title: scRNAseq_d2_EB

source_name: embryoid bodies

value: d2 EB


title: scRNAseq_d4_EB

status: Public on Jul 27 2020

type: SRA

city: Santander

first: Alvaro

last: Rada-Iglesias

state: Cantabria

address: Albert Einstein 22, PCTCAN

country: Spain

institute: Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/University of Cantabria

library_source: transcriptomic

data_processing:
The 10x Genomics scRNA-seq data was generated across different stages of PGCLC differentiation and can be easily explored by opening the cloupe file with the Loupe Cell Browser (https://www.10xgenomics.com). UMIs were counted using NCBI:GCA_000001635.6 and cellranger-2.1.0103. The resulting UMI values were aggregated into a single matrix with default normalization (‘‘–normalize=mapped’’) (UMI_counts).
Genome_build: mm10
Supplementary_files_format_and_content: UMI_counts;cloupe_file

molecule: total RNA

organism: Mus musculus

growth_protocol: ESC were cultured on gelatin-coated plates using Knock-out DMEM supplemented with 15% FBS and LIF. Before each PGCLC differentiation, ESC were adapted to 2i+LIF media (serum-free N2B27 medium supplemented with MEK inhibitor PD0325901 [0.4 μM], GSK3β inhibitor CHIR99021 [3 μM], and LIF) for at least four days in gelatin-coated tissue culture plates. For the EpiLC differentiation, 6-well plates were coated with 15 µg ml-1 Fibronectin and 2 x 105 2i ESC were differentiated in N2B27 media supplemented with 20 ng ml-1 Activin A, 12 ng ml-1 bFGF and 10 µl ml-1 KSR for two days. For EpiSC differentiation, 2i ESC were plated at a density of 2.5 x 105 cells ml-1 on plates coated with 15 µg ml-1 Fibronectin and differentiated into EpiSC by passaging them in N2B27 media supplemented with 20 ng ml-1 Activin A and 12 ng ml-1 bFGF for at least eight days. For the PGCLC differentiation, d2 EpiLC were plated at a density of 2 x 104 cells ml-1 and cultured as embryoid bodies (EB) on Ultra-Low attachment multiwell plates (Corning® Costar®) in GK15 medium supplemented with growth factors (0.5 µg ml-1 BMP4, 0.1 µg ml-1 SCF, 1000 U ml-1 LIF and 50 ng ml-1 EGF, no BMP8a) for four days.

extract_protocol:
For single cell RNA sequencing, EpiSC were dissociated with Accutase and all other cell types with TripleExpress. Cells were then centrifuged and resuspended in PBS containing 0.04 % BSA. Next, cells were passed through a strainer, the cell concentration was determined and the single cell RNA sequencing libraries were prepared using the Chromium Single Cell Gene Expression (10x Genomics) according to the Single Cell 3’ Reagents Kit (v2 chemistry) protocol.
10x Genomics - Single Cell 3’ Reagents Kit (v2)

tag: cell line

value: d4 EB

tag: strain

tag: genotype

value: wild type


title: scRNAseq_EpiSC

source_name: epiblast stem cells

value: EpiSC


title: scRNAseq_SL_ESC

source_name: embryonic stem cells

value: S+L ESC


title: 8mon_sp_gp120

city: Stony Brook

first: Shao-Jun

last: Tang

state: New York

address: 101 Nicolls Road

country: USA

institute: Renaissance School of Medicine at Stony Brook University

data_processing:
Illumina OLB software was used for basecalling
Sequences were processed using the Drop-seq toolkit. Briefly, adaptor sequences and poly-A tails were trimmed
Processed reads were mapped to mm10 genome using Drop-seq_tools v1.12 and STAR v 2.5
Gene expression of each cell was summarized in a gene expresison matrix generated by DigitalExpression, with parameter NUM_CORE_BARCODES=10000
Genome_build: mm10
Supplementary_files_format_and_content: Digital Expression matrix/counts

source_name: Whole spine

extract_protocol:
Brain cortex and whole spinal cord tissue were removed. Tissue was processed using papain and was slowly triturated and filtered using 70μm strainer. Microglia were enriched using density centrifugation. Briefly, A 4mL desnity gradient was prepared in a 15m falcon tube. 6ML of cell suspension was added on top of the OptiPrep media and centrifuged at 800g for 30 minutes. The bottom fraction, enriched with Microglia, was collected, resuspended in HABG media.
Library cDNA was fragmented and amplified using a Nextera XT DNA Sample prep kit with three separate reactions of 600, 1200 and 1800 pg input cDNA. The libraries were pooled and purified twice using 0.7X volume of SPRIselect beads. The purified libraries were sequenced on the Illumina HiSeq 2500

tag: tissue

value: Whole spine

tag: age

value: 8 months


title: 4mon_sp_wt

value: wt

value: 4 months


title: scRNAseq_2i_ESC

value: 2i+LIF ESC


title: 2mon_br_wt

source_name: brain cortex

value: brain cortex

value: 2 months


title: scRNAseq_d1_EpiLC

source_name: epiblast-like cells

value: d1 EpiLC


title: 8mon_sp_wt


title: ChIP_H3K4me2_d8EpiSC_Otx2

library_source: genomic

data_processing:
ChIP-seq single end reads were mapped to the mouse genome (mm10) using BWA. After duplication removal with the MarkDuplicates function from the Picard tools, reads within blacklisted regions were discarded and the aligned reads were normalized with deeptools to 1x sequencing depth (as RPGC: Reads per genomic content).
Genome_build: mm10
Supplementary_files_format_and_content: RPGC normalized bigWig

molecule: genomic DNA

extract_protocol:
Cells were cross-linked with 1 % formaldehyde for 10 min, followed by quenching with 0.125 M glycine, harvesting and washing in PBS containing protease inhibitors. The cells were sequentially lysed in three buffers (lysis buffer 1: 50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TX-100; lysis buffer 2: 10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA; lysis Buffer 3: 10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) with rotation for 10 min in between. Then, the chromatin was sonicated with the Epishear™ Probe Sonicator (Active Motif) with 20 s ON and 30 s OFF for 8 cycles. After centrifugation, the supernatant was divided into input and ChIP samples. The ChIP samples were incubated with specific antibodies (Supplementary Table 4) overnight, followed by immunoprecipitation with Dynabeads Protein G beads (invitrogen). Next, the beads were washed with RIPA buffer (50 mM Hepes, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) on a magnet, eluted (50 mM Tris, 10 mM EDTA, 1% SDS), and reverse crosslinked at 65 °C overnight in parallel with the input. Finally, DNAs were purified with the ChIP DNA Clean & Concentrator (Zymo Research).
Illumina TruSeq

value: d8 EpiSC

tag: antibody


title: ChIP_H3K4me1_d8EpiSC_Otx2


title: ChIP_H3K4me1_d4EpiSC_Otx2

value: d4 EpiSC


title: ChIP_H3K4me1_d2EpiLC_Otx2

value: d2 EpiLC


title: ChIP_H3K4me2_d4EpiSC_WT


title: ChIP_H3K4me1_d4EpiSC_WT


title: ChIP_H3K4me1_d8EpiSC_WT


title: ChIP_H3K4me2_d2EpiLC_Otx2


title: ChIP_H3K4me2_d8EpiSC_WT


title: ChIP_H3K27ac_d2EpiLC_Otx2


title: ChIP_H3K4me2_EpiSC_dCD

value: Mll3/4 dCD


title: ChIP_H3K4me2_EpiLC_dCD


title: ChIP_H3K4me2_EpiLC_WT


title: ChIP_H3K4me2_ESC_dCD


title: ChIP_H3K27ac_d4EB_WT


title: ChIP_H3K4me1_d2EpiLC_WT


title: ChIP_H3K27ac_d4EB_dCD


title: ChIP_H3K27ac_ESC_WT


title: ChIP_H3K4me2_d2EpiLC_WT


title: ChIP_H3K4me1_EpiLC_dCT

value: Mll3/4 dCT


title: ChIP_H3K4me2_d4EpiSC_Otx2


title: ChIP_H3K4me1_EpiLC_WT_rep2


title: ChIP_H3K27ac_ESC_dCT


title: ChIP_H3K4me2_ESC_WT


title: ChIP_H3K4me1_ESC_dCT


title: ChIP_H3K4me1_ESC_WT


title: ChIP_H3K4me2_EpiSC_WT


title: ChIP_H3K27ac_EpiSC_dCD


title: ChIP_H3K27ac_EpiSC_WT


title: ChIP_H3K27ac_EpiLC_dCT


title: ChIP_H3K27ac_EpiLC_WT_rep2


title: ChIP_H3K4me1_EpiSC_dCD


title: ChIP_H3K4me1_EpiSC_WT


title: ChIP_H3K27ac_d2EpiLC_WT


title: ChIP_H3K4me1_EpiLC_WT_rep1


title: ChIP_H3K27ac_EpiLC_WT_rep1


title: ChIP_H3K27ac_EpiLC_dCD


title: ChIP_H3K4me1_EpiLC_dCD


title: HA_EpiSC_WT

data_processing:
Paried or single end reads were mapped to the mouse genome (mm10) using BWA. After duplication removal with the MarkDuplicates function from the Picard tools, reads within blacklisted regions were discarded and the aligned reads were normalized with deeptools to 1x sequencing depth (as RPGC: Reads per genomic content).
Genome_build: mm10
Supplementary_files_format_and_content: RPGC normalized bigWig

value: HA


title: H3K27ac_EpiSC_Dox


title: H3K27ac_EpiSC_ctrl


title: H3K27ac_EpiLC_ctrl


title: HA_EpiLC_Prdm14


title: H3K4me2_EpiLC_ctrl


title: H3K27me3_EpiSC_WT

value: wild type


title: HA_EpiLC_Nanog


title: HA_EpiSC_Nanog_rep2

value: EpiSC


title: HA_EpiSC_Prdm14


title: H3K27ac_EpiLC_Dox


title: H3K27me3_EpiLC_WT


title: HA_EpiSC_Nanog_rep1


title: H3K4me2_EpiSC_Dox


title: H3K4me2_EpiSC_ctrl


title: HA_EpiLC_WT


title: H3K4me2_EpiLC_Dox


title: H3K4me3_EpiLC


title: H3K27me2_EpiSC_WT


title: ChIP_input_ESC


title: H3K4me3_ESC


title: ChIP_input_EpiSC


title: H3K27me2_EpiLC_WT


title: H3K27me2_ESC_WT


title: H3K4me2_EpiSC


title: H3K9me2_EpiSC


title: H3K9me2_ESC


title: H3K27ac_ESC_WT


title: H3K9me3_EpiLC


title: H3K4me1_EpiSC


title: H3K9me3_ESC


title: H3K4me1_EpiLC


title: H3K4me1_ESC


title: H3K27ac_EpiSC_WT


title: H3K27ac_EpiLC_WT


title: ATAC_EpiLC_rep1

data_processing:
Paired end reads were mapped to the mouse genome (mm10) using BWA (Li and Durbin 2009). Read duplicates and reads within blacklisted regions were discarded. BAM files for each stage were converted into bigWig files by normalization to 1x sequencing depth with deeptools-3.3.1 (Ramírez et al. 2016).
Genome_build: mm10
Supplementary_files_format_and_content: RPGC normalized bigWig

source_name: epiblast like cells

growth_protocol: ESC were cultured on gelatin-coated plates using Knock-out DMEM supplemented with 15% FBS and LIF. Before each PGCLC differentiation, ESC were adapted to 2i+LIF media (serum-free N2B27 medium supplemented with MEK inhibitor PD0325901 [0.4 μM], GSK3β inhibitor CHIR99021 [3 μM], and LIF) for at least four days in gelatin-coated tissue culture plates. For the EpiLC differentiation, 6-well plates were coated with 15 µg ml-1 Fibronectin and 2 x 105 2i ESC were differentiated in N2B27 media supplemented with 20 ng ml-1 Activin A, 12 ng ml-1 bFGF and 10 µl ml-1 KSR for two days. For EpiSC differentiation, 2i ESC were plated at a density of 2.5 x 105 cells ml-1 on plates coated with 15 µg ml-1 Fibronectin and differentiated into EpiSC by passaging them in N2B27 media supplemented with 20 ng ml-1 Activin A and 12 ng ml-1 bFGF for at least eight days.

extract_protocol:
The ATAC-seq protocol was adapted from (Buenrostro et al., 2013), using 3.8 x 104 and 5.0 x 104 cells for the two replicates generated for each of the investigated cell types, respectively. Briefly, the cells were lysed in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) supplemented with freshly added protease inhibitor for 15 min. Following centrifugation the pellet was resuspended in a Tn5 transposase reaction mix for 30 min at 37°C. Following DNA purification with the MinElute PCR purification kit.
Illumina Nextera


title: H3K9me2_EpiLC


title: ATAC_EpiSC_rep2


title: ATAC_EpiLC_rep2


title: ATAC_ESC_rep2

source_name: mouse embryonic stem cells


title: H3K4me2_ESC


title: H3K4me2_EpiLC


title: ATAC_ESC_rep1


title: ATAC_input

source_name: ATAC-seq input

value: ATAC-seq input


title: H3K9me3_EpiSC


title: ATAC_EpiSC_rep1


title: E214.ko: KO_no-treatment_rep2

city: Los Angeles

first: Yao Wei

last: Lu

state: CA

address: 2011 Zonal Ave

department: Department of Medicine

institute: University of Southern California

data_processing:
FASTQ files were extracted, and the TruSeq sequencing adapters and low-quality reads were removed from FASTQ files with Cutadapt v.2.3.
The cleaned FASTQ files were quality checked using FastQC (Babraham Bioinformatics).
Cleaned reads were aligned to the mouse genome (Esembl GRCm38 genome obtained from GENCODE) using HISAT2 (v.2.1.0).
Transcript assembly was performed using StringTie (v.1.3.4) with the annotated transcriptome as a reference. The assembled transcriptomes were quantified using prepDE.py script provided by the StringTie developer to generate gene matrix files.
EdgeR (v.3.26.1) was used to compute Counts Per Million (CPM) as a normalized measurement for gene expression. Differentially expressed genes were tested using the Fisher’s exact test, and multiplicity correction is performed with the Benjamini-Hochberg method on the p-values, to control the false discovery rate (FDR).
Genome_build: GRCm38
Supplementary_files_format_and_content: File Type= .txt
Supplementary_files_format_and_content: Reads were normalized and quantified as Count Per Million (CPM) with EdgeR
Supplementary_files_format_and_content: Each file contain CPM from the single-end experiment (CPM_allgene_XinBetaKOvsWT_notreatment.txt) or pair-end experiment(CPM_allgene_XinBetaKOvsWT_withAAV.txt).

