title: input_Bach1-KO_mSC

city: Philadelphia

first: Siqing

state: PENNSYLVANIA

address: 3401 Civic Center Blvd

department: Department of Pediatrics, Division of Hematology

institute: Children’s Hospital of Philadelphia

data_processing:
The FASTQ data were trimmed adaptor by trim_galore (bioinformatics.babraham.ac.uk)
ChIP-seq data mapped to mouse genome mm9 with Bowtie2  in pair-end mode.
ChIP-seq peaks were called by MACS1.4  and MACS2 .
RNA-seq data were mapped to mouse genome mm9 using STAR  (020201) and assembled by StringTie  (1.3.4d) with parameter -eB (did not consider novel isoforms) to get RPKM.
The RPKM value of genes was converted to Transcripts per million (TPM)  to eliminate the statistical biases inherent in the RPKM measure.
Differentially expressed genes were generated by limma  with fold change >1.5 and p <0.05.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-delimited text files for ChIP-seq peaks in sample_1 to sample_13. Tab-delimited text files for RNA-seq RPKM and TPM for sample_18 to sample_24. Excel file for differentially expressed genes for sample_18 to sample_24.

source_name: Bach1 knock out mESCs

growth_protocol: mESCs (E14Tg2A line) were kindly provided by Dr. Fei Lan. mESCs were maintained in 90% Dulbecco’s Modified Eagle Medium (DMEM) (Life technologies, Carlsbad), 10% fetal bovine serum (FBS) (Life technologies, Carlsbad, CA), 1% non-essential amino acid (NEAA), 1% penicillin and streptomycin (100× stock) (Gibco-Thermo Fisher, Waltham, MA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and 200 Units/mL murine leukemia inhibitory factor (LIF) (ESGRO; Merck-Millipore, Germany). mESC expansion was performed in gelatin-coated flasks (BBI Life Sciences, China).

extract_protocol:
Chromatin immunoprecipitation (ChIP) sequencing: Cells were cross-linked with 1% formaldehyde for 10 minutes, quenched with 0.125 M glycine for 10 minutes; then, the cell pellets were resuspended in high salt Lysis buffer (50 mM HEPES/KOH, PH 7.5, 500 mM NaCl, 1% Trixon-X 100, 0.05% SDS, 10 mM EDTA, and proteinase inhibitors), and sonicated with a Bioruptor (Diagenode) for 20 minutes in 30-second intervals to shear the chromatin into 200- to 400-bp lengths.  Chromatin was immunoprecipitated with the indicated antibodies and Dynabeads protein A and protein G (Thermo Fisher, Waltham, MA). For, Bach1-Flag ChIP, DoxBach1 mESCs were treated with 0.5 μg/μL doxycycline (Selleck chemicals, Houston, TX) for 6 hours. Then cells were cross-linked, quenched, and washed as described above. Cell nucleus were extracted before sonication. And the sonicated chromatin was incubated with anti-Flag affinity M2 agarose gel (Sigma-Aldrich, St. Louis, MO) overnight at 4℃. RNA-seq: Non-ribosomal RNA was isolated from 1 μg total RNA by using a TrueLib Poly (A) mRNA Magnetic Isolation Module (Excell Bio, Shanghai, China), and sequencing libraries were prepared with the TrueLib mRNA Library Prep Kit for Illumina (ExCell Bio, Shanghai, China).
ChIP DNA libraries and input DNA samples were prepared as directed by protocol of KAPA Hyper Prep Kit for Illumina (Kapa Biosystems, Wilmington, MA).

treatment_protocol: Bach1-Flag expression was induced by treating DoxBach1 mESCs with 0.5 μg/μL doxycycline (Selleck chemicals, Houston, TX) for 6 hours.

value: mouse embryonic stem cells (mESCs)

tag: genotype/variation

value: Bach1 -/-


title: CRISPRi_ILF3_rep2

status: Public on Jan 12 2021

type: SRA

city: Stanford

first: Howard

middle: Y

last: Chang

state: California

address: 269 Campus Drive

country: USA

institute: Stanford University

library_source: genomic

data_processing:
Library strategy: targeted amplicon sequencing
reads were mapped directly to TLR7 reference Xa and Xi allele and counted at each allele for d score analysis. The co-factors with d scoreco-factor - d scoreCtrl >0.1 were labeled as essential XIST co-factors for TLR7 XCI maintenance
Genome_build: hg19
Supplementary_files_format_and_content: Matrix table with allele-specific read counts of TLR7 for every individual sgRNA perturbation

molecule: genomic DNA

organism: Homo sapiens

source_name: dCas9-Krab+ GM B cell line

growth_protocol: GM related cell lines were cultured in RPMI 1640 (Thermo Fisher Scientific) with 10% FBS (Thermo Fisher Scientific) and 1% Penicillin-Streptomycin (Gibco).

extract_protocol:
RNA was extracted using Direct-zol-96 RNA Kit (Zymo). TLR7 RT-PCR was performed using Stratagene Brilliant II SYBR Green QRT-PCR Master Mix (50°C for 30 min, 95°C for 10 min, then 35 cycles of 95°C for 30 seconds, 60°C for 1 min, and 72°C for  30 seconds, finally 72°C for 5 min).
The second PCR with Ad1_TLR7_F and Ad2_TLR7_R primers was performed using NEBNext Master Mix (2.5uL 1st PCR product with 500 nM primers in program: 98°C for 1 min, 20 cycles of 98°C for 30 seconds, 60°C for 30 seconds, and then 72°C for 40 seconds, finally 72°C for 5 min). The third PCR with indexed primers containing barcodes was performed using  NEBNext Master Mix (2uL of 1:10 diluted 2nd PCR product with 1uM indexed primers in program: 98°C for 1 min, 10 cycles of 98°C for 30 seconds, 72°C for 30 seconds, and then 72°C for 40 seconds, finally 72°C for 5 min). Finally, all the samples were pooled and purified using Zymo DNA clean & concentrator. The library was size-selected to 250-300 bp. The gel slices were cut and the DNA was recovered by Zymo Gel DNA recovery kit (Zymo Research). The concentration of the library was quantified by KAPA Library Quantification Kit for Illumina (Roche) and sequenced on an Illumina Miseq 2X75 cycle.

tag: cell line

tag: cell type

value: GM B cell

tag: genotype


title: CRISPRi_ILF2_rep2


title: G19-A06

city: Vladivostok

first: Valeriia

last: Gulaia

address: 10 Ajax Bay

department: School of Biomedicine

country: Russia

institute: Far Eastern Federal University

library_source: transcriptomic

data_processing:
quality control with FastQC (version 0.11.9)
trimming Nextera sequencing adapters with Trim Galore (parameter “--nextera”)
mapping to GRCh38.p12 human reference genome using STAR-2.6.0c
exclusion of cells expressing 0 genes
normal cells exclusion on the basis of particular cell markers: all hematopoietic cell - CD45 (PTPRC); naïve CD4+ T cells – IL7R, CCR7;  memory CD4+ cells – S100A4; monocytes – CD14, LYZ, FCGR3A, MS4A7; B-cells – MS4A1 (CD20); CD8+ T cells – CD8A; NK-cells – GNLY (NKG5), NKG7; dendritic cells – FCER1A, GST3; platelet – PPBP; stromal cells – CD34; endothelial cells – PECAM1 (CD31)
analysis of copy number variations (CNVs) using CaSpER
cell cycle classification performed by Cyclone
population analysis performed by SingleCellExperiment, Seurat, and Scanpy
Genome_build: GRCh38.p12
Supplementary_files_format_and_content: loom data file containing population analysis from Seurat

molecule: polyA RNA

source_name: SOX2+ glioma nucleus

extract_protocol:
Frozen tumor tissue of approximate size 0.2-0.5 cm3 was placed in cold tissue lysis buffer (5M NaCl, 1M MgCl2, 1M Tris buffer (pH 8.0), 10% NP40 (Sigma, USA), 1uM DTT (Invitrogen), 1x Complete protease inhibitor (Roche), 0.4 U/ul RNAseIn (Takara Bio, Japan), 0.2 U/ul SuperaseIn  (ThermoFisher Scientific) and incubated on ice for 15 min with gentle swirling every 2-3 minutes. Then the tissue was triturated, filtered through 40um cell strainer and centrifuged at 1000g for 5 min at 4°C. The sedimented nuclei were resuspended in cold staining buffer (RNAse-free PBS pH 7.4, 1% BSA, RNAseIn, 1M MgCl2, 1mM DTT) and stained with anti-Sox2 antibody (Invitrogen) or respective IgG isotype control (BD), Alexa Fluor 488 Goat anti-Rabbit IgG antibody (Invitrogen) was used as secondary. Nuclei were visualized with 7-AAD (Sigma) staining. Thereafter, Sox2 positive nuclei were sorted a FACS Aria I or III at 4°C in skirted 96-well PCR plates containing 2 ul cell lysis buffer (2U/ul RNAseIn in 0,2% (vol/vol) Triton X-100 (Sigma)). After sorting plates were immediately sealed and placed on dry ice.
The library preparation was done using Nextera XT DNA Library Preparation Kit (Illumina) according to the manufacturer instructions, the Illumina Nextera Adapters (sequences provided on http://ngs.biodiv.tw/NGSCore/wp-content/uploads/Documents/illumina-adapter-sequences-1000000002694-08.pdf) were synthetized and HPLC purified in Biomers (http://www.biomers.net/).
single cell RNA-seq

tag: tissue

value: patient derived glioma tissue

tag: sample name

value: wt


title: CRISPRi_ILF3_rep1


title: input_WT_mESC

last: Wang

organism: Mus musculus

source_name: mESCs

value: wild type


title: G19-A05


title: Pangasianodon hypophthalmus 9dpf_rep1

city: Auburn University

first: Xu

state: AL

address: 273 CASIC Building, Auburn Research Park, 559 Devall Dr

department: Pathobiology, College of Veterinary Medicine

institute: Auburn University

description: Illumina mRNA-seq

data_processing:
Illumina CASAVA 1.9.1 software was used for basecalling. Fastqc software was used for quality check.
Sequenced reads were trimmed for adaptor sequence using Trimmomatic v 0.36, and masked for low-complexity or low-quality sequence.
The ramaining high-quality clean reads were mapped to pangasius whole genome using STAR v2.7.0
HTSeq-count was conducted to calculate the number of aligned reads of each gene overlapping its exons.
DESeq2 was employed to calculate the log2-fold change (log2FC), a criterion with |log2FC| ≥ 1 and p-value < 0.05 was used as the threshold for evaluating the DEGs.
Genome_build: Pangasianodon hypophthalmus genome GENO_Phyp_1.0 (GenBank assembly accession GCA_009078355.1)
Supplementary_files_format_and_content: Gzipped, tab-delimiited text file inlcuding raw reads counts for every gene in every sample

molecule: total RNA

organism: Pangasianodon hypophthalmus

source_name: whole fry

extract_protocol:
RNA extraction was conducted using the Qiagen RNeasy Plus Kit following the manufacture protocols.
The RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). Ribosomal RNA depletion was conducted using Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA). RNA sequencing library preparation was performed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA). Enriched RNAs were fragmented for 15 minutes at 94 °C. The first strand and second strand cDNA were subsequently synthesized. The cDNA fragments were end-repaired and adenylated at 3'ends, and a universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA) and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA).

tag: age

value: 9 days post fertilization

value: whole-body


title: G19-A04


title: CRISPRi_ILF2_rep1


title: H3K27ac_ChIP_in_Bach1-KO

tag: chip antibody

value: H3K27ac (Millipore, Cat# 07-360)


title: H3K27ac_ChIP_in_WT


title: CRISPRi_HNRNPUL2_rep2


title: P. putida JE1657 NH4 rep 3

city: Richland

first: Joshua

last: Elmore

state: WA

address: 902 Battelle Blvd

institute: PNNL

data_processing:
Reads were trimmed and filtered using Geneious v11.1.5 with an error probability limit of 0.05
Filtered reads were mapped to the reference genome using Geneious v11.1.5 default "Map to Reference: Geneious RNA" parameters
Raw read count and FPKM were calculated for each annotated gene using "Calculate Expression Levels" within Geneious v11.1.5 and exported
Genome_build: NC_002947 with additional sequences for heterologous genes
Supplementary_files_format_and_content: csv files with Raw Read Count and FPKM values for each sample

organism: Pseudomonas putida KT2440

source_name: bacterial cells

growth_protocol: Samples were prepared by pre-cultivating strains in MME medium with glucose and the nitrogen source used in the experiment (ammonium or nitrate) until cells reached stationary phase. Experimental cultures were innoculated to an OD600 of 0.01 and incubated until OD600 reached ~0.2 and harvested by centrifugation at 4 C.

