title: 2177374_TP-190a-NSUN-GTF_2F9_2


title: 2177375_TP-190a-NSUN-GTF_2F9_3

status: Public on Sep 23 2022

type: SRA

city: Memphis

first: Stanislav

last: Zakharenko

state: Tennessee

address: 262 Danny Thomas Place

department: Developmental Neurobiology

country: USA

institute: St. Jude Children's Research Hospital

library_source: transcriptomic

data_processing:
Total stranded RNA sequencing data were processed by  the internal AutoMapper pipeline.
First the raw reads were firs trimmed (Trim-Galore version 0.60),  mapped to human genome assembly (GRCh38) (STAR v2.7)
and then the gene level values were quantified (RSEM v1.31) based on GENCODE annotation (v31) .
Genome_build: hg38
Supplementary_files_format_and_content: gene-level RSEM raw count, TPM, FPKM txt files

molecule: total RNA

organism: Homo sapiens

source_name: Cortical organoid

extract_protocol:
Total RNA was isolated by using mirVana microRNA Isolation Kit (Life Technologies, Carlsbad, CA)
Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Kit according to the manufacturer’s instructions (Illumina, PN 20020599)

tag: tissue

value: Cortical organoid

tag: age

value: 68 days

tag: genotype

value: NSUN5-GTF2ird2 +/-

tag: cell line


title: RNA-Seq frq knockout DD20 rep1

city: College Station

first: Deborah

last: Bell-Pedersen

state: Texas

address: 3258 TAMU

department: Biology

institute: Texas A&M University

data_processing:
For WT, Dfrq, and Dcpc-3 samples, basecalling was performed using the Real Time Analysis (RTA) workflow ver. 2.7.7. with default parameters. For cpc-3c samples, basecalling was performed using Illumina BaseSpace app using the FASTQ generation application ver. 1.0.0 and bcl2fastq.
RNA-seq reads were aligned to the N. crassa genome FungiDB Release 38 using STAR (Dobin et al, 2013).
Mapped RNA-seq reads were normalized to fragments per kilobase of exon model per million mapped reads (FPKM) values using Cufflinks (Trapnell et al., 2010; 2012).
The RNA-seq time series was analyzed using the following Extended Circadian Harmonic Oscillation (ECHO, De Los Santos et al., 2019) settings: two unpaired replicates, smoothing function enabled, unexpressed genes excluded from the analysis at the default 70% threshold, and OE/RE cutoff of 1.25. Genes were considered rhythmic if they had Benjamini-Hochberg p-values < 0.05, and oscillation types harmonic, damped or forced.
Genome_build: N. crassa genome FungiDB Release 38
Supplementary_files_format_and_content: Output from Cufflinks in generic FPKM tracking format saved as plain text files containing the estimated gene-level expression values.

organism: Neurospora crassa

source_name: mycelia

growth_protocol: Mycelial disks in Vogel’s minimal media containing 2% glucose (pH 6.0) were synchronized to the same time of day by a shift from 30°C constant light (LL) to 25°C constant dark (DD). The cultures were grown in LL for a minimum of 4 h and transferred to DD on day 1 (for collection at DD 36, 40, 44, 48, 52), day 2 (for collection at DD 12, 16, 20, 24, 28, 32), day 3 (for collection at DD8), and harvested either at 9:00 a.m. (DD 12, 16, 20, 36, 40, 44) or 5:00 p.m. (DD 8, 24, 28, 32, 48, 52) on day 3.

extract_protocol:
Tissue was harvested in the dark, pressed in between paper towels to remove excess media, and immediately frozen in liquid N2. polyA+ RNA was isolated from 100 μg of total RNA with NEB Oligo d(T)25 Magnetic Beads.
Purified mRNA (8 ng) was used as the input to generate RNA-seq libraries using the Lexogen SENSE Total RNA-seq Library Preparation Kit (Cat. #009).