source_name: Heart Apex

growth_protocol: Mice were housed in specific-pathogen free facility at Boston Children's Hospital in accordance to the approved IACUC protocol.

extract_protocol:
Total RNA from the hearts of 7.5 day old mice was isolated using TRIzol (Thermo Fisher Scientific).
Strand-specific polyA-selected libraries were generated with the TruSeq RNA Library Preparation Kit (Illumina).

treatment_protocol: AAV9 virus (1× 1010 viral genomes(vg)/g) were injected into postnatal day 1 Xinβ-KO pups or their control littermates with subcutaneous injection.

value: Heart Apex

value: Postnatal day 7.5

value: XinBeta KO

tag: aav9 injection

value: No injection


title: E247.wt: WT_no-treatment_rep4

value: Wildtype


title: E235.ko: KO_no-treatment_rep4


title: E415WY: WT_aYAP_rep4


title: E227.ko: KO_no-treatment_rep3


title: E403KY: KO_aYAP_rep3


title: E210.wt: WT_no-treatment_rep2


title: E199.ko: KO_no-treatment_rep1


title: E196.wt: WT_no-treatment_rep1


title: E383KG: KO_GFP_rep3

value: GFP


title: E389WG: WT_GFP_rep4


title: E390WY: WT_aYAP_rep1


title: E217.wt: WT_no-treatment_rep3


title: E385KG: KO_GFP_rep4


title: E380KG: KO_GFP_rep2


title: E392WY: WT_aYAP_rep3


title: E375WG: WT_GFP_rep3


title: E363KY: KO_aYAP_rep1


title: E368KG: KO_GFP_rep1


title: 4C_Prdm14_TSS_EpiLC

data_processing:
Library strategy: 4C-seq
Reads were assigned to samples based on the first 10 bases of the read. Subsequently, the primer sequence was removed from the read and the remaining sequence starting before the restriction site for NlaIII (CATG) was trimmed to 41 bases. These 41 bases were aligned to the mouse reference genome (mm10) using HISAT2 (Kim et al., 2015). From these alignments, RPM (reads per million) normalized bedgraph files were generated for downstream visualization and analysis (Thongjuea et al., 2013).
Genome_build: mm10
Supplementary_files_format_and_content: rpm normalized bedGraph

extract_protocol:
Circular Chromatin Conformation Capture (4C) followed by sequencing for the Prdm14 locus were performed as previously described (Laugsch et al., 2019). Briefly, cells grown to confluence in 10 cm dishes were crosslinked with 1% formaldehyde for 10 min, quenched with 0.125 M glycine, washed with PBS and incubated in lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100 and 1x protease inhibitor) on ice for 10 min. Nuclei were then re-suspended in 0.5 mL 1.2x CutSMART buffer (NEB) with 0.3% SDS and incubated at 37°C with constant shaking for 1h. Then, Triton X-100 was added to a final concentration of 2%, followed by 1h incubation at 37°C with constant shaking. Next, 600 U of NlaIII (NEB) were added to digest chromatin overnight at 37°C with constant shaking (900 rpm). NlaIII was inactivated by adding SDS to a final concentration of 1.6% and incubating the mixture for 20 min at 65°C. The digested chromatin was transferred to 50 mL tubes and 1x ligation buffer (50 mM Tris-HCl pH 7.6, 10 mM MgCl2, 1 mM ATP, 1mM DTT) was added up to a final volume of 7 ml. Triton X-100 was added to a final concentration of 1% and the solution was incubated for 1h at 37°C and 300 rpm. Afterwards, the digested chromatin was ligated using 100 U of T4 DNA ligase (Life Technologies) for 4 hours at 16°C, followed by RNase A treatment (300 μg total, Peqlab) for 45 min at 37°C. Subsequently, chromatin was de-crosslinked by treating with Proteinase K (300 μg total, Peqlab) and incubating at 65°C overnight. DNA was purified by phenol/chloroform extraction followed by ethanol precipitation and re-suspended in 100 μL of water. The purified DNA was digested with 50 U of DpnII (NEB) in 500 μL of 1x NEBuffer at 37°C overnight. DNA samples were again purified (phenol/chloroform extraction and ethanol precipitation), re-suspended in 500 μL of water and ligated with 200 U of T4 DNA Ligase in a final volume of 14 mL 1x ligation buffer overnight at 16°C. DNA samples were subjected to another round of phenol/chloroform extraction and ethanol precipitation, re-suspended in 150 μL of water and purified by QIAquick PCR Purification Kit (QIAGEN).
4C samples were amplified by inverse PCR using primer for the Prdm14 TSS. The primer included illumina adaptor sequences and short barcodes. The inverse PCRs were performed with the expand long template PCR system and 30 amplification cycles (94°C 2 min, 30x [94°C 10 sec, 60°C 1 min, 68°C 3 min], 68°C 5 min). The inverse PCR primers were designed as previously described (Stadhouders et al., 2013). Once amplified, 4C-seq libraries were sequenced on the HiSeq2500.


title: 4C_Prdm14_TSS_ESC

value: 2i+LIF ESC


title: 4C_Prdm14_TSS_EpiSC


title: E364KY: KO_aYAP_rep2


title: H3K4me3_EpiSC


title: E405KY: KO_aYAP_rep4


title: E348WG: WT_GFP_rep2


title: E347WG: WT_GFP_rep1


title: E391WY: WT_aYAP_rep2


title: H3K27me3_ESC_WT


title: Sample_1_Six2pos_14_6_TRI

city: Ramat Gan

first: Tomer

last: Kalisky

address: Max and Anna Webb

department: Electrical Engineering

country: Israel

institute: Bar Ilan University

data_processing:
Mapping reads to mm10 using TopHat v2.1.0 python 2.7.5 using the '-p 8' parameter
Indexing bam file created by tophat:  'samtools index'
Sorting bam file for later use by htseq-count:  'samtools sort -n'
Counting genes using htseq-count  version HTSeq-0.6.1p1-py2.7-linux-x86_64  on python 2.7.5  '  htseq-count -f bam -s no -a 10 '
Normalizing counts using DESeq2
Genome_build: mm10
Supplementary_files_format_and_content: Excel file with raw and norrmalized counts

molecule: polyA RNA

source_name: Mouse Fetal Kidney

growth_protocol: A wild-type female mouse was crossed with a male that was heterozygous for the Six2-GFP transgene.

extract_protocol:
The female was sacrificed at day E18.5 of pregnancy and kidneys from eleven embryos were dissected, placed in PBS on ice, and examined under a fluorescent microscope to check for the presence of GFP. Kidneys from transgenic embryos showed a clear fluorescent pattern marking the cap mesenchyme, whereas those from the non-transgenic embryos showed uniform background fluorescence. Seven out of the eleven embryos contained the Six2-GFP transgene. Keeping the transgenic and non-transgenic kidneys separate, each kidney was then cut into 2-4 small pieces using a surgical razor blade and placed in 1ml of trypsin 0.25% (03-046-1A, Trypsin Solution B (0.25%), Biological Industries) using forceps. Initial tissue trituration was performed using a tissue grinder (D8938, Dounce, Large clearance pestle) followed by up-down pipetting with a P1000 pipette. Tissue fragments were then incubated with trypsin at 37C for 20 minutes, followed by additional trituration by pipetting. After visual confirmation that the majority of cells were fully dissociated, enzyme digestion was stopped by adding 2ml of DMEM with 10% FBS and placed on ice. Cells were first filtered with 70 micron cell strainers (CSS-010-070, Lumitron) and then 40 micron cell strainers (732-2757, VWR), pelleted by centrifugation for 5 minutes at 500 x g, re-suspended in 3ml PBS, and kept on ice until FACS sorting.    Single cell sorting was done using a BD FACSAria III flow cytometer with an 85 micron nozzle. Forward and side scatter were used to filter out red blood cells and to select for live single cells. Gating based on the negative control – the cells from the non-transgenic kidneys - was used to select for cells that were either positive (Six2-high) or negative (Six2-low) for expression of the Six2-GFP transgene.
Six2-high and Six2-low cells were sorted into separate 1.5 ml tubes, each containing RNA purification buffer. This was repeated in three experiments in which we used two types of RNA purification kits: the “single cell RNA Purification Kit” (51800, Norgen Biotek) was used in one experiment where we sorted 50,000 Six2-high and 50,000 Six2-low cells, and the Direct-zol RNA MiniPrep (R2050, Zymo Research) was used in two experiments where we sorted approximately 100,000 Six2-high cells and 400,000 Six2-low cells. Bulk total RNA was purified according to the manufacturer’s instructions and stored in -80C. Altogether, 6 samples of total RNA were collected (3 replicates, each containing total RNA from Six2-high and Six2-low cells).    Sequencing libraries were prepared using the INCPM-Genomics in-house protocol for mRNA sequencing. Briefly, the poly-A fraction (mRNA) was purified from 80-280 ng of total RNA, followed by fragmentation and generation of double-stranded cDNA. Then, end repair, A-tailing, adapter ligation, and PCR amplification steps were performed. Libraries were evaluated using a Qubit (Thermo fisher scientific) and TapeStation (Agilent). Sequencing libraries were constructed with barcodes to allow for multiplexing of 6 libraries in a single lane. All 6 libraries were sequenced paired-end 2 × 126 bases on an Illumina HiSeq 2500 platform in the Israel National Center for Personalized Medicine (G-INCPM). For each sample we obtained 35-42 million reads.
The INCPM-Genomics in-house protocol for mRNA sequencing, which is compatible with Illumina's TrueSeq RNA sequencing protocol (with a few improvements)

value: Kidney

tag: day since gestation

tag: six2 pos/neg

value: Six2 positive


title: Sample_4_Six2negN_17_7_Norgen

value: Six2 negative


title: Six2 Negative

extract_protocol:
The female was sacrificed at day E18.5 of pregnancy and kidneys from eleven embryos were dissected, placed in PBS on ice, and examined under a fluorescent microscope to check for the presence of GFP. Kidneys from transgenic embryos showed a clear fluorescent pattern marking the cap mesenchyme, whereas those from the non-transgenic embryos showed uniform background fluorescence. Seven out of the eleven embryos contained the Six2-GFP transgene. Keeping the transgenic and non-transgenic kidneys separate, each kidney was then cut into 2-4 small pieces using a surgical razor blade and placed in 1ml of trypsin 0.25% (03-046-1A, Trypsin Solution B (0.25%), Biological Industries) using forceps. Initial tissue trituration was performed using a tissue grinder (D8938, Dounce, Large clearance pestle) followed by up-down pipetting with a P1000 pipette. Tissue fragments were then incubated with trypsin at 37C for 20 minutes, followed by additional trituration by pipetting. After visual confirmation that the majority of cells were fully dissociated, enzyme digestion was stopped by adding 2ml of DMEM with 10% FBS and placed on ice. Cells were first filtered with 70 micron cell strainers (CSS-010-070, Lumitron) and then 40 micron cell strainers (732-2757, VWR), pelleted by centrifugation for 5 minutes at 500 x g, re-suspended in 3ml PBS, and kept on ice until FACS sorting.    Single cell sorting was done using a BD FACSAria III flow cytometer with an 85 micron nozzle. Forward and side scatter were used to filter out red blood cells and to select for live single cells. Gating based on the negative control – the cells from the non-transgenic kidneys - was used to select for cells that were either positive (Six2-high) or negative (Six2-low) for expression of the Six2-GFP transgene.
For single cell RNA sequencing, single cells were sorted into 96 individual wells from a 384 well plate that was pre-filled with Smartseq2 cell lysis buffer, RNase inhibitor, oligo-dT primer, ERCC, and dNTP’s. Plates were spun for 1 minute to collect the liquid and cells at the bottom of the wells and immediately frozen. The Smartseq2 protocol was performed by the Israel National Center for Personalized Medicine (G-INCPM) as previously described with 22 amplification cycles. Altogether, 6 plates were processed, each containing 96 individual cells (576 cells total), with 3 plates containing Six2-high cells and 3 plates containing Six2-low cells. Each one of the 6 plates was separately sequenced 1 × 61 bases on the Illumina HiSeq 2500 platform in the Israel National Center for Personalized Medicine (G-INCPM).
The Smartseq2 protocol

value: Six2 Negative


title: Sample_2_Six2neg_14_6_TRI


title: Sample_10_Six2neg_17_7_TRI


title: Sample_9_Six2pos_17_7_TRI


title: Six2 Positive

value: Six2 Positive


title: ph114Hdac8-0PKR--

city: Kansas City

first: Madelaine

last: Gogol

state: MO

address: 1000 E. 50th Street

department: Computational Biology Core

institute: Stowers Institute

description:
Hdac8_KO_PKR_KO_ph114
Hdac8-0PKR-- Rep 4

data_processing:
Data was analyzed by Rosalind (https://rosalind.onramp.bio/), with a HyperScale architecture developed by OnRamp BioInformatics, Inc. (San Diego, CA). Reads were trimmed using cutadapt1. Quality scores were assessed using FastQC. Reads were aligned to the Mus musculus genome build mm10 using STAR. Individual sample reads were quantified using HTseq and normalized via Relative Log Expression (RLE) using DESeq2 R library. Read Distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step using RSeQC. DEseq was also used to calculate fold changes and p-values. Clustering of genes for the final heatmap of differentially expressed genes was done using the PAM (Partitioning Around Medoids) method using the fpc R library. Functional enrichment analysis of pathways, gene ontology, domain structure and other ontologies was performed using HOMER. Several database sources were referenced for enrichment analysis, including Interpro, NCBI, MSigDB, REACTOME, WikiPathways. Enrichment was calculated relative to a set of background genes relevant for the experiment. Additional gene enrichment is available from the following partner institutions: Advaita (http://www.advaitabio.com/ipathwayguide).
Genome_build: mm10
Supplementary_files_format_and_content: Reads on genes were calculated with HTseq and normalized via Relative Log Expression (RLE) using DESeq2 R library.