extract_protocol:
RNAs were extracted using Trizol reagent and QIAGEN Rneasy kit following manufacturer's protocol.
rRNAs were depleted using RiboZero rRNA removal kit, depleted RNAs were purified using ZymoResearch RNA Clean & Concentrator-5 kit, and libraries were prepared using Illumina's ScriptSeq v2 RNA-Seq Library Preparation Kit

tag: strain

tag: nitrogen source

value: ammonium

tag: growth phase harvested

value: mid-log


title: G19-A02


title: Pangasianodon hypophthalmus 11dpf_rep1

description: Illumina mRNA-seq

value: 11 days post fertilization


title: Pangasianodon hypophthalmus 8dpf_rep2

description: Illumina mRNA-seq

value: 8 days post fertilization


title: CRISPRi_HNRNPUL2_rep1


title: H3K4me3_ChIP_in_WT

value: H3K4me3 (Cell Signaling Technology, Cat# 9753S)


title: G19-A01


title: H3K4me3_ChIP_in_Bach1-KO


title: Pangasianodon hypophthalmus 6dpf_rep2

description: Illumina mRNA-seq

value: 6 days post fertilization


title: CRISPRi_HNRNPUL1_rep1


title: G19-A03


title: Nanog_ChIP_in_Bach1-KO_rep2

value: Nanog (Cell Signaling Technology, Cat# 8822)


title: Pangasianodon hypophthalmus 8dpf_rep1

description: Illumina mRNA-seq


title: Pangasianodon hypophthalmus 4dpf_rep1

description: Illumina mRNA-seq

value: 4 days post fertilization


title: G16-H10


title: Pangasianodon hypophthalmus 2dpf_rep2

description: Illumina mRNA-seq

value: 2 days post fertilization


title: HL2

city: Wuxi

first: Jun

last: Qiang

state: Jiangsu

address: 9 Shanshi East road

country: China

institute: Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences

description: The DO of the three treatment tanks was rapidly reduced to 0.7-0.8mg/L by pumping nitrogen into water from a nitrogen gas cylinder.

data_processing:
Using the Illumina paired-end RNA-seq approach
We aligned reads of  all samples to the reference genome using HISAT package
StringTie and Ballgown was used to estimate the expression levels of all transcripts. StringTie was used to perform expression level for mRNAs by calculating FPKM
The differentially expressed mRNAs and genes were selected with log2 (fold change) >1 or log2 (fold change) <-1 and with statistical significance (p value < 0.05) by R package – Ballgown.
Genome_build: http://asia.ensembl.org/Oreochromis_niloticus/Info/Index
Supplementary_files_format_and_content: xls include FPKM values for each Sample

organism: Oreochromis niloticus

source_name: liver tissues

growth_protocol: A total of 90 experimental fish were used for the main acute hypoxic stress experiment. Ninety experimental fish were randomly placed in six tanks (15 fish per tank), three treatment tanks and three 5mg/L control tanks.

extract_protocol:
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: The DO of the three treatment tanks was rapidly reduced to 96h-LH50 by pumping nitrogen into water from a nitrogen gas cylinder. Real-time reading with a DO meter were used to control the DO in water by adjusting oxygen intake. The sampling time was 96h.

value: liver

tag: treatment

value: 0.7-0.8mg/L DO treatment tanks


title: G16-H12

value: patient derived glioma tissue


title: CRISPRi_HNRNPU_rep1

value: GM B cell


title: G16-H08

value: wt


title: CRISPRi_HNRNPUL1_rep2


title: G16-H09


title: HL1


title: Pangasianodon hypophthalmus 6dpf_rep1

description: Illumina mRNA-seq


title: CL3

description: 5mg/L control tank

value: 5mg/L DO control tanks


title: Pangasianodon hypophthalmus 2dpf_rep1

description: Illumina mRNA-seq

value: whole-body


title: G16-H07


title: CL2

description: 5mg/L control tank


title: G16-H06


title: CRISPRi_HNRNPM_rep2


title: A. aculeatus BCC199 condition B [BCC199_lot2_B]

city: Khlong Luang

first: Wuttichai

last: Mhuantong

state: Pathum Thani

address: National Center for Genetic Engineering and Biotechnology (BIOTEC)

country: Thailand

institute: BIOTEC

data_processing:
Sequencing, and bioinformatic analyses were performed by Novogene, Inc.
Illumina CASAVA 1.8 software was used for basecalling.
Sequenced reads for adaptor sequence, and low-quality sequence were trimmed and removed using Fastp.
Genome_build: NOT PROVIDED; REQUESTED
Supplementary_files_format_and_content: FPKM

organism: Aspergillus aculeatus

source_name: Biological replicate of BCC199

growth_protocol: Wheat bran and Soy bean mill

extract_protocol:
Fungal cell were cultured for 3 days at 30 degree, frozen with liquid nitrogen, and RNA was harvested using Trizol reagent, following RNAeasy kit (Qiagen), contaminated DNA was eliminate by RNAase free DNase kit (Qiagen). Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: 72 hours, 30 degree Celsius

tag: medium

value: Wheat bran and soy bean


title: A. aculeatus BCC199 condition C [BCC199_lot2_C]

source_name: Technical replicate of BCC199


title: G16-H11


title: G16-H05


title: Pangasianodon hypophthalmus 4dpf_rep2

description: Illumina mRNA-seq


title: CL1

description: 5mg/L control tank


title: CRISPRi_HNRNPR_rep2


title: HL3


title: G16-H04


title: A. aculeatus BCC199 condition A2 [BCC199_lot1_A2]

source_name: Technical replicate of lot1 BCC199


title: G16-H03


title: Nanog_ChIP_in_WT _rep2

value: mouse embryonic stem cells (mESCs)

value: wild type


title: CRISPRi_HNRNPR_rep1


title: CRISPRi_HNRNPL_rep2


title: CRISPRi_HNRNPL_rep1


title: A. aculeatus HUT2365 condition C [HUT2365_lot2_C]

source_name: Biological replicate of HUT2365


title: CRISPRi_HNRNPK_rep2


title: A. aculeatus HUT2365 condition B [HUT2365_lot2_B]


title: CRISPRi_HNRNPK_rep1


title: G16-H01


title: CRISPRi_HNRNPM_rep1


title: A. aculeatus HUT2365 condition A [HUT2365_lot1_A]


title: CRISPRi_HNRNPDL_rep2


title: A. aculeatus BCC56535 condition D [BCC56535_lot2_D]

source_name: Biological replicate of BCC56535


title: CRISPRi_HNRNPDL_rep1


title: A. aculeatus BCC199 condition A1 [BCC199_lot1_A1]


title: G16-G11


title: A. aculeatus BCC56535 condition C [BCC56535_lot2_C]


title: CRISPRi_HNRNPD_rep2


title: A. aculeatus BCC56535 condition A2 [BCC56535_lot1_A2]

source_name: Technical replicate of lot1 BCC56535


title: CRISPRi_HNRNPD_rep1


title: CRISPRi_HNRNPC_rep2


title: G16-G09


title: G16-G08


title: A. aculeatus BCC56535 condition A1 [BCC56535_lot1_A1]


title: CRISPRi_HNRNPC_rep1


title: G16-G07


title: G16-G12


title: CRISPRi_HNRNPAB_rep2


title: G16-G10


title: G16-G06


title: JiTCS2000-3

city: Brisbane

first: Jianhua

last: Guo

address: Research Road, Level 4 Gehrmann Building

department: Australian Centre for Water and Environmental Biotechnology

country: Australia

institute: University of Queensland

data_processing:
base calling through an integrated primary analysis software called RTA (Real Time Analysis. v1.18).
The NGS QC toolkit (version 2.3.3) was used to treat the raw sequence reads to trim the 3’-end residual adaptors and primers, and the ambiguous characters in the reads were removed. Then, the sequence reads consisting of at least 85% bases were progressively trimmed at the 3’-ends until a quality value ≥ 20 were kept. Downstream analyses were performed using the generated clean reads of no shorter than 75 bp.
The clean reads of each sample were aligned to the E. coli reference genome (NC_000913) using SeqAlto (version 0.5). Cufflinks (version 2.2.1) was used to calculate the strand-specific coverage for each gene, and to analyze the differential expression in triplicate bacterial cell cultures. The statistical analyses and visualization were conducted using CummeRbund package in R (http://compbio.mit.edu/cummeRbund/).
Gene expression was calculated as fragments per kilobase of a gene per million mapped reads (FPKM, a normalized value generated from the frequency of detection and the length of a given gene.
Genome_build: NC_000913.3
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...

organism: Escherichia coli

source_name: E.coli DH5a bacteria

growth_protocol: liquid LB culture at 37 °C

extract_protocol:
Bacterial cells were then harvested by centrifugation at 8000 × g for 10 min. Total RNA was extracted from the mutants using the QIAGEN miRNeasy Mini Kit (QIAGEN, Germany) manufacturer’s protocol with one extra bead-beating step to completely lyse the bacterial cells.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: E.coli DH5a was cultured in 10 mL LB containing 0, 20ug/L and 2 mg/L triclosan for 2 h, in triplicate, at 37 °C, without shaking

value: 2 mg/L triclosan for 2 h


title: A. aculeatus BCC56535 condition B [BCC56535_lot2_B]


title: JiTCS2000-2


title: CRISPRi_HNRNPAB_rep1


title: CT1258 cell line - 24h PDA-66 rep3

city: Hannover

first: Jan Torben

last: Schille

address: Buenteweg 9

department: Small Animal Clinic

country: Germany

institute: University of Veterinary Medicine Hannover

data_processing:
For the human PC-3 and LNCaP cells, mapping and read counting were conducted using the RNA Express (version: 1.0.0) workflow within the Illumina basespace environment. In brief, after mapping to the human HG19 reference genome using the STAR (2.3.1s) aligner read counts for 23710 annotated RefSeq genes were generated.   For CT1258, sequences were aligned to the canine genome (Broad CanFam3.1/canFam3, Sep. 2011) using the Burrows-Wheeler Aligner (BWA). For each of the 24,581 annotated protein-coding canine genes (EMBL gene ID nomenclature), the aligned reads were counted using the R package GAGE.
Differential gene expression analyses for canine and human samples were conducted using the BioconductorR package edgeR 3.14.0. The average total read counts mapped within annotated genes were 12.2M (STD: 0.7M), 15.8M (STD: 1.1M), 13.5M (STD: 0.9M) for CT1258, PC-3 and LNCaP samples, respectively. Each PDA-66 treatment group (12 h or 24 h) was compared to the respective control cells treated with DMSO.
After multidimensional scaling and plotting of the data, one PC-3 DMSO treated control sample (PD1) was identified as outlier and was excluded from further analyses so that the PC-3-24 h-DMSO control group consisted of only two independent samples.
Genes with a false discovery rate adjusted p-value (FDR) of less than 0.001 were considered to have significantly different expressions compared to the control samples. These genes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for functional enrichment analyses using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Annotation Tool.
Genome_build: HG19 and CanFam3.1
Supplementary_files_format_and_content: edgeR gene counts for the three cell lines
Gene tables with fold changes and FDRs for every treatment vs control

organism: Canis lupus familiaris

source_name: Prostate carcinoma cells

growth_protocol: PC-3 cells were cultivated in DMEM/Ham´s F-12 medium (Biochrom GmbH, Berlin, Germany). LNCaP cells were grown in RPMI 1640 medium (Biochrom GmbH). CT1258 cells were cultivated in medium 199 (Live Technologies GmbH, Darmstadt, Germany). All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS Superior, Biochrom GmbH) and 2% penicillin/streptomycin (Biochrom GmbH). All cells were cultivated at 37 °C in a humidified atmosphere of 5% CO2.

extract_protocol:
Total RNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen GmbH, Hilden, Germany) in accordance with the manufacturer’s protocol. RNA integrity numbers (RIN) were determined using a Bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA, USA).
Sequencing libraries were prepared using 1 μg total RNA with RIN > 7. PolyA RNA was enriched and ligated to sequencing adapters using the NEBNext Ultra RNA preparation kit (New England Biolabs Inc., Ipswich, MA, USA) in accordance with the manufacturer’s protocols.

treatment_protocol: Cells were treated with 15 µM PDA-66 for 12 h or 24 h with 3 replicates per cell line and incubation period. Control cells were treated with 0.15% (v/v) DMSO as PDA-66 is dissolved in DMSO.