value: frq knockout

tag: time point

tag: replicate

value: mycelia


title: TU0076_Negative

city: La Jolla

first: Cecilia

middle: Sofie

last: Lindestam

state: CA

address: 9420 Athena Circle

department: Sette

institute: La Jolla Institute for Immunology

data_processing:
Reads passing Illumina filters were further filtered for those mapping to tRNA, rRNA, adapters, and spike-in controls using Bowtie v1.0.0 (-S --fr)
Filtered reads were against the GRCh38 reference genome and Gencode v27 annotations using STAR v2.6.1 (default options)
Reads mapping to the reference genome are further filtered to remove low-complexity reads with PRINSEQ v0.20.3 (-lc_method "dust" -lc_threshold 4)
Sequencing read coverage per gene was counted using HTSeq-count v0.7.1 (-m union -s yes -t exon -i gene_id) and features with 0 counts across all samples were removed
Assembly: GRCh38.gencode.v27B
Supplementary files format and content: read count matrix with rows as genes and columns as cells

source_name: blood - PBMC

growth_protocol: PBMC were thawed and added at a density of 1 million cells per well to a round-bottom 96-well plate.

extract_protocol:
PBMCs were thawed and added at a density of 1x10e6 cells per well to a round-bottom 96-well plate. They were stimulated for 24 hrs at 37C with MTB300, Mav lysate, Mtb lysate, anti-CD28 (1g/ml, CD28.2; eBioscience) together with anti-CD3 (1g/ml pre-coated overnight at 4C, UCHT1; BioLegend), as well as left unstimulated as a negative control. After 24 hrs, cells were resuspended in PBS with 10% (v/v) FBS and incubated at 4C for 10 min. Cells were then stained with fixable viability dye eFluor506 (eBioscience) and an antibody mixture containing anti-human CD3-AF700 (UCHT1; ThermoFisher), CD4-APCeFluor780 (RPA-T4; eBioscience), CD8-V500 (RPA-T8; BD Horizon), CCR7-PerCPCy5.5 (G043H7, Biolegend) CD14-V500 (M5E2; BD Bioscience), CD19-V500 (HIB19; BD Bioscience), CD25-FITC (M-A251; BD Bioscience), CD45RA-eFluor450 (HI100, eBiosciences), CD137-APC (4B4-1; BioLegend), OX40-PECy7 (Ber-ACT35; BioLegend), and PD-L1-PE (29E.2A3; BioLegend) for 20 min at room temperature. Cells were washed and resuspended in PBS before being transferred into a 5 ml polypropylene FACS tube (BD Bioscience). PBMCs were sorted, based on forward and side scatter excluding debris and doublets (Figure S1D), on a FACSAria into TRIzol LS (Thermo Fisher). Acquisition files were analyzed using FlowJo. Total RNA was extracted from ~100,000 cells in TRIzol LS using the miRNeasy Micro Kit (Qiagen) on a QIAcube (Qiagen). Total RNA was amplified according to Smart Seq protocol (31). cDNA was purified using AMPure XP beads. cDNA was used to prepare a standard barcoded sequencing library (Illumina). Samples were sequenced using an Illumina HiSeq2500 to obtain 50-bp single end reads. Samples that failed to be sequenced due to limited sample availability or failed the quality control were eliminated from further sequencing and analysis. The full protocol can be found at protocols.io (http://dx.doi.org/10.17504/protocols.io.bxr6pm9e).
Sequencing was performed according to a paired-end strategy using the NovaSeq sequencer (Illumina). Samples that failed quality control standards were eliminated from further analysis21.

treatment_protocol: PBMCs were stimulated for 24hrs at 37 degree C with MTB300, Mtb or Mav whole cell lysate, as well as left unstimulated as a negative control. After 24hrs cells were stained for sorting.

tag: disease group

value: IGRA-HC

tag: stimulation

value: Unstimulated

tag: cell type

value: PBMC


title: RNA-Seq frq knockout DD16 rep1


title: TU0076_MTB300


title: RNA-Seq frq knockout DD28 rep1


title: 2177373_TP-190a-NSUN-GTF_2F9_1


title: GIST_882, NT#2shRNA-NT

type: RNA

city: Barcelona

first: Margarita

last: Martin

state: Barcelona

address: Casanova 143

department: Biomedicine

country: Spain

institute: University of Barcelona

hyb_protocol: The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY LNA™ microRNA Array 7th Gen (Exiqon, Denmark), which contains capture probes targeting all microRNAs for human, mouse or rat registered in the miRBASE 18.0. Raw dat files: Cy3 is 1_Exiqon*txt, Cy5 is 0_Exiqon*txt.