source_name: Placenta

extract_protocol:
Total RNA was isolated using TRIzol (#15596026, Thermo Fisher Scientific) from placentas at 14.5 dpc.
cDNA libraries for four individual placentas from each genotype  were prepared after ribo-depletion using the Truseq stranded total RNA library prep kit with ribo-zero gold (#RS-122-2302, Illumina).

value: Placenta


title: ph87Hdac8+0PKR--

description:
Hdac8_WT_PKR_KO_ph87
Hdac8+0PKR-- Rep 2


title: ph49Hdac8-0PKR--

description:
Hdac8_KO_PKR_KO_ph49
Hdac8-0PKR-- Rep 1


title: ph150Hdac8+0PKR--

description:
Hdac8_WT_PKR_KO_ph150
Hdac8+0PKR-- Rep 3


title: ph159Hdac8-0PKR++

description:
Hdac8_KO_PKR_WT_ph159
Hdac8-0PKR++ Rep 4


title: ph156Hdac8+0PKR--

description:
Hdac8_WT_PKR_KO_ph156
Hdac8+0PKR-- Rep 4


title: ph121Hdac8-0PKR++

description:
Hdac8_KO_PKR_WT_ph121
Hdac8-0PKR++ Rep 3


title: ph108Hdac8+0PKR++

description:
Hdac8_WT_PKR_WT_ph108
Hdac8+0PKR++ Rep 3


title: ph99Hdac8+0PKR++

description:
Hdac8_WT_PKR_WT_ph99
Hdac8+0PKR++ Rep 2


title: ph101Hdac8-0PKR++

description:
Hdac8_KO_PKR_WT_ph101
Hdac8-0PKR++ Rep 1


title: ph112Hdac8-0PKR++

description:
Hdac8_KO_PKR_WT_ph112
Hdac8-0PKR++ Rep 2


title: ph113Hdac8+0PKR++

description:
Hdac8_WT_PKR_WT_ph113
Hdac8+0PKR++ Rep 4


title: p439Nipbl+-PKR--

description:
Nipbl_Het_PKR_KO_p439
Nipbl+-PKR-- Rep 1

value: CD1 & C57BL/6 Mixed background


title: p473Nipbl+-PKR--

description:
Nipbl_Het_PKR_KO_p473
Nipbl+-PKR-- Rep 3


title: p465Nipbl+-PKR--

description:
Nipbl_Het_PKR_KO_p465
Nipbl+-PKR-- Rep 2


title: ph83Hdac8+0PKR++

description:
Hdac8_WT_PKR_WT_ph83
Hdac8+0PKR++ Rep 1


title: p481Nipbl+-PKR--

description:
Nipbl_Het_PKR_KO_p481
Nipbl+-PKR-- Rep 4


title: Sample_3_Six2posN_17_7_Norgen


title: ph94Hdac8-0PKR--

description:
Hdac8_KO_PKR_KO_ph94
Hdac8-0PKR-- Rep 3


title: ph91Hdac8-0PKR--

description:
Hdac8_KO_PKR_KO_ph91
Hdac8-0PKR-- Rep 2


title: p466Nipbl++PKR--

description:
Nipbl_WT_PKR_KO_p466
Nipbl++PKR-- Rep 4


title: p449Nipbl++PKR--

description:
Nipbl_WT_PKR_KO_p449
Nipbl++PKR-- Rep 3


title: ph42Hdac8+0PKR--

description:
Hdac8_WT_PKR_KO_ph42
Hdac8+0PKR-- Rep 1


title: p445Nipbl++PKR--

description:
Nipbl_WT_PKR_KO_p445
Nipbl++PKR-- Rep 1


title: p433Nipbl+-PKR++

description:
Nipbl_Het_PKR_WT_p433
Nipbl+-PKR++ Rep 3


title: p461Nipbl+-PKR++

description:
Nipbl_Het_PKR_WT_p461
Nipbl+-PKR++ Rep 4


title: p428Nipbl+-PKR++

description:
Nipbl_Het_PKR_WT_p428
Nipbl+-PKR++ Rep 1


title: p447Nipbl++PKR--

description:
Nipbl_WT_PKR_KO_p447
Nipbl++PKR-- Rep 2


title: p431Nipbl+-PKR++

description:
Nipbl_Het_PKR_WT_p431
Nipbl+-PKR++ Rep 2


title: p430Nipbl++PKR++

description:
Nipbl_WT_PKR_WT_p430
Nipbl++PKR++ Rep 2


title: p432Nipbl++PKR++

description:
Nipbl_WT_PKR_WT_p432
Nipbl++PKR++ Rep 3


title: p429Nipbl++PKR++

description:
Nipbl_WT_PKR_WT_p429
Nipbl++PKR++ Rep 1


title: PDX untreated with LY3039484

type: RNA

city: Castellana Grotte (BA)

first: Grazia

last: Serino

address: Via Turi, 27

country: Italy

institute: National Institute of Gastroenterology “S. de Bellis”, Research Hospital

hyb_protocol: The biotinylated DNA was hybridized to the GeneChip Human Transcriptome Array 2.0 Arrays (Affymetrix), containing  containing more than 286263 full length transcripts covering 44,699 coding genes and 22,829 non coding genes selected from Homo sapiens genome databases RefSeq, ENSEMBL and GenBank.

scan_protocol: Chips were washed, stained and scanned on the Affymetrix Complete GeneChip® Instrument System (Affymetrix, Santa Clara, CA), generating digitized image data and raw intensity data.

data_processing: The raw data were preprocessed with background correction and normalization with the Affymetrix Expression Console. The Affymetrix Transcriptome Analysis Console 4.0 was used to identify significantly differential expressed genes using the Limma eBayes method.

label: biotin

organism: Homo sapiens

source_name: iCCA Patient-derived xenograft

label_protocol: One hundred ng of total RNA total RNA were subjected to two cycles of cDNA synthesis with the Affymetrix WT PLUS expression Kit. The first cycle – first strand synthesis is performed using an engineered set of random primers that exclude rRNA-matching equences and include the T7 promoter sequences. After second-strand synthesis, the resulting cDNA is in vitro transcribed with the T7 RNA polymerase to generate a cRNA. This cRNA is subjected to a second cycle – first strand synthesis in the presence of dUTP in a fixed ratio relative to dTTP. Single strand cDNA is then purified and fragmented with a mixture of uracil DNA glycosylase and apurinic/apirimidinic endonuclease 1 (Affymetrix) in correspondence of incorporated dUTPs. DNA fragments are then terminally labeled by terminal deoxynucleotidyl transferase (Affymetrix) with biotin.

extract_protocol: Total RNA isolation from tissue was performed using TRIzol® (Thermo Fisher Scientific,Waltham, MA, USA) in combination with TissueLyser homogenizer (Qiagen, Hilden, Germany) according to manufacturer’s instructions. In addition, a column-based purification step has been done by using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and total RNA was then eluted in ribonuclease-free water.  RNA concentration and quality were determined with the NanoDrop Spectrophotometer (Thermo Fisher Scientific, MA, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), respectively.

value: PDX tissue

tag: treatment

value: 1% Na-CMC, 0.25% Tween-80, 0.05% Antifoam in ddH2O

tag: host strain

value: CD1 immunodeficient nude mouse


title: PDX untreated with LY3039482


title: ELV-treated

city: Taipei

first: Wei-Lun

last: Hwang

address: No.155, Sec.2, Linong Street

department: Department of Biotechnology and Laboratory Science in Medicine

country: Taiwan

institute: National Yang Ming Chiao Tung University

description: Tumor exosome-treated mouse neutrophils

data_processing:
The raw reads were trimmed and adaptors were removed. The quality reads (≧Q30) were aligned to NCBI_mm10 reference genome and expression values were normalized to RPKM (Reads Per Kilobase per Million mapped reads). The CLC Genomics Workbench was used for data processing and analyzing sequencing data.
Genome_build: mm10
Supplementary_files_format_and_content: feature_ID indicates gene symbol and the quantative value is RPKM

source_name: ELV-treated neutrophils

growth_protocol: The CD11b(+)/Ly-6G(High)/Ly-6C(Low-negative) neutrophils were sorted from bone marrows and cultured under complete RPMI-1640 with 10ng/ml GM-CSF for indicated stimulation at 37'C under a humidified chamber.

extract_protocol:
The total RNA is extracted by Trizol (Invitrogen)
The standard Illumia library preparation procedure by TruSeq Standard mRNA sample prep.

treatment_protocol: Tumor exosomes (20ug/ml) for 3 days

value: Bone marrow

tag: cell type

value: neutrophils


title: p453Nipbl++PKR++

description:
Nipbl_WT_PKR_WT_p453
Nipbl++PKR++ Rep 4


title: PDX treated with LY3039481


title: PDX treated with LY3039478


title: Patient Peritumoral tissue

source_name: iCCA peritumoral tissue

value: human iCCA peritumoral tissue

value: no treatment


title: Patient Tumor tissue

source_name: iCCA tumor tissue

value: human iCCA primary tumor


title: PBS-treated

description: PBS-treated mouse neutrophils

source_name: PBS-treated neutrophils

treatment_protocol: PBS for 3 days


title: PDX treated with LY3039479


title: PDX treated with LY3039480


title: PDX untreated with LY3039485


title: Human mass implanted in PDX

value: mass after PDX model generation (7 passage)


title: ChIP_input_EpiLC

value: d2 EpiLC

value: wild type


title: R_T_Treg_Ragpos_r3

city: Rehovot

first: Jakub

last: Abramson

address: 234 Herzl st.

department: Department of Immunology

institute: Weizmann Institute of Science

description: Bulk Mars-Seq (3' end-RNA-seq)

data_processing:
Data was processed using UTAP pipeline (Kohen et al., 2019). Bcl files were converted into fastq by bcl2fastq. Demultiplexing is done using the sample barcode found on R2, the UMI found on R2 is inserted in the read name (in R1 fastq).  R1 Fastq data submitted.
Reads were trimmed using Cutadapt (parameters: -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -a “A{10}” –times 2 -u 3 -u -3 -q 20 -m 25) and mapped to the Mus_musculus genome using STAR v2.4.2a (parameters: –alignEndsType EndToEnd, –outFilterMismatchNoverLmax 0.05, –twopassMode Basic, –alignSoftClipAtReferenceEnds No).
The pipeline quantifies the 3’ of Gencode annotated genes (The 3’ region contains 1,000 bases upstream of the 3’ end and 100 bases downstream). Counting proceeded over genes annotated in RefSeq release using htseq-count (union mode). Genes having minimum 5 UMI-corrected reads in at least one sample, were considered. Normalization of the counts and differential expression analysis was performed using DESeq2 package.
Genome_build: mm10
Supplementary_files_format_and_content: Tab delimited files include raw counts values and normalized abundance measurements for each sample

extract_protocol:
Thymi and spleens were collected and smashed through a 40um mesh. In spleen samples red blood cells were lysated with ACK lysis buffer.Tregs were sorted and RNA was extracted using Dynabeads. MARSseq protocol was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using the MARSseq protocol (Jaitin et. Al. 2014)

value: C57BL/6 x FVB

value: Thymus

value: Foxp3.RFP Rag.GFP


title: R_T_Treg_Ragpos_r4

description: Bulk Mars-Seq (3' end-RNA-seq)


title: PDX untreated with LY3039483


title: R_T_Treg_Ragpos_r2

description: Bulk Mars-Seq (3' end-RNA-seq)


title: R_T_Treg_Ragpos_r1

description: Bulk Mars-Seq (3' end-RNA-seq)


title: PBAT_EpiLC_WT

data_processing:
Genome-wide DNA methylation data was analysed with Bismark (Krueger and Andrews 2011). Briefly, adapter trimming was performed with Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with trim_galore --clip_R1 6 —clip_R2 6 --paired. The resulting reads were mapped with Bismark-v0.16.1 (Krueger and Andrews 2011) and bowtie2-2.2.9 (Langmead and Salzberg 2012), using the “-pbat” setting. Additionally the unmapped reads were remapped as single-end reads and the CpG methylation levels were estimated with the Bismark methylation extractor. Finally only CpGs with a coverage of 3 - 100 reads were considered.
Genome_build: mm10
Supplementary_files_format_and_content: CpG methylation coverage

growth_protocol: ESC were cultured on gelatin-coated plates using Knock-out DMEM supplemented with 15% FBS and LIF. Before each PGCLC differentiation, ESC were adapted to 2i+LIF media (serum-free N2B27 medium supplemented with MEK inhibitor PD0325901 [0.4 μM], GSK3β inhibitor CHIR99021 [3 μM], and LIF) for at least four days in gelatin-coated tissue culture plates. For the EpiLC differentiation, 6-well plates were coated with 15 µg ml-1 Fibronectin and 2 x 105 2i ESC were differentiated in N2B27 media supplemented with 20 ng ml-1 Activin A, 12 ng ml-1 bFGF and 10 µl ml-1 KSR for two days.