value: CT1258 (TihoDProAdcarc1258)

value: prostate adenocarcinoma cell line

value: treated with 15 µM PDA-66 for 24 h


title: CT1258 cell line - 24h PDA-66 rep2


title: G16-G05


title: Mf EVs A

type: RNA

city: Bethesda

first: Sasisekhar

last: Bennuru

state: MD

address: 10 Center Dr, Bldg 10, Rm 11N208

department: LPD

institute: NIH/NIAID

hyb_protocol: The labelled RNAs were then hybridized overnight on the Affymetrix GeneChip miRNA arrays 4.0.

scan_protocol: The .cel files were imported into JMP Genomics Suite (SAS Institute), RMA normalized and ANOVA analysis was performed.

data_processing:
The Genechip miRNA 4.0 Affymetrix arrays were imported into TACX (Transcriptome Analysis Console, Applied Biosystems) and analyzed with default settings of the miRNA 4.0.tac configuration file.
The summary of the results is based on the RMA-DABG algorithm of TACX.
The Robust Multi-chip Analysis takes into consideration background adjustment, quantile normalization, and summarization.
The presence or absence of a miRNA species is based on the criteria of a probe set being detected in at least 50% of the samples above the background signal (DABG: Detected Above Background).
The array comprises miRNA probes from different organisms. Brugia specific miRNAs in the EVs can be missed in this approach and would require a large sequencing approach.
In this approach only well-known miRNAs from B. malayi and miRNAs that are highly similar with other organisms can be identified. Thus, we can expect to see multiple hits for the same miRNA from multiple organisms. Hence, we also grouped them all as unique miRNAs. The data has been deposited in order to make it available to all readers.

label: biotin

organism: Brugia malayi

source_name: Microfilariae

label_protocol: One microgram of total RNA for each replicate was used for labeling with the FlashTag™ Biotin HSR RNA Labeling Kit (Affymetrix-ThermoFisher Scientific, cat#901911, Walthman, MA).

growth_protocol: Mf were incubated at 37 degrees for 24 hours

extract_protocol: RNA (including small RNAs) was extracted using the miRNA easy kit from QIAGEN (Hilden, Germany).

treatment_protocol: After incubation, the parasites were pelleted. Part of the supernatant was collected for supernatant analysis, the other was used to isolated EVs. The parasite pellet was also collected for further analysis.

tag: sample type

value: EVs


title: JiTCS2000-1


title: Mf supernatant C

value: supernatant


title: Mf EVs B


title: Mf pellet B

value: pellet


title: CRISPRi_HNRNPA3_rep2


title: CT1258 cell line - 24h PDA-66 rep1


title: JiTCS20-3

value: 20 ug/L triclosan for 2 h


title: Mf pellet A


title: G16-G04

value: patient derived glioma tissue


title: Mf supernatant B


title: Mf supernatant A


title: Mf EVs C


title: Mf pellet C


title: CRISPRi_HNRNPA3_rep1

value: GM B cell


title: JiTCS20-2


title: G16-G03

value: wt


title: CT1258 cell line - 12h PDA-66 rep3

value: treated with 15 µM PDA-66 for 12 h


title: CT1258 cell line - 12h PDA-66 rep2


title: CT1258 cell line - 12h PDA-66 rep1


title: JiTCS20-1


title: CRISPRi_HNRNPA1_rep1


title: G16-G02


title: CRISPRi_HNRNPA0_rep2


title: CT1258 cell line - control rep3

value: treated with 0.15% (v/v) DMSO for 24 h


title: CRISPRi_HNRNPA1_rep2


title: Jicon-3

value: control


title: G16-G01


title: G16-F12


title: Jicon-2


title: Jicon-1


title: CT1258 cell line - control rep2


title: CRISPRi_FUBP1_rep2


title: CT1258 cell line - control rep1


title: G16-F11


title: G16-F10


title: middle-aged LT-HSC + IGF1 rep 2

city: Bar Harbor

first: Jennifer

last: Trowbridge

state: Maine

address: 600 Main Street

department: Research

institute: The Jackson Laboratory

description:
M2_IGF1_04296
Ad2.8 (CAGAGAGG)
19NGS_002_trowbridge_peak_counts_sub.txt

data_processing:
FASTQ files per each sample were initially analyzed with esATAC v1.12.0 under R v3.6.0 using the mm10 reference genome. Read counts were extracted from peak call and alignment data using DiffBind v2.12.0. edgeR v3.26.4 was used to calculate differential binding of the TMM-normalized counts. If a region did not have at least one sample with cpm > 1, then the region was omitted from the pairwise comparison’s differential analysis. The negative binomial conditional common likelihood was maximized to estimate a common dispersion value across all features/regions. Exact tests were used to elucidate statistical differences between the two sample groups of negative binomially distributed counts producing p-values per test. The Benjamini and Hochberg’s algorithm (p-value adjustment) was used to control the false discovery rate (FDR). Features with an FDR-adjusted p-value < 0.05 were declared statistically significant. Differential binding results were annotated using HOMER v4.10.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: 19NGS_002_trowbridge_peak_counts_sub.txt: Tab-delimited text, counts per peak region.

source_name: bone marrow

extract_protocol:
Method for extraction was adapted from Buenrostro et al. Nat Meth 2013. Briefly, nuclei were prepared, DNA is transposed and purified followed by PCR amplification. Library quality and concentration were evaluated using the Bioanalyzer High Sensitivity DNA Analysis assay (Agilent Technologies) and quantitative PCR (Kapa Biosystems).
ATAC libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: Middle-aged LT-HSCs from 2 independent replicates of pooled mice were sorted directly into a 96-well plate for 18hrs in StemSpan SFEM II, SCF (100 ng/ml), TPO (50 ng/ml), Pen-Strep (1%) with vehicle (.1% BSA/PBS) or IGF1 (100ng/ml) stimulation at 37oC and 5% CO2 before being processed for ATAC-seq.

value: Middle age

value: LT-HSC


title: middle-aged LT-HSC + IGF1 rep 1

description:
M1_IGF1_04295
Ad2.7 (CTCTCTAC)
19NGS_002_trowbridge_peak_counts_sub.txt


title: CRISPRi_ELAVL1_rep2


title: middle-aged LT-HSC rep 2

description:
M2_CNT_04294
Ad2.6 (TAGGCATG)
19NGS_002_trowbridge_peak_counts_sub.txt

value: Control


title: CRISPRi_ELAVL1_rep1


title: G16-F08


title: CRISPRi_HNRNPA0_rep1


title: ZT22_20190219_AR12

city: Waltham

first: Michael

last: Rosbash

address: 415 South street

institute: Brandeis University

data_processing:
bcl2fastq v2.20 was used to convert the raw sequencing files into fastq files
zUMI v2.1.1 was used to align the reads to Drosophila genome
Genome_build: dm6
Supplementary_files_format_and_content: read counts

organism: Drosophila melanogaster

source_name: Adult neurons

growth_protocol: Flies were reared in standard cornmeal medium with yeast under 12:12 h LD cycles.

extract_protocol: The single cell library prep was based on CEL-seq2 with some modifications. Two rounds in vitro transcriptions were used to amplify ploly A mRNA. The RNA from the first round of IVT was purified by 0.8 fold RNAClean XP beads (0.8 fold ratio) and was used as a substrate for another round of first strand synthesis at 42 °C for 2 hours using second round primers (2nd Round Primer: NNNNNN), RNA:DNA hybrids were digested with RNase H (Thermal Fisher #18021014) at 37 °C for 30 minutes. The second round second strand synthesis was carried out at 16 °C with a T7-RA5 primer (GCCGGTAATACGACTCACTATAGGGAGTTCTACAGTCCGACGATC). The resulting cDNA underwent another final second round IVT step at 37 °C overnight and followed EXO-SAP treatment (Affymetrix 78200) for 15 minutes at 37 °C. Other steps were performed as described in the CEL-Seq2 protocol.

treatment_protocol: No treatment

value: Clk856-Gal4 labeled neurons

value: Adult

value: LD


title: ZT22_20190309_AR01


title: middle-aged LT-HSC rep 1

description:
M1_CNT_04293
Ad2.5 (GGACTCCT)
19NGS_002_trowbridge_peak_counts_sub.txt


title: ZT22_20190309_AR02


title: ZT22_20190228_AR012


title: G16-F06


title: G16-F07


title: CRISPRi_EIF4A3_rep1

source_name: dCas9-Krab+ GM B cell line


title: CRISPRi_EIF4A3_rep2


title: G16-F05

source_name: SOX2+ glioma nucleus


title: ZT22_20190219_AR07


title: CRISPRi_FUBP1_rep1


title: G16-F09


title: G16-F04


title: ZT18_20190301_AR12


title: CRISPRi_DHX9_rep2


title: ZT22_20190219_AR04


title: G16-F03


title: CRISPRi_DHX9_rep1


title: 2770-11_Lagor_Doerfler_080318-114892894

city: Houston

first: Alexandria

last: Doerfler

state: TX

address: 1 Baylor Plaza

department: Molecular Physiology and Biophysics

institute: Baylor College of Medicine

data_processing:
Quality control on sequencing reads was done using FastQC software
Paired end reads were merged then aligned using STAR software with the parameters “—outFilterScoreMinOverLread 0 – outFilterMatchNminOverLread 0 – outFilterMismatchNmax 2”
Genome_build: mm9
Supplementary_files_format_and_content: Tab delimited text files with gene counts

source_name: Liver tissue

extract_protocol:
Intestines were isolated from mice then flushed with PBS, cut open to lay flat, seperated into duodenum, jejunum, and ileum, and the bottom two cms were scraped to collect the epithelial cells which were then snap frozen in liquid N2. For liver tissue, the liver was perfused, then 50 mg of tissue was cut and snap frozen in liquid N2
Sequenced RNA samples first passed quality control using the RNA tape on Tapestation 4200 (Agilent) and were quantified with Qubit Fluorometer (Thermo Fisher). Only those samples that passed quality control were used for analysis. The RNA libraries were prepared with QIAseq Stranded Total RNA library Kit (Qiagen) using 100 ng input RNA. Oligo-dT probes attached to Pure mRNA beads were used to enrich for mRNA (Qiagen). RNA was fragmented, reverse transcribed into cDNA, and ligated with Illumina sequencing adaptors. The size selection for libraries was performed using SPRIselect beads (Beckman Coulter) and purity of the libraries was analyzed using the DNA 1000 tape Tapestation 4200 (Agilent). The prepared libraries were pooled and sequenced using NextSeq 500 (Illumina), generating ~20 million 2×76 bp paired-end reads per samples. Two runs were performed on the intestine tissue samples and merged for analysis.

value: Liver

value: 5 weeks

value: VilCre(+)Hmgcr(+/+)


title: CRISPRi_DDX5_rep2


title: 2770-12_Lagor_Doerfler_080318-114892894


title: 2770-8_Lagor_Doerfler_080318-114892894


title: G16-F02


title: ZT18_20190301_AR06


title: ZT18_20190301_AR07


title: G16-F01


title: 2770-7_Lagor_Doerfler_080318-114892894


title: 2770-6_Lagor_Doerfler_080318-114892894

value: VilCre(+)Hmgcr(fl/fl)


title: ZT18_20190301_AR03


title: CRISPRi_DDX39B_rep2


title: G16-E12


title: G16-E11


title: ZT18_20190301_AR02


title: ZT18_20190228_AR07


title: G16-E10


title: 2770-5_Lagor_Doerfler_080318-114892894


title: ZT14_20181231_AR08


title: 2770-9_Lagor_Doerfler_080318-114892894


title: CRISPRi_DDX39A_rep2


title: 2770-2_Lagor_Doerfler_080318-114892894


title: 2770-4_Lagor_Doerfler_080318-114892894


title: G16-E09


title: CRISPRi_DDX5_rep1


title: CRISPRi_HNRNPU_rep2


title: 2770-1_Lagor_Doerfler_080318-114892894


title: CRISPRi_DDX39A_rep1


title: G16-E08


title: CRISPRi_DDX17_rep2


title: Intestine 12

source_name: Intestine epithelial scrapings

value: Intestine


title: ZT14_20181231_AR02


title: G16-E07

value: patient derived glioma tissue


title: Intestine 10


title: CRISPRi_DDX17_rep1

value: GM B cell


title: ZT14_20181231_AR01


title: CRISPRi_DDX1_rep2


title: Intestine 9


title: CRISPRi_DDX1_rep1


title: G16-E04

value: wt


title: CRISPRi_DDX39B_rep1


title: ZT14_20181215_AR07


title: G16-E06


title: ZT14_20181231_AR07


title: ZT14_20181215_AR08


title: ZT10_20190309_AR07


title: Intestine 7


title: CRISPRi_CIZ1_rep1


title: CRISPRi_CIZ1_rep2


title: G16-E02


title: G16-E03


title: ZT10_20190301_AR01


title: CRISPRi_CCAR2_rep2


title: ZT10_20190309_AR06


title: CRISPRi_CELF2_rep2


title: Intestine 8


title: Intestine 5


title: CRISPRi_CELF2_rep1


title: G16-E05


title: Intestine 6


title: G16-E01


title: CRISPRi_CCAR2_rep1


title: Intestine 3


title: G16-D11


title: Intestine 2


title: G16-D12


title: ZT10_20190228_AR03


title: ZT10_20190228_AR05


title: CRISPRi_ATP5B_rep2


title: ZT10_20190228_AR01


title: Intestine 1


title: ZT06_20190309_AR04


title: G16-D09


title: MEL_PROseq_induced_3

city: New Haven

first: Karla

middle: M.