scan_protocol: The miRCURY LNA™ microRNA Array slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA), and the image analysis was carried out using the ImaGene® 9 (miRCURY LNA™ microRNA Array Analysis Software, Exiqon, Denmark).

data_processing: The quantified signals were background corrected (Normexp with offset value 10, see (Ritchie et al., 2007) and normalized using the global Lowess (Locally Weighted Scatterplot Smoothing) regression algorithm.

source_name: Gastrointestinal Stromal Tumor cells

label_protocol: RNA from both sample and reference was labeled with Hy3™ and Hy5™ fluorescent labels, respectively, using the miRCURY LNA™ microRNA Hi-Power Labeling Kit, Hy3™/Hy5™ (Exiqon, Denmark), following the procedure described by the manufacturer

extract_protocol:
Total RNA was extracted with a miRCURY RNA Isolation Kit (Exiqon) from NT control and SH3BP2 knockdown GIST cells
RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA integrity was determined by gel electrophoresis.

value: GIST

tag: phenotype

value: Imatinib sensitive

tag: treatment

value: NTshRNA

source_name: reference RNA

tag: sample type

value: reference RNA


title: GIST_48, NT #1shRNA-NT

value: Imatinib resistant


title: GIST_882, NT#3shRNA-NT


title: GIST_48, NT#3shRNA-NT


title: RNA-Seq frq knockout DD12 rep1


title: 2177372_TP-190a-VGlut1-TdT_4

value: wild type


title: TU0076_Mtb_Lysate

value: Mtb lysate


title: TU0076_MAC_Lysate

value: Mav lysate


title: 2177371_TP-190a-VGlut1-TdT_3


title: RNA-Seq wild-type DD44 rep2

value: wild-type


title: RNA-Seq wild-type DD40 rep2


title: RNA-Seq wild-type DD48 rep2


title: GIST_882, 21#1shRNA-3BP2

value: SH3BP2 shRNA


title: TU0076_aCD3_CD28


title: 2177370_TP-190a-VGlut1-TdT_2

value: Cortical organoid

value: 68 days


title: 2177369_TP-190a-VGlut1-TdT_1


title: TU0071_Negative


title: TU0071_MTB300


title: GIST_882, NT #1shRNA-NT


title: GIST_48, 21#2shRNA-3BP2


title: RNA-Seq wild-type DD36 rep2


title: RNA-Seq cpc-3 kinase active mutant DD40 rep1

value: cpc-3 kinase active mutant


title: RNA-Seq wild-type DD32 rep2

value: mycelia


title: TU0071_MAC_Lysate

value: IGRA-HC


title: TU0071_Mtb_Lysate


title: RNA-Seq cpc-3 kinase active mutant DD32 rep1


title: BL99 (Cancer Detection)

city: Houston

first: Zejuan

last: Li

address: 6565 Fannin St, MC227

department: Department of Pathology and Genomic Medicine

institute: Houston Methodist Hospital

library_source: genomic

data_processing:
Basecalls were converted using bcl2fastq2.
Sequencing quality of raw reads were evaluated using FastQC, and trimmed adaptors and low quality reads (Phred quality score < 15) using Trimmomatic
High quality reads were mapped to hg38  genome using bowtie2 with end-to-end mode
Duplicate reads were removed using samtools.
The reads were counted for overlap with 5 kb non-overlap bins using featureCounts.
Assembly: hg38
Supplementary files format and content: txt file for all sample with raw reads count summarised to genome-wide 5 kb non-overlap bins.