extract_protocol:
Bisulfite sequencing libraries were prepared from column-purified DNA of d2 EpiLC using the PBAT method previously described in (Smallwood et al., 2014). Briefly, for the bisulfite conversion the instructions of the EZ Methylation Direct MagPrep Kit (Zymo) were followed.
After purification, bisulfite converted DNAs were eluted from MagBeads directly into 39 µl of first strand synthesis reaction mastermix (1x Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 µM 6NF preamp oligo (IDT)), heated to 65 °C for 3 minutes and cooled on ice. 50 U of klenow exo- (Enzymatics) was added and the mixture incubated on a thermocycler at 37 °C for 30 minutes after slowly ramping from 4 °C. Reactions were diluted to 100 ml and 20 U of exonuclease I (NEB) added and incubated at 37 °C before purification using a 0.8:1 ratio of AMPure XP beads. Purified products were resuspended in 50 ml of second strand mastermix (1x Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 µM 6NR adaptor 2 oligo (IDT) then heated to 98 °C for 2 minutes and cooled on ice. 50 U of klenow exo- (Enzymatics) was added and the mixture incubated on a thermocycler at 37 °C for 90 minutes after slowly ramping from 4 °C. Second strand products were purified using a 0.8:1 ratio of AMPure XP beads and resuspended in 50 ml of PCR mastermix (1x KAPA HiFi Readymix, 0.2 µM PE1.0 primer, 0.2 µM iTAG index primer) and amplified with 12 cycles. The final libraries were purified using a 0.8:1 volumetric ratio of AMPure XP beads before pooling and sequencing. All libraries were prepared in parallel with the pre-PCR purification steps carried out using a Bravo Workstation pipetting robot (Agilent Technologies).


title: R_T_Treg_Ragneg_r4

description: Bulk Mars-Seq (3' end-RNA-seq)


title: mORF4-NBDY_KO_6

city: Utrecht

first: Eva

last: van Rooij

state: Utrecht

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

data_processing:
Reads were mapped with STAR v2.7.1a against the GRCm38/mm10 reference genome using the ensembl gene annotations
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: Readcounts of genes are present in a matrix format, with rows being genes and columns being individual mice.

source_name: mORF4/NBDY KO_left ventricle

growth_protocol: Mouse were kept unde normal housing conditions

extract_protocol:
RNA was collected using TRIzol reagent according to manufacturer's protocol.
Illumina mRNA sequencing libraries were prepared using Illumina TruSeq Stranded mRNA Library Prep Kit .

treatment_protocol: No treatments was given to the mice

tag: strain background

tag: genotype/variation

tag: Sex

value: Male

value: 8 weeks

value: Cardiac (left ventricle)


title: WT_1

source_name: WT_left ventricle

value: WT


title: WT_2


title: mORF4-NBDY_KO_7


title: WT_3


title: WT_4


title: mORF4-NBDY_KO_5


title: mORF4-NBDY_KO_8


title: R_T_Treg_Ragneg_r1

description: Bulk Mars-Seq (3' end-RNA-seq)


title: R_T_Treg_Ragneg_r2

description: Bulk Mars-Seq (3' end-RNA-seq)


title: R_T_Treg_Ragneg_r3

description: Bulk Mars-Seq (3' end-RNA-seq)


title: R_S_Treg_r2

description: Bulk Mars-Seq (3' end-RNA-seq)

value: Spleen

value: Tregs


title: R_S_Treg_r1

description: Bulk Mars-Seq (3' end-RNA-seq)


title: wt2_Il18_100ngml_96h

data_processing:
Data was processed using UTAP pipeline (Kohen et al., 2019). Bcl files were converted into fastq by bcl2fastq. Demultiplexing is done using the sample barcode found on R2, the UMI found on R2 is inserted in the read name (in R1 fastq).  R1 Fastq data submitted.
Reads were trimmed using Cutadapt (parameters: -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -a “A{10}” –times 2 -u 3 -u -3 -q 20 -m 25) and mapped to the Mus_musculus genome using STAR v2.4.2a (parameters: –alignEndsType EndToEnd, –outFilterMismatchNoverLmax 0.05, –twopassMode Basic, –alignSoftClipAtReferenceEnds No).
The pipeline quantifies the 3’ of Gencode annotated genes (The 3’ region contains 1,000 bases upstream of the 3’ end and 100 bases downstream). Counting proceeded over genes annotated in RefSeq release using htseq-count (union mode). Genes having minimum 5 UMI-corrected reads in at least one sample, were considered. Normalization of the counts and differential expression analysis was performed using DESeq2 package.
Genome_build: mm10
Supplementary_files_format_and_content: Tab delimited files include raw counts values for each sample

growth_protocol: splenocytes from Foxp3.GFP reporter mice were isolated and cultured in vitro in the presence of CD3/CD28 activating beads together with increasing doses of IL18 (0, 10 and 100ng/mL).

extract_protocol:
Treated cells were harvest after 48 and 96 hours. Tregs were sorted and RNA was extracted using Dynabeads. MARSseq protocol was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using the MARSseq protocol (Jaitin et. Al. 2014) (3' end-RNA-seq)

value: spleen


title: R_S_Treg_r3

description: Bulk Mars-Seq (3' end-RNA-seq)


title: wt3_Il18_10ngml_96h


title: wt1_Il18_100ngml_48h


title: wt1_Il18_100ngml_96h


title: wt2_Il18_10ngml_96h


title: PBAT_EpiLC_dCD_Otx2

value: Mll3/4 dCD + Otx2 KO


title: wt1_Il18_10ngml_96h


title: wt2_Il18_10ngml_48h


title: wt3_Il18_10ngml_48h


title: wt2_CD3CD28_96h


title: wt1_CD3CD28_96h


title: wt3_CD3CD28_96h


title: wt1_CD3CD28_48h


title: X18_NT_Ragneg_18Rneg_r2

description: Bulk Mars-Seq (3' end-RNA-seq)

source_name: Tregs

extract_protocol:
Thymi were collected and smashed through a 40um mesh. Tregs were sorted and RNA was extracted using Dynabeads. MARSseq protocol was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using the MARSseq protocol (Jaitin et. Al. 2014)

value: Treg


title: wt3_CD3CD28_48h


title: X18_NT_Ragneg_18Rpos_r2

description: Bulk Mars-Seq (3' end-RNA-seq)


title: X18_NT_Ragneg_18Rneg_r3

description: Bulk Mars-Seq (3' end-RNA-seq)


title: X18_NT_Ragneg_18Rpos_r1

description: Bulk Mars-Seq (3' end-RNA-seq)


title: wt2_Il18_100ngml_48h


title: R_S_Treg_r4

description: Bulk Mars-Seq (3' end-RNA-seq)


title: X18_NT_Ragneg_18Rneg_r1

description: Bulk Mars-Seq (3' end-RNA-seq)


title: X18_NT_Ragpos_r3

description: Bulk Mars-Seq (3' end-RNA-seq)


title: X18_NT_Ragpos_r2

description: Bulk Mars-Seq (3' end-RNA-seq)


title: Adult WT1

city: Cambridge

first: Adrian

last: Liston

address: Babraham Research Campus

country: United Kingdom

institute: The Babraham Institute

data_processing:
Step1: cellranger mkfastq (Cell Ranger V.3.0, 10x Genomics)
Step2: cellranger count (Cell Ranger V.3.0, 10x Genomics)
Step 3: Quality control (Genes expressed in >= 5 cells, cells with gene count more than 200 and less than 5000, cells with less than 10% mitochondrial genes), Seurat V2.2
Genome_build: mm10
Supplementary_files_format_and_content: A matrix of raw UMI counts for each feature (i.e. gene; row) that are detected in each cell (column)  in each sample

source_name: Brain

extract_protocol:
Single cell suspensions were prepared from brain tissue by digestion for 30min at 37ºC with collagenase IV (ThermoFisher), hyaluronidase (Sigma-Aldrich) and DNase I (Sigma-Aldrich). Digested tissue was mechanically disrupted, filtered through 100µm mesh and enriched for leukocytes by centrifugation through 40% Percoll (GE Healthcare). Non-specific binding was blocked using either 2.4G2 supernatant for mouse cells or normal mouse serum for human cells, and dead cells were labelled by fixable viability dye eFluor 780 (ThermoFisher). CD11b+ CD45+ cells were sorted using  BD FACSAria™ III, and resuspended in DPBS 0.04% BSA.Cell viabilityand concentration were assessed by the LUNA-FL Dual Fluorescence Cell Counter.
Single-cell libraries were generated using the GemCode Single-Cell Instrument and Single Cell 3′ Library & Gel Bead Kit v2 and Chip Kit (10x Genomics) according to the manufacturer’s protocol

value: CD11b+ cells


title: X18_NT_Ragneg_18Rpos_r3

description: Bulk Mars-Seq (3' end-RNA-seq)


title: Adult WT2


title: ATAC_mNKT_1

city: Vari

first: Mihalis

last: Verykokakis

address: 34, Fleming street

country: Greece

institute: BSCR Alexander Fleming

data_processing:
Base calling was performed using bcl2fastq software.
Sequenced reads were mapped to the mm10 build using bowtie2 with the --local and --very-sensitive options.
Peak calling was performed for each replicate, using MACS2 on the de-duplicated bam files, with the “--nomodel --shift -100 --extsize 200 --keep-dup all” parameters. Only overlapping peaks between the two replicates of each condition were kept and then were merged resulting in a reproducible peakset (one for each condition). Finally, an “overall” set of 43,317 peaks was generated by combining and merging the peaksets of the four conditions and was used as a reference peakset for the subsequent analysis. Counting of the reads was performed with the DiffBind package and differential accessibility analysis was performed with edgeR (p<0.05). For these analyses, duplicated reads were considered taken into consideration, as it has been shown that their removal can reduce the detection power of differential accessibility analysis in highly abundant regions. Only peaks with at least 10 reads in at least one of the replicates were kept for counting.
Genome_build: mm10
Supplementary_files_format_and_content: BigWig signal files for ATAC-seq data after the removal of multiple aligned reads.

source_name: Thymus

growth_protocol: Mice were maintained under standard dark-light conditions, were euthanized at 4-5 weeks old and thymi were extracted.

extract_protocol:
Single-cell thymocyte suspensions were acquired with mechanical dissociation and cells were stained with appropriate antibodies and tetramers. Samples were flow-sorted in PBS with 0.5% FBS and were then processed for ATAC-seq.
Library was constructed according to standard Illumina protocols

tag: developmental stage

value: mNKT cells


title: MHC class II-deficient mouse with a near-complete absence of CD4 T cells


title: wt3_Il18_100ngml_48h


title: X18_NT_Ragpos_r1

description: Bulk Mars-Seq (3' end-RNA-seq)


title: RNA_Bcl6KO ST1_1

data_processing:
Base calling was performed using bcl2fastq software.
Sequenced reads were mapped to mm10 whole genome using, for RNA-Seq reads, tophat2 with default settings. The reads which remained unmapped were submitted to a second round of mapping using Bowtie2 against mm10 with the --local and --very-sensitive options.
Reads overlapping the Ensembl build 96 mouse exons were counted and filtered for artifacts as follows: if an annotated gene had up to five exons, tag presence was required in at least two exons. If an annotated gene had E >5 exons, tag presence was required in at least E/5 exons. The final gene counts were calculated as the sums of their exonic reads. The counts table was normalized using DESeq normalization and analyzed for differential expression using DESeq and the Bioconductor package metaseqR. The final list of differentially expressed genes was derived by the genes demonstrating a binomial test p-value less than 0.05.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited files containing DESeq normalized counts for each Ensembl gene for RNA-Seq data.

extract_protocol:
Single-cell thymocyte suspensions were acquired with mechanical dissociation and cells were stained with appropriate antibodies and tetramers. Samples were flow-sorted and cells were frozen in RLT buffer, for RNA extraction. RNA was extracted with QIAGEN microRNAeasy kit (Cat No: 74004).
NuGEN Ovation Ultralow Library systems (NuGEN Technologies)

treatment_protocol: No treatment

value: Stage 1 iNKT cells


title: ATAC_mNKT_2


title: wt1_Il18_10ngml_48h


title: RNA_Bcl6KO ST1_2


title: RNA_Bcl6KO ST0_3

value: Stage 0 iNKT cells


title: RNA_Bcl6KO ST0_2


title: RNA_MOLM13_KAT7AID_4h_R3

first: Muxin

last: Gu

email: muxingu@gmail.com, mg445@cam.ac.uk

address: Jeffrey Cheah Biomedical Centre, Puddicombe Way

department: Haematology

institute: University of Cambridge

data_processing:
RNA-seq reads were mapped to the human genome assembly GRCh38 using STAR version 6.43, tolerating mismatch rate of 0.01 and allowing maximal intron lengths of 50kb. Read counts were calculated by STAR using Ensembl annotation of GRCh38 version 91.
Genome_build: GRCh38
Supplementary_files_format_and_content: Read counts

source_name: Human cell line

growth_protocol: MOLM-13 was cultured in RPMI 1640 medium (Life technologies) supplemented with 10% FBS (Life technologies). OCI-AML3 was cultured in MEM-a (Lonza) with 20% FBS (Life technologies) and 1% GlutaMax (Life technologies). To maintain Cas9 expression, blasticidin (ThermoFisher) was added to MOLM13 and OCI-AML3 at 10mg/ml. All cell lines were incubated at 37°C with 5% CO2.

extract_protocol: RNA was extracted from AML cells with RNeasy Plus Mini Kit (Qiagen) according to manufacturer’s instructions. Sequencing was performed on Illumina HiSeq v4 platform with 75-bp paired-end sequencing.