last: Neugebauer

state: CT

address: 333 Cedar Street

department: Molecular Biophysics & Biochemistry

institute: Yale University

description: nascent RNA

data_processing:
For genome-wide LRS: Combined consensus sequence (CCS) reads were generated in FASTQ format, and Porechop was used to separate chimeric reads and trim external adapters with the SMRTer IIA sequences AAGCAGTGGTATCAACGCAGAGTAC and GTACTCTGCGTTGATACCACTGCTT with settings --extra_end_trim 0 --extra_middle_trim_good_side 0 --extra_middle_trim_bad_side 0 --min_split_read_size 100. Cutadapt was used to remove the unique 3′ end adapter on all reads in two rounds of filtering. First any reads with the adapter at the 3′ end were trimmer with settings -a CTGTAGGCACCATCAAT -e 0.1 -m 15 --untrimmed-output=untrimmed.fastq, and any reads which did not contain the full adapter were retained and their reverse complement was generated. Then, a second round of filtering with cutadapt using the settings -a CTGTAGGCACCATCAAT -e 0.1 -m 15 --discard-untrimmed was used to remove adapters from the reverse complement reads, and reads without the 3′ adapter were discarded. This ensures that each read contains a successfully ligated 3′ adapter which marks RNAPII position, and since sequencing occurs in both forward and reverse orientations randomly, it places all reads in the correct 5′ to 3′ orientation. Reads from the two adapter trimming steps were combined into a single file, then Prinseq-lite was used to remove PCR duplicates with settings -derep 1. Prinseq-lite was used again to trim 6 non-templated nucleotides added at the 5′ end by the strand-switching reverse transcriptase and the 5 nucleotides of the 3′ end adapter UMI with settings -trim_left 6 -trim_right 5. Reads were then mapped to the mm10 genome using minimap2 with settings -ax splice -uf -C5 --secondary=no, and the resulting SAM files were converted to BAM and BED files using samtools and bedtools. Reads overlapping the 7SK genomic region (chr9:78175302,78175633 in the mm10 genome) were filtered using samtools before all downstream analyses. Mapped reads in SAM format were filtered to remove reads that contained a polyA tail using a custom script (available at https://github.com/kirstenreimer/MEL_LRS). Briefly, mapped reads that had soft-clipped bases at the 3′ end were discarded if the soft-clipped region of the read contained 4 or more A’s and the fraction of A’s was greater than 0.9. Similarly, reads with soft-clipped bases at the 5′ end (resulting from minus strand reads) containing at least 4 T’s and having a fraction of T’s greater than 0.9 were discarded.
For HBB-targeted LRS: Porechop was used on raw FASTQ reads to remove external adapters and separate chimeric reads with the common forward sequence and the SMRTer IIA reverse sequence GACGTGTGCTCTTCCGATCT and GTACTCTGCGTTGATACCACTGCTT (as well as the reverse complement sequences) with settings --extra_end_trim 0 --extra_middle_trim_good_side 0 --extra_middle_trim_bad_side 0 --min_split_read_size 100 --middle_threshold 75. Reads were filtered and trimmed if they contained the 3′ end adapter as described above using the 3′ end adapter sequence plus the barcode sequence. Prinseq was used to demultiplex and trim reads as above, then cleaned FASTQ files were mapped to a custom annotation of the integrated HBB locus (available at https://github.com/kirstenreimer/MEL_LRS), which is based on the GLOBE vector (Miccio et al., 2008). Additional parameters were added to the above criteria for removing polyA-containing reads from targeted data mapped to the HBB locus based on empirical observation. Since the HBB locus is integrated randomly in the MEL genome, long readthrough transcripts that have coverage past the annotated HBB locus read into random genomic regions and cause long stretches of mismatched soft-clipped bases. A custom script was used to filter polyA-containing reads, but retain readthrough transcripts (available at https://github.com/kirstenreimer/MEL_LRS). Briefly, reads were discarded if: they contained a fraction of A’s or T’s greater than 0.7 in the soft-clipped region that starts past the end of the HBB locus annotation; they contained a fraction of A’s or T’s greater than 0.7 and 4 or more A’s or T’s in the soft-clipped region starting within 50 nucleotides of the annotated polyA site; they contained a stretch of soft-clipped reads greater than 20 nucleotides that starts within the annotated HBB gene.
For PRO-seq: Cutadapt was used to trim paired-end reads to 40 nt, removing adapter sequence and low quality 3′ ends, and discarding reads that were shorter than 20 nucleotides (-m20 -q 1). Additionally, in order to align reads using Bowtie, one nucleotide was removed from the 3′ end of all trimmed reads. Trimmed paired-end reads were first mapped to the dm3 reference genome using Bowtie,  and subsequent uniquely mapped reads to the dm3 genome were used to determine percent spike-in return across all samples. Paired-end reads that failed to align to the dm3 genome were mapped to the mm10 reference genome. Read alignment to the dm3 and mm10 genomes were performed with settings - k1 -v2 –best -X1000 --un. SAM files were sorted using samtools. Read pairs uniquely aligned to the mm10 genome were separated, and strand-specific single nucleotide bedGraphs of the 3′ end mapping positions, corresponding to the biotinylated RNA 3′ end, were generated. Due to the “forward/reverse” orientation of Illuminia paired-end sequencing, “+” and “-“ stranded bedGraph files were switched at the end of the pipeline (Mahat et al., 2016). bedGraph files across replicates in each cell treatment were merged by summing the read counts per nucleotide position.
Genome_build: mm10 or custom HBB locus
Supplementary_files_format_and_content: [bedGraph files]: PROseq reads mapped to the mm10 genome containing combined counts of end 1 3' mapping locations for all replicates in a given induction condition.
Supplementary_files_format_and_content: [coSE_values_per_intron.txt]: coSE values in uninduced and induced conditions for introns in the mm10 genome which have coverage of at least 10 reads (spliced + unspliced).
Supplementary_files_format_and_content: [NIC_and_SSscore_per_intron.txt] NIC values and intron length, GC content, and splice site sequences for introns in the mm10 genome which have coverage of at least 10 reads (spliced + intermediates).

source_name: MEL cells

growth_protocol: Murine Erythroleukemia cells (MEL) were maintained at 37°C and 5% CO2 in DMEM + Glutamax medium (GIBCO) containing 100 U/ml penicillin, 100 μg/ml streptomycin (GIBCO), and 10% fetal bovine serum (GIBCO).

extract_protocol:
All buffers were cooled on ice, all steps were performed on ice, and all samples were spun at 300 xg at 4°C unless otherwise noted. Uninduced or induced MEL cells were washed with PBS and resuspended in 1 ml Buffer W (10 mM Tris-Cl pH 8.0, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 0.5 mM DTT, 10% glycerol), then strained through a 40 μm nylon mesh filter. 9X volume of Buffer P (Buffer W + 0.1% IGEPAL CA-630) was immediately added to each sample, cells were nutated for 2 minutes at room temperature, then spun for 4 minutes. Cells were washed in Buffer F (50 mM Tris-Cl pH 8.3, 40% glycerol, 5 mM MgCl2, 0.5 mM DTT, 1 μL/mL SUPERase.In [ThermoFisher]), then resuspended in Buffer F at a final volume of 1 x 106 permeabilized cells per 40 μL.  Samples were flash frozen in liquid nitrogen and stored at -80°C. One million permeabilized cells were spiked with 5% permeabilized S2 cells for data normalization and used as input for PRO-seq. Nascent RNA was labeled through a biotin-NTP run-on: permeabilized cells was added to an equal volume of a 2X run-on reaction mix (10 mM Tris-HCl pH 8.0, 300 mM KCl, 1% Sarkosyl, 5 mM MgCl2, 1 mM DTT, 200 μM biotin-11-A/C/G/UTP (Perkin-Elmer), 0.8 U/μL SUPERase.In [ThermoFisher]), and incubated at 30°C for 5 mins. RNA was isolated using the Total RNA Purification Kit (Norgen Biotek Corp.) Fragmentation of isolated RNA was performed by base hydrolysis with 0.25 N NaOH for 9 minutes on ice, followed by neutralization with 1X volume of 1 M Tris-HCl pH 6.8. To select for nascent RNAs, 48 μL of washed Streptavidin M-280 magnetic beads (ThermoFisher) in binding buffer (300 mM NaCl, 10 mM Tris-HCl pH 7.4, 0.1% Triton X-100) was added to the fragmented RNA, and samples were rotated at room temperature for 20 minutes. The Streptavidin M-280 magnetic beads were washed twice in each of the following three buffers: high salt buffer (2 M NaCl, 50 mM Tris- HCl pH 7.4, 0.5% Triton X-100), binding buffer (above), and low salt buffer (5 mM Tris-HCl pH 7.4, 0.1% Triton X-100). Beads were resuspended in TRIzol Reagent (ThermoFisher) and heated at 65°C for 5 mins twice to elute the RNA from the beads. A subsequent ethanol precipitation was performed for RNA purification.
Nascent RNA was resuspended in 10 uM of the VRA3 3′ end adapter (/5p/rNrNrNrNrNrNrGrArUrCrGrUrCrGrGrArCrUrGrUrArGrArArCrUrCrUrGrArArC/3InvdT/). 3′ end ligation was performed using 10 units of T4 RNA ligase I (NEB) for 2 hours at room temperature. A second Streptavidin M-280 magnetic bead binding was performed to enrich for ligated nascent RNAs. The beads were subsequently washed twice in high, binding, and low salt buffers, then once in 1X ThermoPol Buffer (NEB). To prepare nascent RNA for 5′ end adapter ligation, the 5′ ends of the RNA were decapped and repaired. 5′ end decapping was performed using RNA 5′ Pyrophosphohydrolase (NEB) at 37°C for 1 hour. The beads were washed once in high and low salt buffer, then once in 1X T4 PNK Reaction Buffer (NEB). Samples were treated with T4 Polynucleotide Kinase (NEB) for 1 hour at 37°C for 5′-hydroxyl repair. Next, T4 RNA ligase I (NEB) was used to ligate the reverse 5′ RNA adapter VRA5 (rCrCrUrUrGrGrCrArCrCrCrGrArGrArArUrUrCrCrA) as described above. Following the 5′ end ligation, beads were washed twice in high, binding, and low salt buffers, then once in 0.25X FS Buffer (ThermoFisher). Reverse transcription was performed using Superscript IV Reverse Transcriptase (ThermoFisher) with 25 pmol of the Illumina TRU-seq RP1 Primer (AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA). The RT product was eluted from the beads by heating the samples at 95°C for 30 seconds. All libraries were amplified by 12 cycles of PCR with 12.5 pmol of Illumina TRU-seq RPI-index primers, excess RP1 primer, and Phusion Polymerase (NEB). The amplified library was purified using the ProNex Size-Selective Purification System (Promega) and sequenced using NextSeq 500 machines in a mid-output 150 bp cycle run.

treatment_protocol: To induce erythroid differentiation, MEL cells were diluted to 50,000 cells/ml in 10 ml fresh culture medium and incubated in growth conditions for 16 hours. DMSO was then added directly to the culture medium to a final concentration of 2% and incubated in growth conditions for 5 days.

