-value < 0.1).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include raw counts

source_name: liver

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

treatment_protocol: Mice were orally gavaged at 50mg/kg with WY in CMC 0.5% or CMC 0.5% and sacrificed 12hours later.

tag: strain

value: liver

tag: genotype

value: SIRT6 heterozygote

value: WY-14643 (50mg/kg for 12 hours)

tag: age

value: 11-13 months


title: wy_hz_2

description: treated wy hz 2


title: 7h-VCP5

value: Vehicle Control for CISP (DMSO)

value: 0 uM


title: 7h-MMS-50

value: 50 uM


title: 7h-ASC-10

value: 10 mM


title: 7h-AMP-1

value: Ampicillin Trihydrate (AMP)


title: 7h-CISP-10

value: Cisplatin (CISP)

value: 10 uM


title: wy_wt_25

description: treated wy wt 25

value: wild type


title: 7h-ASC-1

value: Sodium Ascorbate (ASC)


title: wy_wt_11

description: treated wy wt 11


title: 7h-2NF-250

value: 2-Nitrofluorene (2NF)


title: HEK293T cells - siRNA control rep2

description:
Bulk RNA-seq of fresh live cells with control siRNA
S17-589-siCtrl-2_S17
processed data file: counts_KSHV.txt.gz

tag: cell type

value: Human embryonic kidney cells

value: siRNA control


title: wy_hz_18

description: treated wy hz 18


title: cmc_hz_30

description: control hz 30

value: carboxymethyllcellulose control


title: cmc_hz_38

description: control hz 38

value: liver


title: cmc_wt_32

description: control wt 32


title: cmc_wt_37

description: control wt 37


title: wy_hz_17

description: treated wy hz 17

value: SIRT6 heterozygote


title: wy_wt_20

description: treated wy wt 20

value: WY-14643 (50mg/kg for 12 hours)