treatment_protocol: KAT7 degron-AID system: the region between the BamHI and NotI of pKLV-EF1aGFPKAT7-W was replaced with a gBlock fragment appended with the degron tag using NEBuilder HiFi kit, resulting in pKLV-EF1aGFP2AKAT7-linkermAID-W. A T2A-fused Oryza sativa TIR1 (OsTIR1) fragment was purchased as a gBlock fragment and cloned into the BsrGI site of pKLV-EF1amCherry, resulting in pKVL-EF1a-mCherry-2A-OsTIR1-W. Sequences were verified by Sanger sequencing.


title: RNA_MOLM13_KAT7AID_0h_R3


title: RNA_MOLM13_KAT7AID_2h_R3


title: RNA_Bcl6KO ST1_3


title: RNA_MOLM13_KAT7AID_2h_R2


title: RNA_MOLM13_KAT7AID_4h_R2


title: RNA_WT ST1_3


title: RNA_WT ST1_2


title: RNA_WT ST1_1


title: RNA_WT ST0_3


title: RNA_MOLM13_KAT7AID_0h_R2


title: RNA_MOLM13_KAT7AID_0h_R1


title: RNA_WT ST0_2


title: RNA_MOLM13_KAT7AID_2h_R1


title: RNA_MOLM13_KAT7AID_4h_R1


title: ATAC_Bcl6KO ST1_2


title: RNA_WT ST0_1


title: ATAC_Bcl6KO ST1_1


title: ATAC_Bcl6KO ST0_1


title: ATAC_Bcl6KO ST0_2


title: RNA_OCIAML3_KAT7KO_d5_R1

data_processing:
Reads of ChIP-seq sample and Input DNA were mapped to the human genome GRCh38 using BWA version 0.7.17 using default parameters. Duplicates were marked by Samtools mkdup. Peaks (broad and narrow) were called by MACS2 version 1.4.1 using input DNA as control with parameters --broad-cutoff 0.01 for broad peaks, and –q 0.01 for narrow peaks.
RNA-seq reads were mapped to the human genome assembly GRCh38 using STAR version 6.43, tolerating mismatch rate of 0.01 and allowing maximal intron lengths of 50kb. Read counts were calculated by STAR using Ensembl annotation of GRCh38 version 91.
Genome_build: GRCh38
Supplementary_files_format_and_content: MACS2 peaks, Read counts

extract_protocol: ChIP samples were washed with RIPA wash buffer (50mM Hepes, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) 3 times followed by wash with Annealing buffer (TE+ 50mM NaCl). Elution Buffer (1% SDS, 50mM Tris-HCl, 10mM EDTA) was added to each ChIP samples and heated at 65°C for 30min. Beads were subsequently removed and samples were heated, at 14000rpm, 65°C and supplemented with 0.2 mg/mL RNase A (ThermoFisher). 0.2 mg/mL Proteinase K (Life Technologies) were added the next day to digest proteins by incubating shaking at 450rpm at 65°C for 4h. ChIP purified by PCR purification Kit (Qiagen). PBS and LB1, LB2, LB3 and RIPA wash buffers were all supplemented with Sodium Butyrate (Sigma), cOmplete EDTA-free protease inhibitor cocktail (Roche) and PMSF (Sigma) immediately before use. RNA was extracted from AML cells with RNeasy Plus Mini Kit (Qiagen) according to manufacturer’s instructions. Sequencing was performed on Illumina HiSeq v4 platform with 75-bp paired-end sequencing.

treatment_protocol: Knockout was performed using gRNA targeting KAT7 gene. gRNA was introduced to cells via lentivirus transduction.

tag: days after knockout


title: RNA_OCIAML3_KAT7KO_d5_R2

tag: cell line


title: RNA_OCIAML3_KAT7KO_d3_R2


title: ATAC_WT ST1_2

tag: strain


title: RNA_OCIAML3_Ctrl_d5_R2

tag: genotype

value: wildtype


title: RNA_OCIAML3_Ctrl_d5_R1


title: RNA_Bcl6KO ST0_1

tag: tissue


title: RNA_MOLM13_KAT7KO_d5_R3


title: ATAC_WT ST0_1


title: FXRKO_OCA_0

city: Innsbruck

first: Jose Miguel

last: Ramos Pittol

address: Innrain 80-82

department: Institute of Biochemistry

country: Austria

institute: University of Innsbruck

description:
Replicate 1
FXRko.gene_exp.diff
genes.count_table

data_processing:
Sequencing reads were aligned to the reference genome (mm9 assembly, MGSCv37) using Tophat2.18 with options "--b2-sensitive --microexon-search -p 2 -G Mus_musculus.NCBIM37.67.gtf"
Duplicated reads were removed with Samtools rmdup (v1.9 , using htslib 1.9)
The normalized read numbers per gene were calculated using the cuffnorm feature of Cufflinks (v2.2.1  linked against Boost version 105500) against the Mus_musculus.NCBIM37.67 gene annotation
Differentially expressed genes deffinition and fold-inductions upon OCA treatment were estimated with CuffDiff v2.2.1 with options "--library-norm-method quartile -p 2 Mus_musculus.NCBIM37.67.gtf"
Genome_build: mm9
Supplementary_files_format_and_content: *.diff Table file output from CuffDiff2 with differential gene expression values
Supplementary_files_format_and_content: *.count_table Normalized count values per gene

source_name: Cultured Liver Organoid

growth_protocol: Mouse liver organoids were cultured and differentiated into hepatocype lineage according to Huch et al. 2013. FXR isoforms or H2b-NeonGreen was lentivirally transduced on a pLV(EF1a) IRES-PuroR vector. 2ug/ml puromycin was used for selection of positive clones.

extract_protocol:
Total RNA was extracted using TRIzoL (Invitrogen) reagent according to manufacturer’s instructions. RNA was stored at -80C until further processing
mRNA was isolated using the Poly(A) Beads (NEXTflex). Sequencing libraries were prepared using the Rapid Directional RNA-Seq Kit (NEXTflex)

treatment_protocol: Cells were seeded in 50 ul matrigel drops in 24-well plates before differentiation. After differentiation, the organoids were treated for 8 hours with 5 µM Obeticholic acid or vehicle (DMSO, 0.1% vol/vol) control.

value: FXR-/-, JAX stock #004144

tag: transgene

tag: selection

value: Puromycin

value: 5 uM OCA for 8 hours

tag: library processing date


title: RNA_MOLM13_KAT7KO_d3_R3


title: RNA_MOLM13_KAT7KO_d3_R2


title: FXRKO_DMSO_1

description:
Replicate 2
FXRko.gene_exp.diff
genes.count_table

value: Vehicle (DMSO) for 8 hours


title: RNA_MOLM13_KAT7KO_d5_R1


title: FXRKO_DMSO_2

description:
Replicate 3
FXRko.gene_exp.diff
genes.count_table


title: RNA_MOLM13_KAT7KO_d5_R2


title: FXRa4_OCA_0

description:
Replicate 1
FXRa4.gene_exp.diff
genes.count_table


title: RNA_MOLM13_KAT7KO_d3_R1


title: FXRa4_DMSO_2

description:
Replicate 3
FXRa4.gene_exp.diff
genes.count_table


title: FXRa4_OCA_2


title: FXRKO_DMSO_0


title: FXRa4_OCA_1

description:
Replicate 2
FXRa4.gene_exp.diff
genes.count_table


title: RNA_OCIAML3_Ctrl_d3_R1


title: FXRa4_DMSO_1


title: ATAC_WT ST1_1

value: Thymus


title: RNA_MOLM13_Ctrl_d5_R2


title: RNA_MOLM13_Ctrl_d5_R1


title: RNA_MOLM13_Ctrl_d3_R3


title: RNA_MOLM13_Ctrl_d3_R1


title: FXRKO_OCA_2


title: RNA_OCIAML3_KAT7KO_d3_R1


title: RNA_OCIAML3_Ctrl_d3_R2


title: FXRa4_DMSO_0


title: FXRKO_OCA_1


title: RNA_MOLM13_Ctrl_d3_R2


title: FXRa3_OCA_2

description:
Replicate 3
FXRa3.gene_exp.diff
genes.count_table


title: CHIP_MOLM13_Input-DNA

tag: chip antibody

value: none (input)


title: FXRa3_OCA_0

description:
Replicate 1
FXRa3.gene_exp.diff
genes.count_table


title: CHIP_MOLM13_KAT7

value: anti-KAT7 (Abcam ab70183)


title: FXRa3_DMSO_2


title: RNA_MOLM13_Ctrl_d5_R3


title: FXRa3_OCA_1

description:
Replicate 2
FXRa3.gene_exp.diff
genes.count_table


title: ATAC_WT ST0_2

value: WT


title: FXRa3_DMSO_1


title: LIB12D

city: North Coogee

first: Denise

last: Anderson

state: WA

address: 71 Caledonia Loop

country: Australia

institute: INSiGENe Pty Ltd

data_processing:
Cell Ranger mkfastq pipeline was performed to demultiplex BCL files and to convert barcode and read data to FASTQ files for analysis
Cell Ranger count takes FASTQ files from cellranger mkfastq and performs alignment, filtering, and UMI counting
Genome_build: mm10

source_name: Bone marrow cells

extract_protocol:
Marrow plug were flushed from femurs and tibias. These long bones were crushed and incubated with 337.5U/mL collagenase (Worthington Biochemical Corp., Lakewood, NJ, USA) and 40 U/mL DNase I (Sigma-Aldrich, NSW, Australia) at 37 °C for 60 min in a shaking water bath. The digested marrow cells and the cells from marrow plug were pooled together. Mononucleated cells were treated with red blood cell (RBC) lysis buffer. Cell suspensions were counted using Countess II FL Automated Cell Counter (Thermo Fisher Scientific).
BGI Genomics prepared single cells for 10x Genomics single-cell sequencing
10x Genomics Single-cell RNA sequencing 3' v2

treatment_protocol: Five mice were transplanted with 1000 BCR-ABL1 pre-B ALL cells via tail vein injection. One mouse was euthanized immediately post-transplantation (Day 0). The remaining animals were euthanized 3, 6, 9, and 12 days post-transplantation.

value: Bone marrow

tag: timepoint

value: Day 12


title: FXRa3_DMSO_0

tag: treatment


title: LIB9D

value: Day 9


title: FXRa2_OCA_2

description:
Replicate 3
FXRa2.gene_exp.diff
genes.count_table


title: LIB3D

value: Day 3


title: LIB0D

value: Day 0


title: FXRa2_DMSO_2


title: FXRa2_OCA_0

description:
Replicate 1
FXRa2.gene_exp.diff
genes.count_table


title: FXRa2_DMSO_0


title: CHIP_OCIAML3_Input-DNA


title: DNMT1_W464A/W465A/W796A_eRRBS, biological replicate 3

city: Durham

first: Greg

last: Wang

state: NC

address: 3 Genome Court

department: Duke Department of Pharmacology and Cancer Biology  and Duke Cancer Institute

institute: Duke University School of Medicine, and Department of Biochemistry and Biophysics, UNC at Chapel Hill (adjunct)

data_processing:
Sequence reads from bisulfite-treated libraries were identified using standard Illumina base-calling software.
The last 5 bases were clipped from the 3’ end of each read due to questionable base quality in this region, followed by filtering the clipped reads to retain only those with average base quality score >=20.
Adapter sequence was trimmed from the 3’ end of reads via Cutadapt v1.2.1 (parameters -a AGATCGGAAGAG -O 5 -q 0 -f fastq); reads shorter than 30nt after trimming were discarded.
Filtered and trimmed reads were mapped with Bismark v0.18.1 (parameters: -X 1000 --non_bs_mm), using Bowtie v1.2 as the underlying alignment tool against a reference including the genome sequence of enterobacteria phage λ (NC_001416.1) concatenated to the mm10 reference assembly (GRCm38).
For all mapped read pairs, an additional 3 bases at the 5’ end of read1 and 1 base at the 5’ end of read2 were clipped due to positional methylation bias, as determined from QC plots generated with the ‘bismark_methylation_extractor’ tool (Bismark v0.18.1). To avoid bias in quantification of methylation status, any redundant mapped bases due to overlapping read ends from the same read pair were trimmed.
Read pairs in which either read end had 3 more or methylated cytosines in non-CpG context were discarded, as they were assumed to have escaped bisulfite conversion.  Although the eRRBS data does carry stranded information, data from the plus and minus strands have been collapsed in this analysis.
Methylated and total cytosine base counts per CpG site per sample were tabulated, with data summed over both genomic strands.
Percent methylation per CpG site per sample was calculated as methylated cytosine bases / total cytosine bases, filtered for sites with minimum read depth >=5 for the purposes of genome browser visualization tracks.
Genome_build: mm10
Supplementary_files_format_and_content: Percent methylation at CpG sites with depth 5+ reads; format = bigWig.
Supplementary_files_format_and_content: Count of methylated and total cytosines per CpG site per sample; format = tab-delimited text.

growth_protocol: Mouse ES cells were cultivated on gelatin-coated dishes in the high-glucose DMEM base medium (Invitrogen) supplemented with 15% of fetal bovine serum (FBS, Invitrogen), 1 x nonessential amino acids (Invitrogen), 0.1 mM 2-mercaptoethanol, and 1000 U/ml leukemia inhibitory factor (ESGRO).

extract_protocol:
Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen). eRRBS analysis of different cell samples was carried out in parallel to generate datasets for across-sample comparisons (such as WT vs. its mutant).
eRRBS was carried out with a previously described protocol with slight modification (Garrett-Bakelman et al., 2015). Briefly, ~300 ng of genomic DNA were digested with three different enzymes (~80 units of MspI, 40 units of BfaI and 40 units of MseI) to enhance genomic fragmentation and coverage. The produced fragments were ligated to pre-annealed adapters containing 5’-methyl-cytosine instead of cytosine, followed by overhang fill-in, 3’-terminal-A extension and purification. Bisulfite treatment of the fragments was done using the EZ DNA Methylation–Lightning kit (Zymo Research). Amplified eRRBS libraries was quality checked with Agilent 2200 TapeStation, followed by deep sequencing on the Illumina brand genome analyzer with 150 bp PE parameters.