molecule: genomic DNA

source_name: Plasma

extract_protocol:
Plasma was separated from whole blood by centrifuge 1350 g for 10 minutes at 4 °C. Plasma cfDNA was extracted using QIAamp Circulating Nucleic Acid Kit (QIAGEN, Germantown, MD) following manufacturer’s instructions.
Briefly, cfDNA was ligated with barcode adaptors after end repair and A-tailing. Then cfDNA was purified and was incubated with 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, 100 μM N3-UDP-azide-glucose and 1 μM T4 Phage β-gulcosyltransferase at 37 °C for 1 hour. After purification, cfDNA was incubated with 1 μL DBCO-PEG4-DBCO (a click chemistry tool, 4.5 mM stock in DMSO) at 37 °C for 2 hours. Next, the cfDNA was purified and incubated with 5 μL pre-washed C1 Streptavidin beads (Thermo Fisher Scientific, Waltham, MA) for 15 min. The beads were subsequently washed for 5 minutes eight times. The captured DNA fragments were amplified with 15-18 cycles of PCR. The PCR products were purified with AMPure XP beads according to the manufacturer’s instructions. The concentration of DNA library was measured using a Qubit Fluorometer with dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA) and Bioanalyzer 2100 with Agilent High Sensitivity Kit (Agilent Technologies, Santa Clara, CA).
nano-hmC-Seal

tag: diagnosis

value: Control

tag: training/validation group in bladder diagnostic model

tag: training/validation group in breast diagnostic model

tag: training/validation group in colorectal diagnostic model

tag: training/validation group in kidney diagnostic model

tag: training/validation group in lung diagnostic model

tag: training/validation group in prostate diagnostic model

value: Validation

tag: training/validation group in pan-cancer diagnostic model


title: TU0071_aCD3_CD28

value: PBMC


title: RNA-Seq cpc-3 kinase active mutant DD28 rep1

tag: genotype

tag: tissue


title: RNA-Seq cpc-3 kinase active mutant DD36 rep1

tag: time point

tag: replicate


title: RNA-Seq wild-type DD20 rep2


title: RNA-Seq cpc-3 kinase active mutant DD20 rep1


title: RNA-Seq wild-type DD16 rep2


title: BL98 (Cancer Detection)

value: Training


title: RNA-Seq wild-type DD12 rep2


title: TU0069_MTB300

tag: disease group

tag: stimulation


title: TU0069_Negative

value: Unstimulated

tag: cell type


title: RNA-Seq cpc-3 kinase active mutant DD16 rep1


title: GIST_48, 21#1shRNA-3BP2

tag: cell line

value: GIST

tag: phenotype

tag: treatment

value: reference RNA


title: BL91 (Cancer Detection)

tag: diagnosis

tag: training/validation group in bladder diagnostic model


title: RNA-Seq wild-type DD28 rep2


title: TU0069_Mtb_Lysate


title: BL97 (Cancer Detection)