title: wy_wt_24

description: treated wy wt 24

value: 11-13 months


title: wy_wt_6

description: treated wy wt 6


title: ERCC00048_polyA_ONT

status: Public on Dec 12 2019

city: Sherbrooke

first: Karine

last: Choquet

state: Québec

address: 3201 rue Jean-Mignault

department: Biochemistry and Functional Genomics

institute: Université de Sherbrooke

description: ERCC00048 with poly(A) tail addition

data_processing:
For Illumina RNA-seq datasets, paired-end reads were aligned to reference genomes using STAR (v2.5.1a) with default parameters (except outFilterMultimapNmax=101, outSJfilterOverhangMin=3  1  1  1, outSJfilterDistToOtherSJmin=0  0   0   0, alignIntronMin=11, alignEndsType=EndToEnd). The splicing index counts file was generated for each gene by counting the number of read pairs that span splice junctions by at least 3 nucleotides. '5SS_count' column represents read pair counts over an intron's 5'SS; '3SS_count' column represents read pair counts over an intron's 3'SS; 'splice_count' represents read pair counts that span the exon-exon junction of a spliced intron.
For Oxford Nanopore Technologies direct RNA sequencing datasets, raw signal fast5 files were basecalled using Albacore 2.2.7 (Oxford Nanopore Technologies Ltd.) with the following parameters: read_fast5_basecaller.py --flowcell FLO-MIN106 --kit SQK-RNA001 --recursive --output_format fast5,fastq --worker_threads 8 --save_path ${savePath} --input ${inputPath}. RNA sequences that pass basecalling thresholds were converted into DNA by substituting U to T bases before sequence alignment. Sequences were aligned to the reference genomes using minimap2 (version 2.10-r764-dirty) with recommended parameters for Oxford Nanopore Technologies direct RNA sequencing (-ax splice -uf -k14) and GMAP (version 2018-03-25) with default parameters. All analyses were performed using reads that pass the MINKNOW sequencing threshold (QC>7) and align uniquely to the genome.
For Oxford Nanopore Technologies direct RNA sequencing, the read ends dataset was generated by intersectin the start position of the read (end position of the RNA) to annotated gene features. “Intron” and “Exon” regions refer to their annotated features within protein coding genes. “Poly(A)” sites are defined as regions within 50 nucleotides of the end coordinate of annotated genes. In K562 datasets, the poly(A) region also includes regions that are within 50 nucleotides of RNA-PET annotations from cytoplasm and chromatin fractions in K562 ENCODE data (40). “Post_poly(A)” sites are defined as the region 50-550 nucleotides after the end of annotated genes. “Splice_sites” are defined as 50 nucleotides upstream and 10 nucleotides downstream of annotated 5’ splice sites. “Undetermined” categorizes reads that do not fit into one category and “other” represents read ends that do not align in the sense direction to annotated gene features (e.g. antisense transcripts, noncoding RNAs, intergenic transcription, etc.).
For Oxford Nanopore Technologies direct RNA sequencing, the splice dataset was generated by first intersecting reads with constitutively spliced introns. Constitutively spliced introns with “medium stringency” included in this dataset have at least 20 reads total RNA-seq dataset that span either splice junction by at least 4 nt overlap and more than 80% of the spanning reads are spliced. For reads mapping to constitutive introns, the features of read cigar strings were extracted for the 50 nt around the intron 5’ and 3’ splice sites and the entirety of the intron. Reads were called as ‘not spliced’ if the alignment file shows no indication of splicing within the 50 nt around each splice site, mapped portions of the read (rather than deletions) represent greater than 50% of the 50 nt around each splice site, and at least 75% of the read within the intron is aligned to the reference. Reads were called as ‘spliced’ if the alignment file displays the start or end of a splicing event within the 50nt around both splice sites and the size of the aligned splicing event is within 90-110% and 100 nt of the intron size. If aligned reads that map to introns do not meet these qualifications, the splicing event is characterized as ‘undetermined’ and not used in subsequent analyses. Other information included in this file include the soft clipped length at the start of the read alignment, the distance between the read start (or aligned RNA end) and the 3’SS of the intron, and whether or not the read start (or aligned RNA end) is close to the poly(A) site of a gene (within 150 nt upstream or any distance downstream of the annotated poly(A) site of the gene)
For Oxford Nanopore Technologies direct RNA sequencing, the intron pairs dataset was generated by identifying reads that span two or more introns. The coordinates and splicing status (from the splice dataset) of the two introns is recorded in the file.
For Oxford Nnaopore Technologies ERCC00048 direct RNA sequencing, read end difference was generated by calculating the distance between the aligned read end to the known 3' end of the in vitro transcribed RNA.
Genome_build: Reference genomes for human K562 and Drosophila S2 sequence alignments were obtained from ENSEMBLE GRCh38 (release-86) and FlyBase dm6 (r6.19), respectively.
Tab delimited ‘*SI_counts.txt’ files contain contain read pair counts spanning intron junctions. Tab delimited '*end_stats.txt' files contain the read name and feature that the start of the read (end of the RNA) maps to. Tab delimited ‘*medIntrons_discarded_splice_df.txt’ files contain the read name, coordinates of the intron, read length, alignment error rate, read start clipped length, distance from the read start (or RNA end) to the 3’SS of the intron, the determined splicing status, gene and poly(A) site separated by ‘_’, and a column for if the RNA ends near or after the gene’s poly(A) site. Tab delimited ‘*intron_pairs_df.txt’ files contain the read name, intron coordinates, and splicing status of two introns that one read maps to. Tab delimited "read_end_difference.txt" files contain the read name and distance from aligned sequence end to ERCC00048 transcript end position.
Supplementary_files_format_and_content: Tab delimited "read_end_difference.txt" files contain the read name and distance from aligned sequence end to ERCC00048 transcript end position.

organism: synthetic construct

source_name: ERCC

extract_protocol:
ERCC-00048 was synthesized as a G-block with a T7 promoter (IDT) and in vitro transcribed using the HiScribe T7 High Yield RNA Synthesis kit (NEB, E2040S). The resulting RNA was purified by gel extraction and tailed using E. coli poly(A) polymerase (Clontech/Takara, 2180) with ATP.
Direct RNA sequencing protocol using the SQK-RNA002 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software on the MinION instrument for ~2 hr with default settings for direct RNA sequencing.
direct RNA nanopore sequencing

treatment_protocol: Cells were labeled in media containing 500 uM 4-thiouridine for 8 minutes.

tag: cell treatment

value: in vitro transcribed ERCC-00048

tag: library prep

value: Oxford Nanopore Technologies direct RNA sequencing with SQK-RNA002 kit


title: ERCC00048_polyI_ONT

description: ERCC00048 with poly(I) tail addition

extract_protocol:
ERCC-00048 was synthesized as a G-block with a T7 promoter (IDT) and in vitro transcribed using the HiScribe T7 High Yield RNA Synthesis kit (NEB, E2040S). The resulting RNA was purified by gel extraction and tailed using yeast poly(A) polymerase (ThermoFisher, 74225Z25KU) with ITP.
Direct RNA sequencing protocol using the SQK-RNA002 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to a custom C10 adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software on the MinION instrument for ~2 hr with default settings for direct RNA sequencing.
direct RNA nanopore sequencing