value: Murine Erythroleukemia cells (MEL)

tag: erythroid induction

value: 2% DMSO 5 days

tag: purification

value: biotin-NTP purification

value: wild type


title: CRISPRi_ATP5B_rep1


title: G16-D10


title: MEL_PROseq_induced_2

description: nascent RNA


title: ZT06_20190309_AR03


title: MEL_PROseq_induced_1

description: nascent RNA


title: CRISPRi_ALYREF_rep1


title: CRISPRi_ALYREF_rep2


title: G16-D07


title: G16-D08


title: MS_Rv2872

city: chongqing

first: yu

middle: xiao

last: wang

address: beibei tiansheng 2

institute: Southwest University

data_processing:
Illumina Hiseq Xten used for basecalling
Sequenced reads were filtered for Seqtk, and get clean Reads that can be used for data analysis, using the bowtie2 (version: 2-2.0.5), the Genome mapping was performed on post-pretreatment reads, which was more suitable for the sequencing data of the prokaryotic transcriptome. The comparison result generated by Mapping is the BAM file
Fragments Per Kilobase of exon model per Million mapped reads.We first apply HTSeq in TopHat than after each gene Fragments could count to count, and then use TMM (trimmed mean of M values) normalization method, finally calculated using perl script FPKM value of each gene.
Genome_build: Mycobacterium_smegmatis_MC2_155_uid57701
Supplementary_files_format_and_content: tab-delimited text file includes Counts and FPKMs for each Sample

organism: Mycolicibacterium smegmatis MC2 155

source_name: Mycobacterium smegmatis C2 155

growth_protocol: MS_Vec, MS_Rv2872 strains were grown in Middlebrook 7H9 broth in 250ml bottles (supplemented with 0.05% Tween80) .

extract_protocol:
Total RNA was isolated using RNeasy mini kit (Qiagen, Germany). Paired-end libraries were synthesized by using the TruSeq® RNA Sample Preparation Kit (Illumina, USA) following TruSeq® RNA Sample Preparation Guide.
The cDNA fragments afterwards through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library.

treatment_protocol: When recombination MS_Vec and recombination MS_Rv2872 strains were grown until OD600 =0.4 and subsequently add to 1% acetamide, 9 h induction.


title: ZT06_20190228_AR06


title: G16-D05


title: G16-D06


title: MEL_PROseq_uninduced_3

description: nascent RNA


title: MEL_PROseq_uninduced_1

description: nascent RNA


title: MEL_PROseq_uninduced_2

description: nascent RNA


title: G16-D03


title: ZT06_20190228_AR04


title: ZT02_20181231_AR05


title: 3'mRNA-seq of Satellite Stem Cells treated with lnc-023  GAPMERS rep4

city: Rome

first: Andrea

last: Cipriano

state: Italy

address: Piazzale Aldo Moro 5

department: Biologia e Biotecnologue C. Darwin

country: Italy

institute: La Sapienza

data_processing:
Illumina novaSeq base call (BCL) files were converted in fastq file through bcl2fastq (version v2.20.0.422).
Sequence reads were trimmed using trimgalore (v0.4.1) to remove adapter sequences and low-quality end bases (regions with average quality below Phred score  20).
Alignment was performed with STAR 2.5.3a on mm10 reference.
The expression levels of genes were determined with htseq-count 0.9.1 by using mm10 Ensembl assembly (release 90).
Genes having < 1 Count Per Million (CPM) in at least 4 samples and those with a percentage of multi-mapping reads > 20% were filtered out.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file including Count Per Million (CPM) values for each Sample.

source_name: Satellite Stem Cells

growth_protocol: Muscle satellite cells from murine hindlimb muscles were isolated through a FACS sorting analysis and plated on ECM Gel (Sigma)-coated dishes in Cyto-grow (Resnova) complete medium as a growth medium (D-MEM 20% FBS) .

extract_protocol:
Total RNA from myoblasts/myotubes and SCs was extracted with TriReagent (Sigma). 0.5–1ug of RNA were treated with RNase-free DNase I enzyme (Thermo scientific).
Libraries were prepared from 100 ng of total RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen GmbH). Quality of libraries was assessed by using screen tape High sentisivity DNA D1000 (Agilent Technologies).
3'mRNA-seq

treatment_protocol: Downregulation of Lnc023 expression, was performed with 75nM of LNA GapmeRs (Euroclone), by transfection in Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Negative Control A (Euroclone) was used as negative (Scramble) control. Cells were transfected at  ̴ 50% confluence, for two overnights

value: Satellite Stem Cells


title: MS_Vec


title: Treated

city: Boulder

first: Chengzhe

last: Tian

state: Colorado

address: 3415 Colorado Ave, JSC Biotech Building, Spencer Lab

department: Biochemistry

institute: University of Colorado Boulder

data_processing:
Gene expression matrices from the untreated and treated conditions were computed by Cell Ranger (10X Genomics) and combined into a single dataset by ‘cellranger aggr’.
Genome_build: h38
Supplementary_files_format_and_content: Files were generated using Cell Ranger (10X Genomics). matrix.mtx stores the number of transcripts detected for each gene in each cell. barcodes.tsv and genes.tsv store the Cell IDs and Gene IDs, respectively.

source_name: A375 cells

growth_protocol: The A375 melanoma cell line (#CRL-1619) was purchased from American Type Culture Collection (ATCC). A375 cells were cultured at 37 °C with 5% CO2 in DMEM (Thermo Fisher, #12800-082) supplemented with 10% FBS, 1.5g/L sodium bicarbonate (Fisher Chemical, #S233-500), and 1X penicillin/streptomycin.

extract_protocol:
As per the protocol "Single-cell suspensions from cultured cell lines for single-cell RNA sequencing" (10X Genomics)
Performed by Microarray and Genomics Core at the University of Colorado Anschutz Medical Campus.

treatment_protocol: A375 cells were seeded in 6 cm plates (30% confluence) 24 hr prior to the start of treatment. Cells were cultured with or without 1 µM dabrafenib in full-growth medium for 72 hr.

value: Melanoma skin cancer

tag: mutation

value: Treated with 1 uM dabrafenib


title: G16-D02


title: G16-D04


title: ZT02_20181231_AR06

value: Clk856-Gal4 labeled neurons

value: Adult


title: ZT02_20181231_AR04

value: LD


title: Untreated

value: Untreated


title: 3'mRNA-seq of Satellite Stem Cells treated with lnc-023  GAPMERS rep3


title: ZT02_20181231_AR03


title: CTRL10

city: Los Angeles

first: Noah

last: Zaitlen

state: CA

address: 675 Charles E Young Dr S

department: Neurology

institute: UCLA

description: validation control sample

data_processing:
Library strategy: cfDNA-seq
trim adapters with TrimGalore
align to hg38 with Bismark in paired end mode
deduplication using Bismark
CpG extraction using MethylDackel
Genome_build: hg38
Supplementary_files_format_and_content: bed files in the format chrom, start, end, methylated read counts, depth

molecule: other

source_name: blood

extract_protocol:
QIAamp Circulating Nucleic Acid kit (Qiagen, Cat. No. 55114).
Accel-NGS R Methyl-Seq DNA Library Kit, (Swift Biosciences, Cat. No. 30024) and Accel-NGS Methyl-Seq Dual Indexing kit (Swift Biosciences, Cat. No. 38096)

tag: batch

value: validation

tag: disease status

value: ctrl

value: blood

tag: molecule

value: cell-free DNA


title: ZT02_20190309_AR12

tag: cell type


title: G16-D01

tag: tissue

value: patient derived glioma tissue

tag: genotype


title: ZT06_20190228_AR08

tag: age


title: CTRL12

description: validation control sample


title: G16-C12

tag: sample name

value: wt


title: 3'mRNA-seq of Satellite Stem Cells treated with lnc-023  GAPMERS rep2

tag: strain


title: 3'mRNA-seq of Satellite Stem Cells treated with Scramble GAPMER rep3


title: 3'mRNA-seq of Satellite Stem Cells treated with lnc-023  GAPMERS rep1


title: 3'mRNA-seq of Satellite Stem Cells treated with Scramble GAPMER rep4


title: G16-C11


title: 3'mRNA-seq of Satellite Stem Cells treated with Scramble GAPMER rep2


title: G16-C09


title: CTRL9

description: validation control sample


title: 3'mRNA-seq of Satellite Stem Cells treated with Scramble GAPMER rep1


title: ZT06_20190228_AR02


title: ZT02_20190309_AR08


title: CTRL8

description: validation control sample


title: CTRL11

description: validation control sample


title: G16-C10


title: CT22_20190704_AR13


title: CT22_20190704_AR12


title: CTRL5

description: validation control sample


title: G16-C08


title: CTRL7

description: validation control sample


title: 4 dpci wild-type cardiac ventricles- Sample 2

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

data_processing:
Reaper version 13-100 was employed to trim reads after a quality drop below a mean of Q20 in a window of 10 nucleotides. Only reads between 30 and 150 nucleotides were cleared for further analyses.
Trimmed and filtered reads were aligned versus the Ensembl Zebrafish genome version DanRer10 (GRCz10.87) using STAR 2.4.0a with the parameter “--outFilterMismatchNoverLmax 0.1” to increase the maximum ratio of mismatches to mapped length to 10%.
The number of reads aligning to genes was counted with featureCounts 1.4.5-p1 tool from the Subread package. Only reads mapping at least partially inside exons were admitted and aggregated per gene. Reads overlapping multiple genes or aligning to multiple regions were excluded.
Differentially expressed genes were identified using DESeq2 version 1.6.2.
Genome_build: DanRer10
Supplementary_files_format_and_content: library size normalized counts

organism: Danio rerio

source_name: 4 dpci cardiac ventricles from wild-type

extract_protocol:
RNA was isolated from 4 days post injured cardiac ventricles (bulbus arteriosus and atrium were dissected out from each heart) using an RNeasy micro Kit (Qiagen). To avoid genomic DNA contamination, samples were treated by on-column DNase digestion (DNase-Free DNase Set, Qiagen) during isolation.
2.2µg of total RNA was used as input for Truseq Stranded mRNA Library preparation following the low sample protocol (Illumina).

value: cardiac ventricles


title: G16-C07

city: Vladivostok

first: Valeriia

last: Gulaia

address: 10 Ajax Bay

institute: Far Eastern Federal University

data_processing:
quality control with FastQC (version 0.11.9)
trimming Nextera sequencing adapters with Trim Galore (parameter “--nextera”)
mapping to GRCh38.p12 human reference genome using STAR-2.6.0c
exclusion of cells expressing 0 genes
normal cells exclusion on the basis of particular cell markers: all hematopoietic cell - CD45 (PTPRC); naïve CD4+ T cells – IL7R, CCR7;  memory CD4+ cells – S100A4; monocytes – CD14, LYZ, FCGR3A, MS4A7; B-cells – MS4A1 (CD20); CD8+ T cells – CD8A; NK-cells – GNLY (NKG5), NKG7; dendritic cells – FCER1A, GST3; platelet – PPBP; stromal cells – CD34; endothelial cells – PECAM1 (CD31)
analysis of copy number variations (CNVs) using CaSpER
cell cycle classification performed by Cyclone
population analysis performed by SingleCellExperiment, Seurat, and Scanpy
Genome_build: GRCh38.p12
Supplementary_files_format_and_content: loom data file containing population analysis from Seurat

extract_protocol:
Frozen tumor tissue of approximate size 0.2-0.5 cm3 was placed in cold tissue lysis buffer (5M NaCl, 1M MgCl2, 1M Tris buffer (pH 8.0), 10% NP40 (Sigma, USA), 1uM DTT (Invitrogen), 1x Complete protease inhibitor (Roche), 0.4 U/ul RNAseIn (Takara Bio, Japan), 0.2 U/ul SuperaseIn  (ThermoFisher Scientific) and incubated on ice for 15 min with gentle swirling every 2-3 minutes. Then the tissue was triturated, filtered through 40um cell strainer and centrifuged at 1000g for 5 min at 4°C. The sedimented nuclei were resuspended in cold staining buffer (RNAse-free PBS pH 7.4, 1% BSA, RNAseIn, 1M MgCl2, 1mM DTT) and stained with anti-Sox2 antibody (Invitrogen) or respective IgG isotype control (BD), Alexa Fluor 488 Goat anti-Rabbit IgG antibody (Invitrogen) was used as secondary. Nuclei were visualized with 7-AAD (Sigma) staining. Thereafter, Sox2 positive nuclei were sorted a FACS Aria I or III at 4°C in skirted 96-well PCR plates containing 2 ul cell lysis buffer (2U/ul RNAseIn in 0,2% (vol/vol) Triton X-100 (Sigma)). After sorting plates were immediately sealed and placed on dry ice.
The library preparation was done using Nextera XT DNA Library Preparation Kit (Illumina) according to the manufacturer instructions, the Illumina Nextera Adapters (sequences provided on http://ngs.biodiv.tw/NGSCore/wp-content/uploads/Documents/illumina-adapter-sequences-1000000002694-08.pdf) were synthetized and HPLC purified in Biomers (http://www.biomers.net/).
single cell RNA-seq