treatment_protocol: ES cells were transfected by Lipofectamine 2000 (Invitrogen) with the pPyCAGIP empty vector (EV) or that carrying wild-type (WT) or mutant DNMT1. Forty-eight hours post-transfection, the transduced ES cells were selected out in culture medium with 1μg/mL puromycin for over two weeks. Multiple stable-expression cell lines were established for each group, followed by further characterizations such as immunoblotting of DNMT1 and eRRBS profiling.

value: ES Cells


title: FXRa1_OCA_2

description:
Replicate 3
FXRa1.gene_exp.diff
genes.count_table


title: DNMT1_W465A/W796A_eRRBS, biological replicate 3

value: DNMT1 W465A/W796A


title: DNMT1_W464A/W465A/W796A_eRRBS, biological replicate 2


title: DNMT1_W465A/W796A_eRRBS, biological replicate 2


title: DNMT1_W464A/W465A/W796A_eRRBS, biological replicate 1


title: CHIP_OCIAML3_KAT7


title: FXRa1_OCA_1

description:
Replicate 2
FXRa1.gene_exp.diff
genes.count_table


title: FXRa1_OCA_0

description:
Replicate 1
FXRa1.gene_exp.diff
genes.count_table


title: DNMT1_W465A/W796A_eRRBS, biological replicate 1


title: FXRa1_DMSO_2


title: DNMT1_W464A/W465A_eRRBS, biological replicate 1

value: DNMT1 W464A/W465A


title: FXRa1_DMSO_0


title: FXRa1_DMSO_1


title: DNMT1_W464A/W465A_eRRBS, biological replicate 3


title: FXRa2_OCA_1

description:
Replicate 2
FXRa2.gene_exp.diff
genes.count_table


title: DNMT1_W464A/W465A_eRRBS, biological replicate 2


title: FXRko_Input

description:
KO_Sub.bw
KO_peaks.bed

data_processing:
Sequencing reads were aligned to the reference genome (mm9 assembly) using Bowtie2
Duplicated reads were removed with Samtools rmdup (v1.9 , using htslib 1.9)
FXR enriched regions were determined using MACS v 1.4.2 on ChIP datasets using their respective inputs as background control.
Genomic coverage files were generated with deepTools2 bamCoverage with options "--binSize 50 --normalizeUsing RPGC --effectiveGenomeSize 2620345972 --ignoreForNormalization chrX chrM chrY --extendReads 300"
For visualization, Genomic coverage files from Input material was substracted from their paired ChIP sample using deepTools2 bigwigCompare with options "--operation subtract -bs 50 --skipNA"
Genome_build: mm9
Supplementary_files_format_and_content: *.bw BigWig format containing substracted genomic coverage per condition.
Supplementary_files_format_and_content: *.bed FXR enriched regions, according to MACS

extract_protocol:
Chromatin lysates were prepared as follows: Pooled organoids from 12 wells (approx. 5 mill cells) were mechanically disrupted in PBS and centrifugated to remove the matrigel. Followed by fixation in 1% Formaldehyde in PBS, rotating at room temperature for 15 minutes, and quenched with Glycine at 250 mM final concentration. The following steps, including ChIP, were performed as previously described (Ramos-Pittol et al. 2018).
Sequencing libraries were prepared using the Rapid DNA-Seq Kit (NEXTflex)

treatment_protocol: Cells were seeded in 50 ul matrigel drops in 24-well plates before differentiation. After differentiation, the organoids were treated for 2 hours with 5 µM Obeticholic acid. An estimated 5 million cells were used per chromatin preparation.

tag: antibody


title: LIB6D

value: Day 6


title: DNMT1_W465A_eRRBS, biological replicate 2

tag: cell type

value: DNMT1 W465A


title: FXRa2_DMSO_1

value: FXR-/-, JAX stock #004144

value: Puromycin


title: DNMT1_W465A_eRRBS, biological replicate 1


title: DNMT1_W465A_eRRBS, biological replicate 3


title: FXRa4_Input

description:
a4_Sub.bw
a4_peaks.bed


title: DNMT1_W796A_eRRBS, biological replicate 3

value: DNMT1 W796A


title: FXRa1_Input

description:
a1_Sub.bw
a1_peaks.bed


title: FXRa2_Input

description:
a2_Sub.bw
a2_peaks.bed


title: FXRko_ChIP

description:
KO_Sub.bw
KO_peaks.bed

value: FXR(H-130) (SantaCruz Biotech, sc-13063)


title: FXRa4_ChIP

description:
a4_Sub.bw
a4_peaks.bed


title: DNMT1_WT_eRRBS, biological replicate 2

value: DNMT1 WT


title: DNMT1_W796A_eRRBS, biological replicate 1


title: DNMT1_WT_eRRBS, biological replicate 3


title: FXRa3_ChIP

description:
a3_Sub.bw
a3_peaks.bed


title: DNMT1_W796A_eRRBS, biological replicate 2


title: FXRa3_Input

description:
a3_Sub.bw
a3_peaks.bed


title: FXRa1_ChIP

description:
a1_Sub.bw
a1_peaks.bed


title: DNMT1_WT_eRRBS, biological replicate 1


title: CT26-sphere

description: The miRNA profile of ELVs from CT26-derived spheres.

data_processing:
Initially, the sequences generated went through a filtering process to obtain quality reads (Phred Score >20). Trimmomatics was implemented to trim or remove the reads according to the quality score.
Qualified reads were analyzed using miRDeep2 to clip the 3' adapter sequence and discarding reads shorter than 18 nucleotides. Reads were then aligned  to mm10 genome and MiRBase (version 21).
MiRDeep2 was used to estimate expression levels of known miRNAs and expression values were normalized as reads-per-million (RPM).
Genome_build: mm10
Supplementary_files_format_and_content: miRNA_ID represents miRNA identity; RPM value represents the normalized expression value.

source_name: Colon cancer cell, CT26

growth_protocol:
The CT26 spheres were expanded from the defined stem cell medium cultivation (10ng/ml bFGF, 10ng/ml EGF, N2 supplements in DMEM/F12 medium) for 2 weeks. Atmosphere: air, 95%; Carbon dioxide (CO2), 5%. Temperature, 37.0°C.
The condition medium from cancer spheres was collected for ELV purification by untracentrifugation.

extract_protocol:
Small RNAs were isolated by miRNeasy kit (Quiagene).
RNA samples were prepared using Illumina sample preparation kit (Illumina, San Diego, CA) according to the TruSeq Small RNA Sample Preparation Guide. The 3′ and 5′ adaptors were ligated to total RNA and reverse transcribed followed by PCR amplification. The enriched cDNA was size-fractionated and purified on a 6% polyacrylamide gel electrophoresis.  Bands containing the 18-40 nucleotide RNA fragments (140-155 nucleotides in length with both adapters) were excised as the small RNA library.

value: Balb/C

tag: cellular component

value: Secreted microvesicles

value: Colon cancer


title: FXRa2_ChIP

description:
a2_Sub.bw
a2_peaks.bed


title: CT26-WT-ex-del

description: The miRNA profile of ELVs from parental CT26 cells.

growth_protocol:
The parental CT26 cells were cultured under complete DMEM medium. Atmosphere: air, 95%; Carbon dioxide (CO2), 5%. Temperature, 37.0°C.
The exosome-depleted complete DMEM medium was used to collect condition medium for ELV purification by ultracentrifugation.


title: Kidney_Elderly_DO_Female_Mice_G16_ID_136

city: Davis

first: Brian

middle: J

last: Bennett

address: 430 West Health Sciences Drive

department: Obesity and Metabolism

institute: Western Human Nutrition Research Center, ARS

description: Gene expression in kidney tissue obtained from elderly female DO mouse

scan_protocol: Done according to Affymetrix MoGene 2.1 ST Array Protocol  using the Affymetrix GeneTitan instrument

data_processing: Probes containing known SNPs from the founder strains were masked. We used the robust multiarray average method (RMA) implemented in the Affymetrix gene expression console with default settings (median polish and sketch-quantile normalization) to estimate the normalized expression levels of transcripts.

source_name: Kidney form 56 weeks old DO female mice

growth_protocol: 4 weeks old mice were obtained from Jackson laboratory and maintained on chow diets until 56 week of age

extract_protocol: Total RNA was extracted from about 25 mg of kidney tissue using automated Maxwell 16 Tissue LEV Total RNA Purification Kit, Promega,

treatment_protocol: Mice were injected intraperitoneally with a volume of sterile isotonic saline equivalent to 10 percent of their body weight on the day before tissue collection

tag: gender

value: Female

value: DO mice G16

value: Kidney

tag: age

value: 56 weeks


title: Kidney_Elderly_DO_Female_Mice_G16_ID_132

hyb_protocol: Done according to Affymetrix MoGene 2.1 ST Array Protocol


title: Kidney_Elderly_DO_Female_Mice_G16_ID_124

label_protocol: Done according to Affymetrix MoGene 2.1 ST Array Protocol


title: Kidney_Elderly_DO_Female_Mice_G16_ID_135


title: Kidney_Elderly_DO_Female_Mice_G16_ID_137


title: Kidney_Elderly_DO_Female_Mice_G16_ID_130


title: hESC_untreated_TrAEL-seq

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

institute: Altos Labs

description: hesc_reverse_read_percentages_in_250kb_windows_spaced_10kb.txt

data_processing:
Sequencing data was processed with a custom script (location available from the manuscript) to transfer the first 8bp of a each sequence to the readID of the FastQ file (UMI, unique molecular identifier). In addition, up to and including 3 T bases were removed from the 5'-end following the UMI transfer. The resulting FastQ files were trimmed with Trim Galore (v0.6.5) to remove read-through contamination with the Illumina standard adapter as well as poor base call qualities (cutoff: Phred 20). Trimmed reads were then aligned to the relevant genomes using Bowtie2 (v2.4.1) in --local mode. Following the alignments, reads were de-duplicated taking UMIs as well as mapping position into account, using UmiBam (v0.2.0; https://github.com/FelixKrueger/Umi-Grinder).
Samples were imported into Seqmonk v1.47, truncated to a single base and quantified in windows: for dmc1 total read counts in 20bp windows, for hESC %reverse reads in 250kb windows every 10kb, for SfiI digests total read counts at 13bp cleavage sites, for other samples %reverse reads in 1kb windows
Genome_build: S. cerevisiae R64-1-1 or GRCh38