title: wy_hz_5

description: treated wy hz 5

tag: treatment


title: wy_wt_10

description: treated wy wt 10


title: cmc_hz_36

description: control hz 36


title: wy_hz_15

description: treated wy hz 15


title: cmc_hz_35

description: control hz 35


title: S2_ONT_PlaB_1

description:
4sU labeled chromatin-associated RNA
S2 PlaB 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 1a

data_processing:
For Illumina RNA-seq datasets, paired-end reads were aligned to reference genomes using STAR (v2.5.1a) with default parameters (except outFilterMultimapNmax=101, outSJfilterOverhangMin=3  1  1  1, outSJfilterDistToOtherSJmin=0  0   0   0, alignIntronMin=11, alignEndsType=EndToEnd). The splicing index counts file was generated for each gene by counting the number of read pairs that span splice junctions by at least 3 nucleotides. '5SS_count' column represents read pair counts over an intron's 5'SS; '3SS_count' column represents read pair counts over an intron's 3'SS; 'splice_count' represents read pair counts that span the exon-exon junction of a spliced intron.
For Oxford Nanopore Technologies direct RNA sequencing datasets, raw signal fast5 files were basecalled using Albacore 2.2.7 (Oxford Nanopore Technologies Ltd.) with the following parameters: read_fast5_basecaller.py --flowcell FLO-MIN106 --kit SQK-RNA001 --recursive --output_format fast5,fastq --worker_threads 8 --save_path ${savePath} --input ${inputPath}. RNA sequences that pass basecalling thresholds were converted into DNA by substituting U to T bases before sequence alignment. Sequences were aligned to the reference genomes using minimap2 (version 2.10-r764-dirty) with recommended parameters for Oxford Nanopore Technologies direct RNA sequencing (-ax splice -uf -k14) and GMAP (version 2018-03-25) with default parameters. All analyses were performed using reads that pass the MINKNOW sequencing threshold (QC>7) and align uniquely to the genome.
For Oxford Nanopore Technologies direct RNA sequencing, the read ends dataset was generated by intersectin the start position of the read (end position of the RNA) to annotated gene features. “Intron” and “Exon” regions refer to their annotated features within protein coding genes. “Poly(A)” sites are defined as regions within 50 nucleotides of the end coordinate of annotated genes. In K562 datasets, the poly(A) region also includes regions that are within 50 nucleotides of RNA-PET annotations from cytoplasm and chromatin fractions in K562 ENCODE data (40). “Post_poly(A)” sites are defined as the region 50-550 nucleotides after the end of annotated genes. “Splice_sites” are defined as 50 nucleotides upstream and 10 nucleotides downstream of annotated 5’ splice sites. “Undetermined” categorizes reads that do not fit into one category and “other” represents read ends that do not align in the sense direction to annotated gene features (e.g. antisense transcripts, noncoding RNAs, intergenic transcription, etc.).
For Oxford Nanopore Technologies direct RNA sequencing, the splice dataset was generated by first intersecting reads with constitutively spliced introns. Constitutively spliced introns with “medium stringency” included in this dataset have at least 20 reads total RNA-seq dataset that span either splice junction by at least 4 nt overlap and more than 80% of the spanning reads are spliced. For reads mapping to constitutive introns, the features of read cigar strings were extracted for the 50 nt around the intron 5’ and 3’ splice sites and the entirety of the intron. Reads were called as ‘not spliced’ if the alignment file shows no indication of splicing within the 50 nt around each splice site, mapped portions of the read (rather than deletions) represent greater than 50% of the 50 nt around each splice site, and at least 75% of the read within the intron is aligned to the reference. Reads were called as ‘spliced’ if the alignment file displays the start or end of a splicing event within the 50nt around both splice sites and the size of the aligned splicing event is within 90-110% and 100 nt of the intron size. If aligned reads that map to introns do not meet these qualifications, the splicing event is characterized as ‘undetermined’ and not used in subsequent analyses. Other information included in this file include the soft clipped length at the start of the read alignment, the distance between the read start (or aligned RNA end) and the 3’SS of the intron, and whether or not the read start (or aligned RNA end) is close to the poly(A) site of a gene (within 150 nt upstream or any distance downstream of the annotated poly(A) site of the gene)
For Oxford Nanopore Technologies direct RNA sequencing, the intron pairs dataset was generated by identifying reads that span two or more introns. The coordinates and splicing status (from the splice dataset) of the two introns is recorded in the file.
Genome_build: Reference genomes for human K562 and Drosophila S2 sequence alignments were obtained from ENSEMBLE GRCh38 (release-86) and FlyBase dm6 (r6.19), respectively.
Supplementary_files_format_and_content: Tab delimited ‘*SI_counts.txt’ files contain contain read pair counts spanning intron junctions. Tab delimited '*end_stats.txt' files contain the read name and feature that the start of the read (end of the RNA) maps to. Tab delimited ‘*medIntrons_discarded_splice_df.txt’ files contain the read name, coordinates of the intron, read length, alignment error rate, read start clipped length, distance from the read start (or RNA end) to the 3’SS of the intron, the determined splicing status, gene and poly(A) site separated by ‘_’, and a column for if the RNA ends near or after the gene’s poly(A) site. Tab delimited ‘*intron_pairs_df.txt’ files contain the read name, intron coordinates, and splicing status of two introns that one read maps to.