title: CT22_20190704_AR14


title: ALS11

description: validation als sample

value: als


title: CT22_20190704_AR08


title: CT22_20190613_AR16


title: 4 dpci wild-type cardiac ventricles- Sample 1


title: G16-C06


title: ALS12

description: validation als sample


title: 4 dpci ccn2a-/- cardiac ventricles- Sample 2

source_name: 4 dpci cardiac ventricles from ccn2a-/-


title: G16-C05


title: G16-C04


title: CTRL6

description: validation control sample


title: ALS10

description: validation als sample


title: G16-C03


title: ALS9

description: validation als sample


title: CT22_20190613_AR14


title: 4 dpci ccn2a-/- cardiac ventricles- Sample 1


title: CT22_20190613_AR13


title: ALS8

description: validation als sample


title: G16-C02


title: CT18_20190710_AR20


title: G16-C01


title: ALS7

description: validation als sample


title: CT18_20190710_AR19


title: G16-B12


title: mbnl1,2,3 knockdown replicate 3

city: Gainesville

first: Jared

last: Richardson

state: Floirda

address: 2033 Mowry Rd

institute: University of Florida

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
NA (Control in all splicing files)
z3pool_5_S5

data_processing:
Reads basecalled using NextSeq Control Software V2.0
BCL files converted to Fastq using BCL2FASTQ V2.16.0.10
Fastq files checked for quality using FastQC V0.11.8
Aligned to genome using STAR V2.5.1B
Alternative splicing analyzed using rMATS V3.2.5
Genome_build: GRCz11
Supplementary_files_format_and_content: rMATS output:
A3SS.MATS.ReadsOnTargetAndJunctionCounts.txt = Alternative 3' splicing events rMATS output file
A5SS.MATS.ReadsOnTargetAndJunctionCounts.txt = Alternative 5' splicing events rMATS output file
MXE.MATS.ReadsOnTargetAndJunctionCounts.txt = Mutually exclusive splicing events rMATS output file
RI.MATS.ReadsOnTargetAndJunctionCounts.txt = Retained intron splicing events rMATS output file
SE.MATS.ReadsOnTargetAndJunctionCounts.txt = Skipped exon splicing events rMATS output file

source_name: Skeletal muscle

growth_protocol: Danio rerio grown to four months of age

extract_protocol:
Tissue was lypholized with beads and then column purified
Neb-Next Ultra RNA Kit

treatment_protocol: CRISPR-Cas9 knockout mbnl paralogs

value: Skeletal muscle

value: 4 months


title: mbnl3 knockdown replicate 2

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z123.tar
z3pool_7_S7


title: CT22_20190613_AR15


title: CT18_20190710_AR17


title: CTRL4

description: discovery control sample

value: discovery


title: mbnl1,2,3 knockdown replicate 1

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
NA (Control in all splicing files)
z3pool_3_S3


title: mbnl3 knockdown replicate 3

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z123.tar
z3pool_8_S8


title: G16-B10


title: CT18_20190710_AR18


title: CT18_20190613_AR12


title: wt replicate 3

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z23.tar
z3pool_2_S2

value: WT


title: CTRL3

description: discovery control sample


title: CTRL2

description: discovery control sample


title: CT18_20190613_AR08


title: mbnl3 knockdown replicate 1

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z123.tar
z3pool_6_S6


title: G16-B08


title: CT18_20190613_AR07


title: CT18_20190613_AR03


title: G16-B07


title: mbnl2,3 knockdown replicate 3

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z13.tar
z2pool_7_S7


title: wt replicate 1

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z23.tar
z2pool_8_S8


title: ALS6

description: validation als sample


title: mbnl1,2,3 knockdown replicate 2

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
NA (Control in all splicing files)
z3pool_4_S4


title: ALS5

description: validation als sample


title: G16-B11


title: CTRL1

description: discovery control sample


title: CT14_20190702_AR20


title: ALS3

description: discovery als sample


title: CT14_20190702_AR19


title: ALS4

description: discovery als sample


title: G16-B05


title: G16-B09


title: mbnl1,3 knockdown replicate 3

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z12.tar
z2pool_4_S4


title: wt replicate 2

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z23.tar
z3pool_1_S1


title: CT14_20190702_AR17


title: G16-B03


title: mbnl1,3 knockdown replicate 2

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z12.tar
z2pool_3_S3


title: mbnl2,3 knockdown replicate 1

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z13.tar
z2pool_5_S5


title: CT14_20190702_AR18


title: NPC11 PDX 064

city: Singapore

first: Lucky

middle: Swarnalatha

last: Sasidharan

state: Singapore

address: 31 Biopolis Way

department: Institute of Bioengineering and Nanotechnology

country: Singapore

institute: A*STAR Research Entities

description: Long non-coding RNA sequencing

data_processing:
Raw .fastq sequencing results are separated into human and mouse reads using Xenome with hg19 human genome and GRCm38 p6 mouse genome. Reads mapped exclusively to human are aligned to Gencode hg19 transcriptome using STAR and quantified by RSEM to obtain FPKM (Fragments Per Kilobase per Million mapped reads) and raw counts per gene for downstream analyses. Only the pr otein coding genes (n = 20330) were considered.
Gene ontology (GO) analysis was performed with Gene Set Enrichment Analysis (GSEA) when inferring from the whole protein-coding transcriptome, or with g:Profiler when inferring from a subset of labelled genes. We separately performed GO analysis using the GSEA Hallmark gene set, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, ontology of biological processes (GO:BP) and cellular components (GO:CC)
For GSEA, the input matrix was the read counts per gene calculated from RSEM, which was then normalized by size factor estimation from DESeq2 on a per-sample basis. Due to the limited number of available samples, false-discovery rate was controlled by permuting the gene sets instead of phenotypes. Ontologies with a family-wise error rate (FWER) < 0.05 were deemed significantly different between phenotypes.
Principal component analysis (PCA) was used in discerning the inter-sample differences. Principal components were calculated from the top 1000 most dispersed protein-coding genes, measured by the log mean-variance ratio (logVMR) of the log-FPKM expression values from RSEM. The ontology of genes contributing to the top principal components (PC) are further considered using g:Profiler. The analysis was separated by the positive and negative contributions to the PC, and ontology were deemed significantly different with adjusted p-value < 0.05.

Genome_build: hg19 human genome and GRCm38 p6 mouse genome
Supplementary_files_format_and_content: RSEM output

source_name: NPC patient tumor biopsy

growth_protocol:
NPC patient participants and samples: Eighteen NPC tissue samples were obtained from patients who underwent biopsy or surgical resection at the National University Hospital Singapore between March 2015 and April 2019. Specimen collection and experimental use were approved by the Institutional Review Board of National Healthcare Group (DSRB Reference: 2015/00098-SRF0004). One part of the tissue collected from patients were immediately transferred to RPMI-1460 media with HEPES and L-Glutamine and 5X antibiotic/antimycotic and 5 µg/ml Metronidazole at 4°C. Other part was fixed in 10% neutral buffered formalin (10% NBF) for routine Hematoxylin and Eosin (H&E) staining and remaining tissues were snap frozen in liquid nitrogen for DNA and RNA extraction.
NPC PDX implantation: NPC patient sample was cut into 4-5 mm pieces and immersed in 1:1 Geltrex:HBSS (Gibco). For subcutaneous implantation, 2-3 mm nick was made on the skin of an anaesthetized NSG mice to insert the explanted tissue. For renal capsular implantation, an incision was made in the mouse kidney to insert the tumor tissue in the subcapsular space. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee, A*Star Research Entities, Singapore.
Establishment of Organoid cultures:
A modified cancer tissue-originated spheroid (CTOS) method was adopted for establishment of NPC organoids from xenograft tumors (Fig. 1A). [16] Briefly, PDX tissues were manually minced into 1-2 mm3 pieces and subjected to enzymatic digestion using 0.28 U/mL Liberase DH (Roche) in RPMI-1460 containing 5% Penicillin-Streptomycin, 20 mM HEPES, 5 U/mL DNase1 and 30 µM Y-27632 (Stemcell Technologies) for 1.5 h at 37°C. The tissue digest was then resuspended in ice-cold HBSS with 5 U/mL DNase1 and 30 µM Y-27632. To remove large undigested pieces, the cell suspension was filtered through 100 µm cell strainer, followed by isolation of the 70-100 µm and 30-70 µm fractions using 70 µm and 30 µm MACS Smart Strainer (Miltenyi Biotech) respectively. Organoids were first counted and then gently mixed with Geltrex (Gibco) at 1.2% (w/v). 50 µl of the organoid suspension was seeded in 96 well ultra-low attachment plates (Corning). The gel matrix was then overlaid with 200 µl of complete PneumaCult™-ALI (P-ALI) media (Stemcell Technologies) supplemented with niche factors (NF): 4 µg/ml Heparin, 125 ng/ml Hydrocortisone, 50 ng/ml EGF , 50 ng/ml bFGF,  20 ng/ml FGF-10,  10 µM SB 202190, 500 nM A83-01, 10 µM Y-27632, and 2 mM Glutamax.
C666-1 cell monolayer and spheroids culture:
C666-1 cells (RRID:CVCL_7949) were authenticated using STR (Short tandem repeat) profiling and were free of mycoplasma contamination. The cells were cultured in RPMI‐1640 medium supplemented with 10% FBS at 37°C in a humidified incubator under 5% CO2. Confluent cells (70–80%) were sub-passaged by incubation with 0.05% trypsin for 5 min at 37°C and a splitting ratio of 1:3 was used. For monolayer cultures of C666-1 in 96 well high binding flat bottom plates, cells were seeded at a concentration of 5000 cells /well. For spheroid cultures, cells were seeded in 96 well flat bottomed ultra-low attachment plates at a concentration of 8000 cells/well.

extract_protocol:
RNA was isolated using the RNeasy Mini Kit (Qiagen) and processed as per manufacturer’s instructions.
Strand-specific RNA-seq libraries were prepared using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, UK), as per manufacturer’s instructions. Sequencing was performed on an Illumina NovaSeq 6000 (Illumina, USA) to generate 150 bp paired-end reads. For patient samples, coding and long non-coding RNA was sequenced, whereas only the coding mRNA was sequenced for all other samples.

treatment_protocol: Normoxic Organoids were culture at 21% oxygen where as the hypoxic ones were cultured in 1% oxygen concentration. All irradiation was performed using Gammacell with 137Cs source (Nordion, Canada) at a dose rate of 1 Gy/min with ±15% error. The organoids were iraddiated on 3 consecutive days (day 5-day 7) at 5 Gy and harvested at day 12, followed by RNA extraction.