source_name: hESC line H9

extract_protocol:
For yeast: Cells in ethanol (1-3x107 per plug) were pelleted in round bottom 2 ml tubes by centrifuging 30 s 20,000 g, washed once in 1 ml PFGE wash buffer (10 mM Tris HCl pH 7.5, 50 mM EDTA) and resuspended in 60 µl same with 1 µl lyticase (17 U/ µl in 10 mM KPO4 pH7, 50% glycerol, Merck >2000 U/mg L2524). Samples were heated to 50°C for 1-10 min before addition of 40 µl molten CleanCut agarose (Bio-Rad 1703594), vortexing vigorously for 5 s before pipetting in plug mould (Bio-Rad 1703713) and solidifying 15-30 min at 4°C. Each plug was transferred to a 2 ml tube containing 500 µl PFGE was buffer containing 10 µl 17 U/µl lyticase and incubated 1 hour at 37°. Solution was replaced with 500 µl PK buffer (100 mM EDTA pH 8, 0.2% sodium deoxycholate, 1% sodium N-lauroyl sarcosine, 1 mg/ml Proteinase K) and incubated overnight at 50°. Plugs were rinsed with 1 ml TE, then washed three times with 1 ml TE for 1-2 hours at room temperature with rocking, 10 mM PMSF was added to the second and third washes from 100 mM stock (Merck 93482). Plugs were then digested 1 h at 37°C with 1 µl 1000 U/ml RNase T1 (Thermo EN0541) in 200 µl TE. RNase A was not used as it binds strongly to single stranded DNA [76]. For hESC: 1x106 cells were washed once in 5 ml L buffer (10 mM Tris HCl pH 7.5, 100 mM EDTA, 20 mM NaCl) and resuspended in 60 µl L buffer in a 2 ml tube. Samples were heated to 50°C for 2-3 min before addition of 40 µl molten CleanCut agarose (Bio-Rad 1703594), vortexing vigorously for 5 s before pipetting in plug mould (Bio-Rad 1703713) and solidifying 15-30 min at 4°C. Each plug was transferred to a 2 ml tube containing 500 µl digestion buffer (10 mM Tris HCl pH 7.5, 100 mM EDTA, 20 mM NaCl, 1% sodium N-lauroyl sarcosine, 0.1 mg/ml Proteinase K) and incubated overnight at 50°. Washing as for yeast.
TrAEL-seq: ½ an agarose plug of cells was equilibrated once in 100 µl 1x TdT buffer (NEB) for 30 min at room temperature, then incubated for 2 h at 37°C in 100 µl 1x TdT buffer containing 4 µl 10 mM ATP and 2 µl Terminal Transferase (NEB M0315L). Plugs were rinsed with 1 ml tris buffer (10 mM Tris HCl pH 8.0), equilibrated in 100 µl 1x T4 RNA ligase buffer (NEB) containing 40 µl 50% PEG 8000 for 1 hour at room temperature then incubated overnight at 25°C in 100 µl 1x T4 RNA ligase buffer (NEB) containing 40 µl 50% PEG 8000, 1 µl 10 pM/µl TrAEL-seq adaptor 1 ([App]NNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTUGCGCAGGCCATTGGCC[BtndT]GCGCUACACTCTTTCCCTACACGACGCT) and 1 µl T4 RNA ligase 2 truncated KQ (NEB M0373L). Plugs were then rinsed with 1 ml tris buffer, transferred to 15 ml tubes and washed three times in 10 ml tris buffer with rocking at room temperature for 1-2 hours each, then washed again overnight under the same conditions. Plugs were equilibrated for 15 min with 1 ml agarase buffer (10 mM Bis-Tris-HCl, 1 mM EDTA pH 6.5), then the supernatant removed and 50 µl agarase buffer added. Plugs were melted for 20 min at 65°C, transferred for 5 min to a heating block pre-heated to 42°C, 1 µl β-agarase (NEB M0392S) was added and mixed by flicking without allowing sample to cool, and incubation continued at 42°C for 1 h. DNA was ethanol precipitated with 25 µl 10 M NH4OAc, 1 µl GlycoBlue, 330 µl of ethanol and resuspended in 10 µl 0.1xTE. 40 µl reaction mix containing 5µl Isothermal amplification buffer (NEB), 3 µl 100 mM MgSO4, 2µl 10 mM dNTPs and 1µl Bst 2 WarmStart DNA polymerase (NEB M0538S) was added and sample incubated 30 min at 65°C before precipitation with 12.5 µl 10 M NH4OAc, 1 µl GlycoBlue, 160 µl ethanol and re-dissolving pellet in 130 µl 1xTE.  END-seq: ½ an agarose plug of cells was equilibrated for 1 h at room temperature in 100µl NEBuffer 2 with 0.1mM dNTPs, then overnight at 37°C with 100 µl NEBuffer 2 with 0.1 mM dNTPs and 1 µl Klenow (NEB M0210S). Note that this is not the same blunting regimen used in END-seq [11] as Klenow performed best for SfiI-digested yeast genomic DNA in our hands. After rinsing twice with 1 ml tris buffer, plug was transferred to a 15 ml tube and washed three times for 15 min each with 10 ml tris buffer on rocker at room temperature before transfer to a new 2 ml tube. The plug was equilibrated with 100 µl CutSmart buffer containing 5 mM DTT and 1 mM dATP for 1 hour at room temperature before incubation for 2 h at 37°C in another 100 µl of the same buffer containing 1 µl Klenow exo- (NEB M0212S) and 1 µl T4 PNK (NEB M0201S). Plug was rinsed twice with 1 ml tris buffer then washed once with 10 ml of tris buffer for 15 min as above then returned to a 2 ml tube. The plug was equilibrated for 1 h at room temperature in 100 µl 1x Quick Ligation buffer (NEB B6058S) containing 2.7 µl END-seq adaptor 1 ([Phos]GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGUU[BtndT]U[BtndT]UUACACTCTTTCCCTACACGACGCTCTTCCGATC∗T), then overnight at 25°C with another 100 µl of the same buffer containing  2.7 µl END-seq adaptor 1 and 1 µl high concentration T4 DNA Ligase (NEB M0202M). After rinsing twice with 1 ml tris buffer, plug was transferred to a 15 ml tube and washed three times for 1-2 h each with 10 ml tris buffer on rocker at room temperature, then again overnight.  The plug was transferred to a 1.5 ml tube and equilibrated 15 min with 1 ml agarase buffer (10 mM Bis-Tris-HCl, 1 mM EDTA pH 6.5), then the supernatant removed and 50 µl agarase buffer added to the plug. Plug was melted 20 min at 65°C then transferred for 5 min to a heating block pre-heated to 42°C, 1 µl beta-agarase (NEB M0392S) was added and mixed by flicking without allowing sample to cool, and incubation continued at 42°C for 1 h. DNA was ethanol precipitated with 25 µl 10 M NH4OAc, 1 µl GlycoBlue, 330 µl of ethanol and resuspended in 130 µl 1x TE, 15 min at 65°C.
The DNA was transferred to an AFA microTUBE (Covaris 520045) and fragmented in a Covaris E220 using duty factor 10, PIP 175, Cycles 200, Temp 11, then transferred to a 1.5 ml tube containing 8 µl pre-washed Dynabeads MyOne streptavidin C1 beads (Thermo, 65001) re-suspended in 300 µl 2xTN (10 mM Tris pH 8, 2 M NaCl) along with 170 µl water (total volume 600 µl) and incubated 30 min at room temperature on a rotating wheel. Beads were washed once with 500 µl 5 mM Tris pH 8, 0.5 mM EDTA, 1 M NaCl, 5 min on wheel and once with 500 µl 0.1x TE, 5 min on wheel before re-suspension in 25 µl 0.1x TE. 3.5 μl NEBNext Ultra II End Prep buffer, 1 µl 1 ng/µl sonicated salmon sperm DNA (this is added as a carrier) and 1.5 μl NEBNext Ultra II End Prep enzyme were added and reaction incubated 30 min at room temperature and 30 min at 65°C. After cooling, 1.25 µl 10 pM/µl TrAEL-seq adaptor 2 ([Phos]GATCGGAAGAGCACACGTCTGAACTCCAGTCUUUUGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T ) or END-seq adaptor 2c ([Phos]GATCGGAAGAGCTATTATTTAAATTTTAATTUGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T), 0.5 µl NEBNext ligation enhancer and 15 µl NEBNext Ultra II ligation mix were added and incubated 30 min at room temperature. The reaction mix was removed and discarded and beads were rinsed with 500 µl wash buffer (5 mM Tris pH 8, 0.5 mM EDTA, 1 M NaCl) then washed twice with 1 ml wash buffer for 10 min on wheel at room temperature and once for 10 min with 1 ml 0.1x TE. Libraries were eluted from beads with 11 µl 1x TE and 1.5 µl USER enzyme (NEB) for 15 min at 37°C, then again with 10.5 µl 1x TE and 1.5 µl USER enzyme (NEB) for 15 min at 37°, and the two eluates combined. An initial test amplification was used to determine the optimal cycle number for each library. For this, 1.25 μl library was amplified in 10 µl total volume with 0.4 µl each of the NEBNext Universal and any NEBNext Index primers with 5 µl NEBNext Ultra II Q5 PCR master mix. Cycling program: 98°C 30s then 18 cycles of [98°C 10 s, 65°C 75 s], 65°C 5 min. Test PCR was cleaned with 8 µl AMPure XP beads (Beckman A63881) and eluted with 2.5 µl 0.1x TE, of which 1 µl was examines on a Bioanalyser high sensitivity DNA

value: hESC line H9

tag: growth and treatment

value: N2B27 plus 3uM retinoic acid

tag: fix and post-processing


title: hESC_Retinoic_acid_TrAEL-seq

value: Maintained in E8 media


title: Kidney_Elderly_DO_Female_Mice_G16_ID_134


title: Kidney_Elderly_DO_Female_Mice_G16_ID_129


title: Kidney_Elderly_DO_Female_Mice_G16_ID_140


title: Kidney_Elderly_DO_Female_Mice_G16_ID_133


title: Kidney_Elderly_DO_Female_Mice_G16_ID_128


title: Kidney_Elderly_DO_Female_Mice_G16_ID_126


title: Kidney_Elderly_DO_Female_Mice_G16_ID_138


title: Kidney_Elderly_DO_Female_Mice_G16_ID_127


title: Kidney_Elderly_DO_Female_Mice_G16_ID_120


title: Kidney_Elderly_DO_Female_Mice_G16_ID_122


title: Kidney_Elderly_DO_Female_Mice_G16_ID_116


title: Kidney_Elderly_DO_Female_Mice_G16_ID_131


title: Kidney_Elderly_DO_Female_Mice_G16_ID_110


title: Kidney_Elderly_DO_Female_Mice_G16_ID_111


title: Kidney_Elderly_DO_Female_Mice_G16_ID_115


title: Kidney_Elderly_DO_Female_Mice_G16_ID_117


title: Kidney_Elderly_DO_Female_Mice_G16_ID_104


title: Kidney_Elderly_DO_Female_Mice_G16_ID_106


title: Kidney_Elderly_DO_Female_Mice_G16_ID_099


title: Kidney_Elderly_DO_Female_Mice_G16_ID_109


title: Kidney_Elderly_DO_Female_Mice_G16_ID_102


title: colon cancer tumor, 90494

city: Notre Dame

first: Steven

middle: A

last: Buechler

state: IN

address: 102B Crowley Hall

department: Appl. Comp. Math & Statistics

institute: University of Notre Dame

data_processing:
FASTQ files were aligned to the Ensembl v90 genome build using the Rsubread package
gene counts computed using the feature counts algorithm
transcript per million (TPM) values were computed
Genome_build: Ensembl v90
Supplementary_files_format_and_content: raw counts - individual sample file
matrix table containing raw counts for every gene and sample (tab-delimited text file)
matrix table containing transcripts per million (TPM) values for every gene and sample (tab-delimited text file)
table of gene symbols and entrez ids (tab-delimited text file)

source_name: colon cancer tumor

extract_protocol:
Tumor tissue was obtained from tumor-rich regions of unstained slides (1 or 2 per patient) and deparaffinized by Xylene wash. mRNA was then extracted with RecoverAll Total Nucleic Acid Isolation Kit.
Libraries were constructed using the NEBNext Ultra II Directional Library Prep kit with NEBNext rRNA Depletion module (New England Biolabs) (Cat. #E7765S, E6350L) following manufacturers recommendations.  Libraries were quality assessed on the Bioanalyzer DNA 7500 chip and quantified by qPCR using the Illumina Kapa Library Quantification Kit (Roche Diagnostics). The libraries were normalized, pooled in equal molar amounts, and sequenced on 6 lanes of a NextSeq v2.5 High Output flowcell using paired 75bp reads.

tag: Stage

value: II

tag: t-stage

tag: location

value: transverse


title: colon cancer tumor, 90523


title: colon cancer tumor, 90429

value: ascending


title: colon cancer tumor, 90498

value: sigmoid


title: colon cancer tumor, 90419


title: Kidney_Elderly_DO_Female_Mice_G16_ID_091

tag: strain

tag: tissue


title: Kidney_Elderly_DO_Female_Mice_G16_ID_084


title: Kidney_Elderly_DO_Female_Mice_G16_ID_093


title: Kidney_Elderly_DO_Female_Mice_G16_ID_094


title: Kidney_Elderly_DO_Female_Mice_G16_ID_076


title: colon cancer tumor, 90414


title: Kidney_Elderly_DO_Female_Mice_G16_ID_087


title: Kidney_Elderly_DO_Female_Mice_G16_ID_098


title: Kidney_Elderly_DO_Female_Mice_G16_ID_089


title: Kidney_Elderly_DO_Female_Mice_G16_ID_083


title: Kidney_Elderly_DO_Female_Mice_G16_ID_081


title: Kidney_Elderly_DO_Female_Mice_G16_ID_121


title: Kidney_Elderly_DO_Female_Mice_G16_ID_075


title: Kidney_Elderly_DO_Female_Mice_G16_ID_088


title: Kidney_Elderly_DO_Female_Mice_G16_ID_082


title: colon cancer tumor, 90473


title: colon cancer tumor, 90409


title: colon cancer tumor, 90467


title: Kidney_Elderly_DO_Female_Mice_G16_ID_085


title: colon cancer tumor, 90396


title: colon cancer tumor, 90401


title: Kidney_Elderly_DO_Female_Mice_G16_ID_072


title: Kidney_Elderly_DO_Female_Mice_G16_ID_073


title: Kidney_Elderly_DO_Female_Mice_G16_ID_077


title: colon cancer tumor, 90388


title: colon cancer tumor, 90412


title: colon cancer tumor, 90384


title: colon cancer tumor, 90337


title: Kidney_Elderly_DO_Female_Mice_G16_ID_070


title: colon cancer tumor, 90323


title: colon cancer tumor, 90322


title: Kidney_Elderly_DO_Female_Mice_G16_ID_068


title: Kidney_Elderly_DO_Female_Mice_G16_ID_071


title: colon cancer tumor, 90315


title: colon cancer tumor, 90321


title: colon cancer tumor, 90269


title: Kidney_Elderly_DO_Female_Mice_G16_ID_067


title: Kidney_Elderly_DO_Female_Mice_G16_ID_056


title: colon cancer tumor, 90279


title: Kidney_Elderly_DO_Female_Mice_G16_ID_060


title: colon cancer tumor, 90237


title: colon cancer tumor, 90222


title: Kidney_Elderly_DO_Female_Mice_G16_ID_061


title: colon cancer tumor, 90289


title: Kidney_Elderly_DO_Female_Mice_G16_ID_074


title: colon cancer tumor, 90178


title: colon cancer tumor, 90209


title: colon cancer tumor, 90175


title: colon cancer tumor, 90197


title: colon cancer tumor, 90172


title: Kidney_Elderly_DO_Female_Mice_G16_ID_052


title: Kidney_Elderly_DO_Female_Mice_G16_ID_054


title: Kidney_Elderly_DO_Female_Mice_G16_ID_047


title: Kidney_Elderly_DO_Female_Mice_G16_ID_053


title: colon cancer tumor, 90152


title: colon cancer tumor, 90150


title: Kidney_Elderly_DO_Female_Mice_G16_ID_049


title: Kidney_Elderly_DO_Female_Mice_G16_ID_045


title: Kidney_Elderly_DO_Female_Mice_G16_ID_043


title: Kidney_Elderly_DO_Female_Mice_G16_ID_046


title: Kidney_Elderly_DO_Female_Mice_G16_ID_048


title: Kidney_Elderly_DO_Female_Mice_G16_ID_042


title: colon cancer tumor, 90149


title: Kidney_Elderly_DO_Female_Mice_G16_ID_041


title: Kidney_Elderly_DO_Female_Mice_G16_ID_040


title: Kidney_Elderly_DO_Female_Mice_G16_ID_038


title: Kidney_Elderly_DO_Female_Mice_G16_ID_039


title: Kidney_Elderly_DO_Female_Mice_G16_ID_037


title: colon cancer tumor, 90146


title: Kidney_Elderly_DO_Female_Mice_G16_ID_051


title: colon cancer tumor, 90185

value: descending


title: colon cancer tumor, 90221


title: colon cancer tumor, 90114


title: colon cancer tumor, 90112


title: colon cancer tumor, 90106


title: Kidney_Elderly_DO_Female_Mice_G16_ID_035


title: Kidney_Elderly_DO_Female_Mice_G16_ID_034


title: Kidney_Elderly_DO_Female_Mice_G16_ID_033


title: colon cancer tumor, 90090


title: colon cancer tumor, 90086


title: colon cancer tumor, 90081


title: Kidney_Elderly_DO_Female_Mice_G16_ID_031


title: colon cancer tumor, 90071


title: colon cancer tumor, 90074


title: colon cancer tumor, 90124


title: Kidney_Elderly_DO_Female_Mice_G16_ID_057


title: Kidney_Elderly_DO_Female_Mice_G16_ID_030


title: colon cancer tumor, 90056


title: colon cancer tumor, 90032


title: colon cancer tumor, 90018


title: colon cancer tumor, 90017


title: colon cancer tumor, 90097


title: Kidney_Elderly_DO_Female_Mice_G16_ID_024


title: T2-22

city: Mahasset

first: Andrew

last: Shih

state: NY

address: 350 Community Dirive

institute: Feinstein Institute - Northwell Health

data_processing:
align with STAR2 to human GENCODE reference GRCh37
gene counts with htseq-counts using GENCODE genes using gene names
Genome_build: GRCh37
Supplementary_files_format_and_content: RCC_htseq_counts.txt are the digital counts from the gene boundaries as previously described