source_name: S2 cells

growth_protocol: S2 cells (Expression Systems, 94-005) were maintained at 25°C in Schneider’s Drosophila medium containing 10% heat inactivated FBS, 50 U/ml penicillin and 50 ug/ml streptomycin.

extract_protocol:
S2 cells in an adherent layer at 95% confluency were lifted with gentle pipetting and proceeded imediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 50 million S2 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylacohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254).  Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using RiboMinus Eukaryotic Kit v2 (ThermoFisher, A15020). A poly(A) tail was added to the 3’ ends of the RNA sample with E. coli poly(A) polymerase (Clontech/Takara, 2180), incubating for 7.5 min at 37°C.
Direct RNA sequencing protocol using the SQK-RNA002 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software for 48 hours with default settings for direct RNA sequencing.

value: 100 nM PlaB 1 hour; 4-thiouridine (4sU), 500 uM, 8 minutes

tag: fractionation

value: chromatin purification

tag: 4su selection

value: 4sU biotinylation and streptavidin pulldown


title: S2_ONT_DMSO_1

description:
4sU labeled chromatin-associated RNA
S2 DMSO 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 1

extract_protocol:
S2 cells in an adherent layer at 95% confluency were lifted with gentle pipetting and proceeded imediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 50 million S2 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylacohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using RiboMinus Eukaryotic Kit v2 (ThermoFisher, A15020). A poly(A) tail was added to the 3’ ends of the RNA sample with E. coli poly(A) polymerase (Clontech/Takara, 2180), incubating for 7.5 min at 37°C.
Direct RNA sequencing protocol using the SQK-RNA002 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software for 48 hours with default settings for direct RNA sequencing.

value: 0.01% DMSO 1 hour; 4-thiouridine (4sU), 500 uM, 8 minutes


title: cmc_hz_71

description: control hz 71


title: cmc_hz_39

description: control hz 39


title: cmc_wt_31

description: control wt 31


title: S2_4sUchr_ONT_2

description:
S2 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 2.
Direct RNA nanopore sequencing.
4sU labeled chromatin-associated RNA.
Oxford Nanopore Technologies MinION