value: NPC patient tumor biopsy

value: wild-type

value: Fresh frozen

tag: pdx success

value: Not successful


title: mbnl1,3 knockdown replicate 1

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z12.tar
z2pool_2_S2


title: G16-B02


title: CT14_20190001_AR04

value: Clk856-Gal4 labeled neurons

value: Adult


title: G16-B06


title: TLR296

description: Long non-coding RNA sequencing

value: Successful


title: mbnl1,2 knockdown replicate 2

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z3.tar
z1pool_8_S8


title: TLR250-P10

description: Stranded mRNA sequencing

source_name: 250T PDX passage 10

value: Human cells in mouse xenograft model

value: Passage 10


title: G16-A12

value: patient derived glioma tissue


title: CT14_20190001_AR03


title: mbnl1,2 knockdown replicate 3

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z3.tar
z2pool_1_S1


title: mbnl1,2 knockdown replicate 1

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z3.tar
z1pool_7_S7


title: TLR250-P9

description: Stranded mRNA sequencing

source_name: 250T PDX passage 9

value: Passage 9


title: CT14_20190001_AR01


title: TLR296-P18

description: Stranded mRNA sequencing

source_name: 296T PDX passage 18

value: Passage 18


title: G16-B04

value: wt


title: G16-A10


title: G16-A09


title: CT10_20190704_AR07

source_name: Adult neurons


title: mbnl2 knockout replicate 3

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z2.tar
z1pool_6_S6

value: Skeletal muscle


title: G16-A08


title: CT10_20190704_AR06


title: G16-B01


title: mbnl2 knockout replicate 2

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z2.tar
z1pool_5_S5

value: 4 months


title: mbnl2,3 knockdown replicate 2

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z13.tar
z2pool_6_S6


title: CT14_20190001_AR02


title: mbnl2 knockout replicate 1

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z2.tar
z1pool_4_S4


title: #419

description: Stranded mRNA sequencing

source_name: Organoid

value: Normoxia


title: G16-A05

source_name: SOX2+ glioma nucleus


title: CT10_20190704_AR05


title: mbnl1 knockout replicate 3

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z1.tar
z1pool_3_S3


title: CT10_20190610_AR02


title: TLR296-P16

description: Stranded mRNA sequencing

source_name: 296T PDX passage 16

value: Passage 16


title: #503

description: Stranded mRNA sequencing


title: 250T P7 #503

description: Stranded mRNA sequencing

source_name: PDX tumor used to make organoid #503

value: 250 PDX Passage 7


title: mbnl1 knockout replicate 1

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z1.tar
z1pool_1_S1


title: ALS2

description: discovery als sample

value: discovery

value: als

value: blood

value: cell-free DNA


title: CT10_20190610_AR01

value: Clk856-Gal4 labeled neurons

value: Adult


title: mbnl1 knockout replicate 2

description:
Ribosomal depleted RNA
Alternative splicing files from rMATS in processed file data
processed data files for this sample are available in:
MATS_z1.tar
z1pool_2_S2


title: #430

description: Stranded mRNA sequencing

value: Human cells in mouse xenograft model

value: wild-type


title: G16-A07

value: patient derived glioma tissue


title: 250T P7 #419

description: Stranded mRNA sequencing

source_name: PDX tumor used to make organoid #419


title: G16-A06


title: 250T P7 #430

description: Stranded mRNA sequencing

source_name: PDX tumor used to make organoid #430


title: CT10_20190704_AR04


title: G16-A03


title: siXBP1-3

city: Wuhan

first: Faming

last: Zhao

state: Hubei

address: Hangkong road 13

department: Tongji Medical College

institute: Huazhong University of Science and Technology

description: Sample_6-HCC-3.Aligned.sortedByCoord.out.bam

data_processing:
Sequenced reads were mapped to human reference genome (hg38) using STAR (2.5.2a) with default parameters.
Aligned reads were summarized into gene counts using the featureCounts (v1.5.0) command of the Subread package with default parameters in conjunction with the human gencode annotation file (hg38 release 85).
Genome_build: hg38
tab-delimited text file containing raw read counts for all samples
Genome_build: hg38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

growth_protocol: HepG2 cells were cultured in regular growth medium (DMEM + 10% FBS + 1%P/S)

extract_protocol:
The TruSeq stranded RNA sample prep kit was used for library generation according to Illumina's protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: For samples 1-6, cells were transfected with either Allstar control siRNA or XBP1-specific siRNA. After two days, the cells were treated with 30 nM thapsigargin for 3 hours.

tag: cell line

value: human HCC cell line

tag: transfected with

value: XBP1-specific siRNA


title: CT10_20190524_AR03

tag: treatment


title: CT10_20190524_AR04


title: G16-A01

value: wt


title: ALS1

description: discovery als sample


title: siXBP1-1

description: Sample_4-HCC-1.Aligned.sortedByCoord.out.bam


title: 250T P7 #429

description: Stranded mRNA sequencing

source_name: PDX tumor used to make organoid #429

tag: sample type


title: G16-A02


title: siXBP1-2

description: Sample_5-HCC-2.Aligned.sortedByCoord.out.bam


title: #429

description: Stranded mRNA sequencing


title: CT06_20190814_AR19


title: CT06_20190814_AR20


title: G16-A04


title: TTseq_TotalRNA_K562_Rep1

city: Goettingen

last: Lidschreiber

address: Am Fassberg 11

institute: Max-Planck-Institute for Multidisciplinary Scienes

description:
K562_eRNAs.gtf
K562_enhancer.gtf
K562_eRNAs_conservative.gtf
K562_enhancer_conservative.gtf

data_processing:
Paired-end 50 bp reads were first mapped to a single copy of the rDNA locus to remove rRNA related sequences. Reads that did not map to the rDNA were then aligned to the hg20/hg38 (GRCh38) genome assembly (Human Genome Reference Consortium) using STAR 2.6.0c (Dobin 2013) with the following specifications: outFilterMismatchNoverLmax 0.05, outFilterMultimapScoreRange 0 and alignIntronMax 500000. Bam files were filtered with Samtools (Li 2009) to remove alignments with MAPQ smaller than 7 (-q 7) and only proper pairs (f99, f147, f83, f163) were selected. Fragment counts for different features were calculated with HTSeq (Anders 2015). Further data processing was carried out using the R/Bioconductor environment. To normalize TT-seq samples for sequencing depth and make them comparable between cell lines we used the median of ratios approach described in the DESeq2 R/Bioconductor package (Anders 2010). Transcription unit (TU) annotation and classification was done using the 4sU-labeled (L) samples as described (Michel, Demel 2017) with a few modifications. Briefly, strandspecific coverage was calculated from fragment midpoints in consecutive 200bp bins throughout the genome for all TTseq samples. Binning reduced the number of uncovered positions within expressed transcripts and increased the sensitivity for detection of lowly synthesized transcripts. The GenoSTAN R/Bioconductor package (Zacher 2017) was used to learn a twostate hidden Markov model with a PoissonLogNormal emission distribution in order to segment the genome into transcribed and untranscribed states, which resulted in 51,198  134,528 TUs per cell line. TUs that overlapped at least 25% of a proteincoding gene annotated in RefSeq (Release 109.20190607) or GENCODE (v31) and overlapped with an annotated exon of the corresponding gene were classified as mRNAs. Remaining TUs were annotated as non-coding (nc)RNAs. In order to overcome low expression or mappability issues, ncRNAs that were only 200 bp (1 bin) apart were merged. Subsequently, TU start and end sites were refined to nucleotide precision by finding borders of abrupt coverage increase or decrease between two consecutive segments in the two 200bp bins located around the initially assigned start and stop sites via fitting a piecewise constant curve to the coverage profiles (whole fragments) for all TTseq samples using the segmentation method from the R/Bioconductor package tilingArray (Huber 2006). In order to filter spurious predictions, TUs were further filtered with a minimal expression threshold of replicate-averaged normalized FPK >= 20 as described above (requiring at least 15 normalized FPK per replicate). ncRNAs overlapping with GENCODE (v31) annotated small non-coding RNA classes (snRNA, snoRNA, tRNA) were omitted from further analysis. The remaining ncRNAs were further classified according to their genomic location relative to protein-coding genes into four categories: upstream antisense RNA (uaRNA), convergent RNA (conRNA), antisense RNA (asRNA) and intergenic RNA. ncRNAs located on the opposite strand of an mRNA were classified as asRNA if the TSS was located > 1 kb downstream of the sense TSS, as uaRNA if the TSS was located < 1 kb upstream of the sense TSS, and as conRNA if the TSS was located < 1 kb downstream of the sense TSS. Each of the remaining ncRNAs was classified as intergenic.
eRNA annotation: Of the ncRNA classes defined above, we further classified intergenic and asRNAs as eRNAs, if their TSS +/- 500 bp overlapped with an enhancer state annotated by GenoSTAN (Zacher 2017) based on patterns of five histone modifications (H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3) in any of 127 cell types and tissues covering 111 datasets of the Roadmap Epigenomics project (Roadmap Epigenomics Consortium et al., 2015) as well as 16 datasets of the Encyclopedia of DNA Elements (ENCODE) Project (Dunham et al., 2012). Long antisense eRNAs that overlapped with the promoter region of the respective protein-coding gene (TSS +/- 1 kbp) were annotated to end 1 kbp downstream of the protein-coding TSS. Next, we introduced additional filtering criteria, as highly transcribed TUs can give rise to spurious downstream TUs, which do not represent independent TUs, but ongoing, gradually decreasing transcription by Pol II downstream of the main TU. To avoid misclassification of such downstream TUs as putative eRNAs, we did the following:
First, we restricted our set of intergenic eRNAs to only those originating from regions with TT-seq detected transcription on both strands, with an allowed gap of up to 750 bp between divergently transcribed eRNA pairs. To this end, a non-coding TU had to be annotated on the opposite strand, but it was not required to lie above the expression threshold. Second, we also restricted our set of antisense eRNAs. To this end, non-coding TUs downstream of mRNA/uaRNA TUs were sequentially merged with the upstream mRNA/uaRNA if the gap between TUs was less than 5 kbp. Subsequently, non-coding TUs, which had been classified as antisense eRNAs and were constituents of such merged regions were excluded from further analyses, unless their TT-seq signal (normalized FPK) was at least 2-fold higher than TT-seq signal over the end (last 1 kbp) of the upstream mRNA/uaRNA.
Enhancer annotation: To define intergenic enhancer regions, we selected the region between the TSSs of each pair of intergenic bidirectional eRNAs and extended it by 500 bp in both directions. To define intragenic enhancer regions, we selected the region covering 750 bp upstream to 500 bp downstream of the TSS of antisense eRNAs. Since we allowed a gap of maximum 750 bp between bidirectional intergenic eRNAs, we selected 750 bp also as putative enhancer region upstream of antisense eRNAs. Based on this definition all intragenic enhancer regions were 1250 bp long whereas the length of intergenic enhancer regions was dependent on the gap or overlap between bidirectional eRNAs. Strongly transcribed intergenic enhancer regions gave rise to uninterrupted bidirectional TT-seq signal over long genomic stretches, which could not be resolved at the resolution of individual enhancers, and did therefore result in the definition of some enhancer regions covering several kbp.
Dealing with annotated lncRNAs: The FANTOM consortium has shown that the majority of intergenic lncRNAs originate from enhancer regions, and thus we only excluded eRNAs/enhancers if they overlapped a functional lncRNA originating from an intergenic promoter DHS (24 lncRNAs, e.g. Malat1; taken from Supplementary Table 5 in (Hon 2017) selecting CAT_geneCategory = p_lncRNA_intergenic).
The resulting annotations of eRNAs and enhancer regions reported in Lidschreiber et al. are provided as eRNAs/enhancer .gtf files. In addition, conservative subsets of the eRNAs/enhancer regions reported in Lidschreiber et al. are provided. The conservative annotations contain only intragenic enhancer regions defined by eRNAs >= 300 nt and with normalized FPK >= 30; intergenic enhancer regions / eRNAs are the same as in Lidschreiber et al.
processed data files format and content: eRNA and transcribed enhancer annotations are provided for each of the cell lines as .gtf files. Strand-specific bigWig files contain size factor normalized fragment coverages for each TT-seq labeled RNA sample.

source_name: human cancer cell line

growth_protocol: Cells were cultured in cell culture media supplemented with 1% penicillin/streptomycin (GE Healthcare) and 10% fetal bovine serum (Sigma) at 37°C in a humidified 5% CO2 incubator. BT-20 (ATCC HTB-19), DU 145 (ATCC® HTB-81) and SK-UT-1 (ATCC HTB114) cells were cultured in MEM (Gibco) supplemented with 0.1 mM NEAA (Gibco) and 1 mM sodium pyruvat (Gibco). 769-P (ATCC CRL-1933) and 786-O (ATCC CRL-1932) cells were cultured in RPMI-1640 (Gibco A1049101). MDA-MB-231 (ATCC HTB-26) and LoVo (ATCC CCL-229) cells were cultured in DMEM-GlutaMAX (Gibco 21885-25). U-118 MG (ATCC HTB-15) and GP5d (Sigma) cells were cultured in DMEM (Gibco). GP5d cells were cultivated in cell culture flasks coated with poly-L-lysine (Sigma). M059J (ATCC CRL-2366) cells were cultivated in DMEM/F-12 (Gibco), HCT 116 (ATCC CCL-247) cells in McCoy's 5a Medium Modified (Gibco) and PC-3 (ATCC CRL-1435) cells in F-12K (ATCC 30-2004).

extract_protocol:
Total RNA was extracted using TRIzol (Life Technologies), 4sU-labeled RNA was extracted according to Gressel at al., protocols.io, 2019.
Sequencing libraries were prepared with the Ovation Universal RNA-Seq System (NuGEN) following the manufacturer's’ instructions

treatment_protocol: 48 hours before experiment cells were seeded in 15 cm dishes to obtain 70 % confluent cells at time of 4sU labeling. Cells were labeled with 500 µM 4-thiouridine (4sU) (Sigma-Aldrich) for 5 min at 37 °C under 5% CO2. Cells were harvested using TRIzol (Life Technologies).