source_name: Renal Cell Carcinoma

extract_protocol:
A genitourinary pathologist (O. Yaskiv) reviewed FFPE tissue blocks and their corresponding H&E slides. Slides were matched up with tissue blocks and ~35 mg of the pre-identified tumor were removed from the block. RNA was extracted using the RecoverAllTM Total Nucleic Acid Isolation Kit according to manufacturer’s instructions.
Libraries were prepared using the Illumina TruSeq RNA Access Library kit and sequenced on a NextSeq 500 following manufacturer’s instructions.

value: Renal Cell Carcinoma

tag: tissue preparation

value: nephrectomy specimens preserved in FFPE

tag: metastatic

value: Yes

tag: tumor stage

value: M


title: T2-12

value: No


title: T2-2


title: T2-4


title: T2-6


title: Kidney_Elderly_DO_Female_Mice_G16_ID_023

value: Female


title: Kidney_Elderly_DO_Female_Mice_G16_ID_029

value: DO mice G16


title: T2-9


title: Kidney_Elderly_DO_Female_Mice_G16_ID_028

value: Kidney


title: Kidney_Elderly_DO_Female_Mice_G16_ID_026

value: 56 weeks


title: Kidney_Elderly_DO_Female_Mice_G16_ID_027


title: Kidney_Elderly_DO_Female_Mice_G16_ID_021


title: Kidney_Elderly_DO_Female_Mice_G16_ID_017


title: T2-8


title: Kidney_Elderly_DO_Female_Mice_G16_ID_012


title: Kidney_Elderly_DO_Female_Mice_G16_ID_022


title: Kidney_Elderly_DO_Female_Mice_G16_ID_010


title: T2-26

value: F


title: T2-7


title: T2-14


title: Kidney_Elderly_DO_Female_Mice_G16_ID_006


title: Kidney_Elderly_DO_Female_Mice_G16_ID_016


title: Kidney_Elderly_DO_Female_Mice_G16_ID_025


title: Kidney_Elderly_DO_Female_Mice_G16_ID_019


title: T2-23


title: Kidney_Elderly_DO_Female_Mice_G16_ID_011


title: T2-3


title: RNAseq_N2_dauer

city: Basel

first: Helge

last: Grosshans

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedriche Miescher Institute for Biomedical Research (FMI)

description: BSSE_QGF_14002_130718_SN792_0249_AD1V60ACXX_6_CGATGT_L006

data_processing:
RNA-seq data (including the dauer exit samples from https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52910) were mapped to the C. elegans genome (ce10) using the qAlign function (splicedAlignment=TRUE) from the QuasR package in R.
Gene expression was quantified using qCount function from the QuasR package in R.
Counts were scaled by total mapped library size for each sample.
A pseudocount of 8 was added and counts were log2-transformed.
Genome_build: ce10
Supplementary_files_format_and_content: Tab-separated: First column contains gene names (WormBase identifiers), second column contains summed width of exons per gene, following columns contain raw counts of reads overlapping each gene for each sample.

organism: Caenorhabditis elegans

source_name: synchronized population

growth_protocol: Dauer animals were synchronously release from dauer by feeding at 25 degree. Worms were collected hourly and washed 3 times in M9.

extract_protocol:
RNA was extracted using Trizol and conventional Phenol/choloform extraction
TruSeq-illumina-mRNA seq followed by Hiseq 50-cycle-single-end-reads on HiSeq2500

tag: developmental stage


title: Kidney_Elderly_DO_Female_Mice_G16_ID_003


title: RNAseq_tm548_23h

description:
tm548_TP23_2139F47
tm548_timepoint_23

data_processing:
3' adapters are trimmed with cutadapt=2.3 (--error-rate = 0.1, --minimum-length = 15, --overlap = 3)
RNA-seq data were mapped to the C. elegans ce10 genome using STAR with default parameters (version 2.7.0f)
reads are counted using htseq-count (version = 0.11.2)
Genome_build: ce10
Supplementary_files_format_and_content: Tab-separated: First column contains gene names (Wormbase identifiers), second column contains summed width of exons per gene, following columns contain raw counts of reads overlapping each gene for each sample.

growth_protocol: a population of L1 synchronized by egg prep and M9 incubation was grown on OP50 containing agar plates at 25 degrees. Worms were collected hourly by washing of worms with M9 and washing 3 times in M9.

extract_protocol:
RNA was extracted using Trizol and conventional Phenol / Chlorophorm extraction.
Illumina TruSeq mRNA-Seq Sample Prep Kit (Stranded - high input), followed by the Hiseq50 Cycle Single-end reads on HiSeq2500 protocol

tag: genotype/variation

value: blmp-1(tm548) mutant

value: 23 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_21h

description:
tm548_TP21_2139F45
tm548_timepoint_21

value: 21 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_22h

description:
tm548_TP22_2139F46
tm548_timepoint_22

value: 22 h after plating L1-arrested C. elegans larvae


title: T2-21


title: Kidney_Elderly_DO_Female_Mice_G16_ID_013


title: Kidney_Elderly_DO_Female_Mice_G16_ID_009


title: T2-19


title: RNAseq_tm548_20h

description:
tm548_TP20_2139F44
tm548_timepoint_20

value: 20 h after plating L1-arrested C. elegans larvae


title: T2-20


title: T2-13


title: RNAseq_tm548_17h

description:
tm548_TP17_2139F41
tm548_timepoint_17

value: 17 h after plating L1-arrested C. elegans larvae


title: T2-17


title: RNAseq_tm548_24h

description:
tm548_TP24_2139F48
tm548_timepoint_24

value: 24 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_19h

description:
tm548_TP19_2139F43
tm548_timepoint_19

value: 19 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_16h

description:
tm548_TP16_2139F40
tm548_timepoint_16

value: 16 h after plating L1-arrested C. elegans larvae


title: T1-23

value: Renal Cell Carcinoma

value: nephrectomy specimens preserved in FFPE

value: Yes


title: T2-1

value: No

value: M


title: T2-10


title: RNAseq_tm548_18h

description:
tm548_TP18_2139F42
tm548_timepoint_18

value: 18 h after plating L1-arrested C. elegans larvae


title: T1-24


title: RNAseq_tm548_15h

description:
tm548_TP15_2139F39
tm548_timepoint_15

value: 15 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_12h

description:
tm548_TP12_2139F36
tm548_timepoint_12

value: 12 h after plating L1-arrested C. elegans larvae


title: T1-22


title: RNAseq_tm548_13h

description:
tm548_TP13_2139F37
tm548_timepoint_13

value: blmp-1(tm548) mutant

value: 13 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_11h

description:
tm548_TP11_2139F35
tm548_timepoint_11

value: 11 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_10h

description:
tm548_TP10_2139F34
tm548_timepoint_10

value: 10 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_8h

description:
tm548_TP8_2139F32
tm548_timepoint_8

value: 8 h after plating L1-arrested C. elegans larvae


title: T1-21

value: F


title: RNAseq_tm548_7h

description:
tm548_TP7_2139F31
tm548_timepoint_7

value: 7 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_9h

description:
tm548_TP9_2139F33
tm548_timepoint_9

value: 9 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_6h

description:
tm548_TP6_2139F30
tm548_timepoint_6

value: 6 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_3h

description:
tm548_TP3_2139F27
tm548_timepoint_3

value: 3 h after plating L1-arrested C. elegans larvae


title: T2-11

value: Renal Cell Carcinoma

value: nephrectomy specimens preserved in FFPE


title: RNAseq_tm548_14h

description:
tm548_TP14_2139F38
tm548_timepoint_14

value: 14 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_4h

description:
tm548_TP4_2139F28
tm548_timepoint_4

value: 4 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_5h

description:
tm548_TP5_2139F29
tm548_timepoint_5

value: 5 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_2h

description:
tm548_TP2_2139F26
tm548_timepoint_2

value: 2 h after plating L1-arrested C. elegans larvae


title: RNAseq_tm548_1h

description:
tm548_TP1_2139F25
tm548_timepoint_1

value: 1 h after plating L1-arrested C. elegans larvae


title: RNAseq_N2_larvae_23h


title: RNAseq_N2_larvae_21h


title: RNAseq_N2_larvae_22h


title: T1-6

value: No

value: M


title: RNAseq_N2_larvae_20h


title: T1-4

value: Yes


title: RNAseq_N2_larvae_19h


title: RNAseq_N2_larvae_18h


title: T1-3


title: RNAseq_N2_larvae_17h


title: RNAseq_N2_larvae_24h


title: RNAseq_N2_larvae_16h


title: Kidney_Elderly_DO_Female_Mice_G16_ID_007

value: Female

value: DO mice G16

value: Kidney

value: 56 weeks


title: T1-1


title: RNAseq_N2_larvae_14h


title: T1-5


title: PiSZ2 Liver H7

city: Boston

first: Carlos

last: Villacorta-Martin

state: MA

address: 670 Albany St

institute: Center for Regenerative Medicine at Boston University

description:
PiSZ2 Liver
H7

data_processing:
Reads were aligned to the ENSEMBL human reference genome GRCh38.91 using STAR
We used the Bioconductor package edgeR to import, filter and normalize the count matrix, followed by the limma package and voom, for linear model fitting and differential expression testing, using empirical Bayes moderation for estimating gene-wise variability prior to significance testing based on the moderated t-statistic.
Genome_build: GRCh38.91
Supplementary_files_format_and_content: EdgeR normalized, counts per million

source_name: human iPSC

growth_protocol: Undifferentiated iPSCs were maintained on murine embryonic fibroblast feeders with knockout serum replacement supplemented media or in feeder free conditions with mTeSR-1 media.

extract_protocol:
PHH were freshly thawed and RNA was immediately isolated. Total RNA was isolated using miRNeasy kit (Qiagen) per manufacturer’s instruction.
For digital gene expression (DGE) the concentration of all samples was diluted to 5ng/mL in 10mL total volume and sent to the Broad Institute for library construction and sequencing on the Illumina NextSeq500.

treatment_protocol: As previously described we performed iPSC directed differentiation into NKX2-1+ lung progenitors or iHeps using our published protocols (Jacob A et al. Cell Stem Cell 2017, Wilson AA et al STEMCR 2015).

tag: cell line

tag: target differentiation type

value: Liver


title: RNAseq_N2_larvae_12h


title: PiZZ6 Undiff H9

description:
PiZZ6 Undiff
H9

value: Undiff


title: T1-2


title: PiZZ2 Undiff H8

description:
PiZZ2 Undiff
H8


title: RNAseq_N2_larvae_13h


title: MA002 Liver H6

description:
MA002 Liver
H6


title: RNAseq_N2_larvae_11h


title: RNAseq_N2_larvae_10h


title: RNAseq_N2_larvae_15h


title: PiZZ6 Liver H5

description:
PiZZ6 Liver
H5


title: PiZZ1 Liver H4

description:
PiZZ1 Liver
H4


title: RNAseq_N2_larvae_9h

value: 9 h


title: PiSZ1 Lung H3

description:
PiSZ1 Lung
H3

value: Lung


title: T1-16

value: Renal Cell Carcinoma

value: nephrectomy specimens preserved in FFPE


title: T1-14


title: T1-20


title: T1-13


title: T1-8


title: T1-19

value: F


title: T1-9


title: T1-18


title: T1-17


title: T1-7


title: T1-15


title: T1-12


title: T1-10


title: T1-11


title: MA002 Lung H2

description:
MA002 Lung
H2


title: RNAseq_N2_larvae_3h

value: 3 h


title: PiSZ3 Undiff H11

description:
PiSZ3 Undiff
H11


title: RNAseq_N2_larvae_5h

value: 5 h


title: MA011 Undiff H10

description:
MA011 Undiff
H10


title: PHH H12

description:
PHH
H12

source_name: Primary Hepatocytes

value: PHH


title: RNAseq_N2_larvae_2h

value: 2 h


title: PiZZ3 Lung H1

description:
PiZZ3 Lung
H1


title: RNAseq_N2_larvae_6h

value: 6 h