data_processing:
For Illumina RNA-seq datasets, paired-end reads were aligned to reference genomes using STAR (v2.5.1a) with default parameters (except outFilterMultimapNmax=101, outSJfilterOverhangMin=3 1 1 1, outSJfilterDistToOtherSJmin=0 0 0 0, alignIntronMin=11, alignEndsType=EndToEnd). The splicing index counts file was generated for each gene by counting the number of read pairs that span splice junctions by at least 3 nucleotides. '5SS_count' column represents read pair counts over an intron's 5'SS; '3SS_count' column represents read pair counts over an intron's 3'SS; 'splice_count' represents read pair counts that span the exon-exon junction of a spliced intron.
For Oxford Nanopore Technologies direct RNA sequencing datasets, raw signal fast5 files were basecalled using Albacore 2.2.7 (Oxford Nanopore Technologies Ltd.) with the following parameters: read_fast5_basecaller.py --flowcell FLO-MIN106 --kit SQK-RNA001 --recursive --output_format fast5,fastq --worker_threads 8 --save_path ${savePath} --input ${inputPath}. RNA sequences that pass basecalling thresholds were converted into DNA by substituting U to T bases before sequence alignment. Sequences were aligned to the reference genomes using minimap2 (version 2.10-r764-dirty) with recommended parameters for Oxford Nanopore Technologies direct RNA sequencing (-ax splice -uf -k14) and GMAP (version 2018-03-25) with default parameters. All analyses were performed using reads that pass the MINKNOW sequencing threshold (QC>7) and align uniquely to the genome.
For Oxford Nanopore Technologies direct RNA sequencing, the read ends dataset was generated by intersecting the start position of the read (end position of the RNA) to annotated gene features. “Intron” and “Exon” regions refer to their annotated features within protein coding genes. “Poly(A)” sites are defined as regions within 50 nucleotides of the end coordinate of annotated genes. In K562 datasets, the poly(A) region also includes regions that are within 50 nucleotides of RNA-PET annotations from cytoplasm and chromatin fractions in K562 ENCODE data (40). “Post_poly(A)” sites are defined as the region 50-550 nucleotides after the end of annotated genes. “Splice_sites” are defined as 50 nucleotides upstream and 10 nucleotides downstream of annotated 5’ splice sites. “Undetermined” categorizes reads that do not fit into one category and “other” represents read ends that do not align in the sense direction to annotated gene features (e.g. antisense transcripts, noncoding RNAs, intergenic transcription, etc.).
For Oxford Nanopore Technologies direct RNA sequencing, the splice dataset was generated by first intersecting reads with constitutively spliced introns. Constitutively spliced introns with “medium stringency” included in this dataset have at least 20 reads total RNA-seq dataset that span either splice junction by at least 4 nt overlap and more than 80% of the spanning reads are spliced. For reads mapping to constitutive introns, the features of read cigar strings were extracted for the 50 nt around the intron 5’ and 3’ splice sites and the entirety of the intron. Reads were called as ‘not spliced’ if the alignment file shows no indication of splicing within the 50 nt around each splice site, mapped portions of the read (rather than deletions) represent greater than 50% of the 50 nt around each splice site, and at least 75% of the read within the intron is aligned to the reference. Reads were called as ‘spliced’ if the alignment file displays the start or end of a splicing event within the 50nt around both splice sites and the size of the aligned splicing event is within 90-110% and 100 nt of the intron size. If aligned reads that map to introns do not meet these qualifications, the splicing event is characterized as ‘undetermined’ and not used in subsequent analyses. Other information included in this file include the soft clipped length at the start of the read alignment, the distance between the read start (or aligned RNA end) and the 3’SS of the intron, and whether or not the read start (or aligned RNA end) is close to the poly(A) site of a gene (within 150 nt upstream or any distance downstream of the annotated poly(A) site of the gene).
For Oxford Nanopore Technologies direct RNA sequencing, the intron pairs dataset was generated by identifying reads that span two or more introns. The coordinates and splicing status (from the splice dataset) of the two introns is recorded in the file.
Genome_build: Reference genomes for human K562 and Drosophila S2 sequence alignments were obtained from ENSEMBLE GRCh38 (release-86) and FlyBase dm6 (r6.19), respectively.
Supplementary_files_format_and_content: Tab-delimited ‘*SI_counts.txt’ files contain read pair counts spanning intron junctions. Tab-delimited '*end_stats.txt' files contain the read name and feature that the start of the read (end of the RNA) maps to. Tab-delimited ‘*medIntrons_discarded_splice_df.txt’ files contain the read name, coordinates of the intron, read length, alignment error rate, read start clipped length, distance from the read start (or RNA end) to the 3’SS of the intron, the determined splicing status, gene and poly(A) site separated by ‘_’, and a column for if the RNA ends near or after the gene’s poly(A) site. Tab-delimited ‘*intron_pairs_df.txt’ files contain the read name, intron coordinates, and splicing status of two introns that one read maps to.

extract_protocol:
S2 cells in an adherent layer at 95% confluency were lifted with gentle pipetting and proceeded immediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 50 million S2 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylalcohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using Ribo-Zero Gold rRNA Removal kit (Illumina, MRZG126). A poly(A) tail was added to the 3’ ends of the RNA sample with E. coli poly(A) polymerase (New England Biolabs, M0276S), incubating for 1 hour at 37°C.
Direct RNA sequencing protocol using the SQK-RNA001 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software for 48 hours with default settings for direct RNA sequencing.

tag: cell line

value: 4-thiouridine (4sU), 500 uM, 8 minutes

value: Oxford Nanopore Technologies direct RNA sequencing with SQK-RNA001 kit


title: S2_4sUchr_ONT_3

description:
S2 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 3.
Direct RNA nanopore sequencing.
4sU labeled chromatin-associated RNA.
Oxford Nanopore Technologies MinION


title: S2_4sUchr_ONT_1b

description:
S2 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 1 technical rep b.
Direct RNA nanopore sequencing.
4sU labeled chromatin-associated RNA.
Oxford Nanopore Technologies MinION


title: cmc_hz_34

description: control hz 34


title: wy_hz_14

description: treated wy hz 14


title: cmc_wt_78

description: control wt 78

value: wild type


title: wy_wt_19

description: treated wy wt 19


title: cmc_wt_76

description: control wt 76


title: cmc_wt_29

description: control wt 29