value: cancer

tag: 4su labelling

value: 5min 4sU

tag: method

value: TT-seq

tag: labeled molecule

value: Total RNA


title: TTseq_TotalRNA_K562_Rep2


title: siNC-2

description: Sample_2-HCC-2.Aligned.sortedByCoord.out.bam

value: Allstar control siRNA


title: CT06_20190719_AR17


title: TTseq_TotalRNA_Jurkat

description:
Jurkat_eRNAs.gtf
Jurkat_enhancer.gtf
Jurkat_eRNAs_conservative.gtf
Jurkat_enhancer_conservative.gtf

value: Jurkat


title: SC #753 Hyp Con

description: Stranded mRNA sequencing

value: Hypoxia


title: CT06_20190719_AR18


title: siNC-3

description: Sample_3-HCC-3.Aligned.sortedByCoord.out.bam


title: SC #753 Hyp Rad

description: Stranded mRNA sequencing

value: Hypoxia+Radiation


title: CT06_20190710_AR04


title: SC #753 Rad

description: Stranded mRNA sequencing

value: Normoxia+Radiation


title: TTseq_TotalRNA_SKUT1_Rep2

description:
SKUT1_eRNAs.gtf
SKUT1_enhancer.gtf
SKUT1_eRNAs_conservative.gtf
SKUT1_enhancer_conservative.gtf


title: SC #753

description: Stranded mRNA sequencing

value: Normoxia


title: COVID-19 patient 4

city: Daejeon

first: JUNGMO

last: KIM

state: -- None selected --

address: 291 Daehak-ro, Yuseong-gu

department: Center for Vascular Research

country: South Korea

institute: Institute for Basic Science

description: single cell RNA

data_processing:
STAR(v2.7.3a) was used to process single cell 3' RNA-seq outputs. And UMI-tools(v1.1.1) extract UMI from fastq files.
Then, using featureCount(Subread v2.0.1), reads were aligned to human reference genome plus SARS-CoV2 reference genome
Aligned reads were then read by Seurat (v3.2.3) and cell-gene raw expression matrices were made. Then, low quality cells (expressing less than 200 genes and more than 4000 genes)  were removed. Also, genes that are only expressed in less than 3 cells were discarded.
After quality control, expression matrices were log normalized and scaled with Seurat to produce processed data files.
Identification of differentially expressed genes and dimensionality reductions were performed by Seurat.
Genome_build: a customized reference genome in which the SARS-CoV-2 genome (NC_045512.2) was added as an additional chromosome to the human reference genome GRCh38.p13 (GCA_000001405.28).
Supplementary_files_format_and_content: Cell-gene expression matrix of UMI counts for human nasal swab single cell data from COVID-19 patients

source_name: nasal swab

extract_protocol:
After tissue preparation and cell digestion, single-cell suspensions were processed on the BD Rhapsody system following the manufacturer's protocols.
cDNA libraries were constructed using BD Rhapsody WTA Kit

value: nasal swab

tag: disease state

tag: sampling post confirm

value: day 1

tag: gender

value: male


title: 296T P13 #753

description: Stranded mRNA sequencing

source_name: PDX tumor used to make organoid #753

value: Human cells in mouse xenograft model

value: wild-type

value: 296 PDX Passage 13


title: CT06_20190710_AR03

tag: cell type

value: Clk856-Gal4 labeled neurons

value: Adult


title: COVID-19 patient 3

description: single cell RNA

tag: tissue

value: female


title: COVID-19 patient 2, Day6

description: single cell RNA

value: day 6


title: CT06_20190710_AR01


title: COVID-19 patient 2, Day3

description: single cell RNA

value: day 3


title: CT06_20190710_AR02


title: SC #499 Hyp Rad

description: Stranded mRNA sequencing

tag: genotype


title: TTseq_TotalRNA_PC3_Rep2

description:
PC3_eRNAs.gtf
PC3_enhancer.gtf
PC3_eRNAs_conservative.gtf
PC3_enhancer_conservative.gtf

value: cancer

value: 5min 4sU

value: TT-seq

value: Total RNA


title: TTseq_TotalRNA_PC3_Rep1


title: TTseq_TotalRNA_SKUT1_Rep1


title: COVID-19 patient 1

description: single cell RNA

tag: age


title: SC #499 Hyp Con

description: Stranded mRNA sequencing

value: Hypoxia


title: COVID-19 patient 2

description: single cell RNA


title: CT02_20190702_AR16


title: SC #499 Rad

description: Stranded mRNA sequencing

value: Normoxia+Radiation


title: TTseq_TotalRNA_DU145_Rep1

description:
DU145_eRNAs.gtf
DU145_enhancer.gtf
DU145_eRNAs_conservative.gtf
DU145_enhancer_conservative.gtf


title: TTseq_TotalRNA_786O_Rep2

description:
786O_eRNAs.gtf
786O_enhancer.gtf
786O_eRNAs_conservative.gtf
786O_enhancer_conservative.gtf


title: CT02_20190702_AR14


title: siNC-1

description: Sample_1-HCC-1.Aligned.sortedByCoord.out.bam

value: human HCC cell line

value: Allstar control siRNA


title: SC #497 Hyp Rad

description: Stranded mRNA sequencing

value: Hypoxia+Radiation


title: TTseq_TotalRNA_769P_Rep2

description:
769P_eRNAs.gtf
769P_enhancer.gtf
769P_eRNAs_conservative.gtf
769P_enhancer_conservative.gtf


title: TTseq_TotalRNA_DU145_Rep2


title: SC #499

description: Stranded mRNA sequencing

value: Human cells in mouse xenograft model

value: Normoxia


title: CT02_20190528_AR07


title: TTseq_TotalRNA_LoVo_Rep2

description:
LoVo_eRNAs.gtf
LoVo_enhancer.gtf
LoVo_eRNAs_conservative.gtf
LoVo_enhancer_conservative.gtf

value: LoVo


title: TTseq_TotalRNA_786O_Rep1


title: TTseq_TotalRNA_HCT116_Rep2

description:
HCT116_eRNAs.gtf
HCT116_enhancer.gtf
HCT116_eRNAs_conservative.gtf
HCT116_enhancer_conservative.gtf

value: HCT 116


title: CT02_20190702_AR13

value: Clk856-Gal4 labeled neurons

value: Adult


title: TTseq_TotalRNA_LoVo_Rep1


title: SC #497 Hyp Con

description: Stranded mRNA sequencing

value: wild-type


title: CT02_20190528_AR08


title: #499 Hyp Con

description: Stranded mRNA sequencing


title: SC #497 Rad

description: Stranded mRNA sequencing


title: CT02_20190528_AR06


title: TTseq_TotalRNA_HCT116_Rep1


title: #499 Hyp Rad

description: Stranded mRNA sequencing


title: TTseq_TotalRNA_GP5d_Rep2

description:
GP5d_eRNAs.gtf
GP5d_enhancer.gtf
GP5d_eRNAs_conservative.gtf
GP5d_enhancer_conservative.gtf


title: CT02_20190528_AR05


title: TTseq_TotalRNA_769P_Rep1


title: rep1 RNAseq araP

city: Piscataway

first: Premal

last: Shah

state: NJ

address: 145 Bevier Rd

department: Genetics

institute: Rutgers University

data_processing:
adapter removal performed with cutadapt v2.8
demultiplexing using the fastX toolkit script fastx_barcode_splitter.pl
deduplication with the BBtools script dedupe.sh
removal of UMIs and barcodes with cutadapt v2.8
in silico rRNA depletion with hisat2 v2.1.0
transcript quantification with kallisto v0.46.2
alignment with hisat2 v2.1.0
Genome_build: Each cell lines individual genome was determined in Tenaillon et al 2016
Supplementary_files_format_and_content: A single csv file contains the counts and TPMs for each of the samples. These are not the original counts and TPMs. Counts were first rounded, and new TPMs were calculated from rounded counts. Analysis was based on rounded counts and the TPMs generated from them.

source_name: Long Term Evolution Experiment

growth_protocol: Bacteria were grown in the DM25 medium supplemented with 4g/L of glucose.

extract_protocol:
Cells were collected via vacuum filtration, flash frozen in liquid nitrogen, ground to a fine powder with frozen lysis buffer, then allowed to thaw to complete lysis. Lysis buffer contained 20 mM Tris pH 8, 10 mM MgCl2, 100 mM NH4Cl, 5 mM CaCl2, 1 mM chloramphenicol, 0.1% v/v sodium deoxycholate, 0.4% v/v Triton X-100, 100 U/mL DNase I, 1 uL/mL SUPERase-In (Thermo Fisher Scientific AM2694)
Each sample produced a matched set of RNA-seq and ribo-seq fragments which were size selected to a range of 18-50nt. These were ligated with a 5' linker (containing a 4nt UMI) and a 3' linker (containing a 5nt UMI and a 5nt sample barcode). rRNA depletion was completed with the Illumina Ribo-Zero rRNA Depletion Kit for bacteria.
RNA-seq and ribo-seq in E. coli cell lines from the Long Term Evolution Experiment

treatment_protocol: No treatments were applied to the samples.


title: SC #497

description: Stranded mRNA sequencing


title: rep2 RNAseq araP


title: #499

description: Stranded mRNA sequencing


title: rep2 RNAseq araM


title: rep2 ribo-seq araP

data_processing:
Library strategy: Ribo-seq
adapter removal performed with cutadapt v2.8
demultiplexing using the fastX toolkit script fastx_barcode_splitter.pl
deduplication with the BBtools script dedupe.sh
removal of UMIs and barcodes with cutadapt v2.8
in silico rRNA depletion with hisat2 v2.1.0
transcript quantification with kallisto v0.46.2
alignment with hisat2 v2.1.0
Genome_build: Each cell lines individual genome was determined in Tenaillon et al 2016
Supplementary_files_format_and_content: A single csv file contains the counts and TPMs for each of the samples. These are not the original counts and TPMs. Counts were first rounded, and new TPMs were calculated from rounded counts. Analysis was based on rounded counts and the TPMs generated from them.


title: CT02_20190702_AR15


title: 296T P12 #499

description: Stranded mRNA sequencing

source_name: PDX tumor used to make organoid #499

value: 296 PDX Passage 12


title: #497 Hyp Con

description: Stranded mRNA sequencing


title: #497 Hyp Rad

description: Stranded mRNA sequencing


title: #499 Rad

description: Stranded mRNA sequencing


title: rep1 RNAseq araM


title: TTseq_TotalRNA_GP5d_Rep1

value: cancer


title: rep2 ribo-seq araM


title: TTseq_TotalRNA_BT20_Rep2

description:
BT20_eRNAs.gtf
BT20_enhancer.gtf
BT20_eRNAs_conservative.gtf
BT20_enhancer_conservative.gtf

value: 5min 4sU


title: BRAC1235.6h_PLASMA [WT.PLASMA.4]

city: London

first: Dinis

middle: Pedro

last: Calado

address: Midland Road

country: United Kingdom

institute: The Francis Crick Institute

data_processing:
The RSEM package (version 1.3.0) and STAR (2.5.2a) were used to align reads to the mouse Ensembl GRCm38 transcriptome.
Raw counts were normalised and differential expression analysis was performed with the DESeq2 package (version 1.18.1) within the R programming (version 3.4.3)
Genome_build: mouse Ensembl GRCm38
Supplementary_files_format_and_content: Transcripts per million (TPM) values. Rows represent Ensembl mouse genes and columns represent individual samples.

source_name: Plasma cells_wild type mouse

extract_protocol:
Spleen were disrupted using a 70uM mesh strainer. Samples were then washed with FACS buffer (2% FBS and 2 mM EDTA in PBS), incubated for 30 min on ice  with antibodies and  CD43 MicroBeads (Cat# 130-049-801, Miltenyi Biotec). Red blood cells were then  lysed for 5 min at room temperature with 5mL of Gey's buffer. After a wash , samples were passed through a 70 um filter LS columns (Miltenyi) for magnetic separation and B cell enrichment. After washing,  single-cell suspension was  sorted by flow cytometry. Samples were collected in eppendorffs containing RTL Buffer Plus (Qiagen) with 1% beta-mercaptoethanol. Samples were purified with AllPrep DNA/RNA Mini Kits (Cat# 80204, Qiagen) as per  manufacturer’s instructions.
Samples were prepared with Nugen cDNA kit and sequenced using Illumina HiSeq system

treatment_protocol: 8-12 week old female mice were intravenously immunised with 5x10^10 sheep red blood cells in 200uL of PBS and analysed 10 days after.

value: 8-12 weeks

tag: Sex

value: female

tag: genotype/variation

value: wild type

value: 10 days after sheep red blood cells immunisation of mice

tag: tissue/cell type

value: Spleen Plasma cells

tag: cell population

value: B220lo CD138+ IgD- cells