tag: training/validation group in breast diagnostic model

tag: training/validation group in colorectal diagnostic model


title: GIST_48, 21#3 shRNA-3BP2

value: Imatinib resistant

tag: sample type


title: GIST_882, 21#3shRNA-3BP2

value: Imatinib sensitive


title: TU0069_MAC_Lysate

value: Mav lysate


title: GIST_882, 21#2shRNA-3BP2


title: RNA-Seq cpc-3 kinase active mutant DD12 rep1


title: RNA-Seq wild-type DD44 rep1

value: wild-type


title: Ribo-seq cpc-3 kinase active mutant DD48 rep2

data_processing:
Library strategy: Ribo-seq
For WT, Dfrq, and Dcpc-3 samples, basecalling was performed using the Real Time Analysis (RTA) workflow ver. 2.7.7. with default parameters. For cpc-3c samples, basecalling was performed using Illumina BaseSpace app using the FASTQ generation application ver. 1.0.0 and bcl2fastq.
Ribosome profiling (ribo-seq) reads were trimmed using Cutadapt (Martin, 2011) to remove the 3′ adapter of the reads (adapter sequence – CTGTAGGCACCATCAAT).
Adapter-trimmed ribo-seq reads were size-filtered to the 28-34 nt range using Trimmomatic (Bolger et al., 2014).
Ribo-seq reads were aligned to the N. crassa genome FungiDB Release 38 using STAR (Dobin et al., 2013).
To calculate the ribo-seq read coverage over the 5’ UTR, 3’ UTR, or CDS regions, the geneBody_coverage2.py program from the RSeQC package (Wnag et al., 2012) was used.
Ribosome-protected footprint (RPF) features such as read length distribution and sub-codon phasing were determined using NGS toolkit Plastid (Dunn et al, 2016).
The RPF originating only from the coding sequences (CDS) were extracted and quantified using HTSeq-count (Anders et al., 2015). The counted reads were normalized using the median of ratios method employed in the DESeq2 analysis workflow (Love et al., 2014).
The normalized RPF time series was analyzed using the following Extended Circadian Harmonic Oscillation (ECHO, De Los Santos et al., 2019) settings: two unpaired replicates, smoothing function enabled, unexpressed genes excluded from the analysis at the default 70% threshold. Genes were considered rhythmic if they had Benjamini-Hochberg p-values < 0.05, and oscillation types harmonic, damped or forced.
Genome_build: N. crassa genome FungiDB Release 38
Supplementary_files_format_and_content: Output from normalized HTSeq-count done on the reads originating from the CDS and saved as plain text files containing ribosome-occupancy reads.

molecule: polyA RNA

extract_protocol:
Tissue was harvested in the dark, pressed in between paper towels to remove excess media, and immediately frozen in liquid N2. Ribosomes were pelleted by sucrose-density centrifugation and ribosome-protected mRNAs were isolated from the pelleted cell extracts using the Qiagen miRNeasy Mini Kit (Cat. # 217004).
All subsequent steps up to step 46 in the published ribosome profiling protocol (Ingolia et al., 2012) were followed as described. An rRNA depletion step was not performed, but instead after heat-inactivation of CircLigase, 2.0 μL of GlycoBlueTM, 6.0 μL 5 M NaCl, 74 μL of DEPC water, and 150 μL of isopropanol were added to each tube and precipitation was carried out overnight at -80°C. The DNA was pelleted by centrifugation for 30 min at 20,000 g at 4°C. The pellets were washed with 70% ethanol and allowed to air-dry for 10 min. The pellet was then resuspended in 5.0 μL of 10 mM Tris (pH 8.0), and cDNA synthesis using SuperScript™ III Reverse Transcriptase, followed by barcode addition by PCR amplification using Phusion Hot Start High-Fidelity DNA Polymerase were performed as described (101). Sequencing libraries were quantified and checked for quality on a 2100 Bioanalyzer using a DNA high sensitivity chip per the manufacturer’s instructions.

tag: molecule subtype

value: ribosome-protected mRNA


title: TU0069_aCD3_CD28


title: BL89 (Cancer Detection)

tag: training/validation group in kidney diagnostic model

tag: training/validation group in lung diagnostic model

tag: training/validation group in prostate diagnostic model

tag: training/validation group in pan-cancer diagnostic model


title: RNA-Seq wild-type DD40 rep1

tag: genotype

tag: time point

tag: replicate

tag: tissue


title: BL90 (Cancer Detection)


title: BL88 (Cancer Detection)


title: Ribo-seq cpc-3 kinase active mutant DD40 rep2


title: BL87 (Cancer Detection)


title: RNA-Seq wild-type DD32 rep1


title: RNA-Seq wild-type DD48 rep1


title: BL82 (Cancer Detection)


title: BL86 (Cancer Detection)


title: RNA-Seq wild-type DD28 rep1


title: RNA-Seq wild-type DD24 rep1


title: TU0064_Negative

tag: disease group

value: IGRA+HC

tag: stimulation

tag: cell type


title: TU0064_MTB300


title: TU0064_MAC_Lysate


title: TU0064_aCD3_CD28


title: BL8 (Cancer Detection)

tag: diagnosis


title: BL79 (Cancer Detection)

tag: training/validation group in bladder diagnostic model


title: TU0063_Negative


title: RNA-Seq wild-type DD36 rep1


title: RNA-Seq wild-type DD16 rep1


title: BL78 (Cancer Detection)

tag: training/validation group in breast diagnostic model


title: TU0063_MTB300


title: Ribo-seq cpc-3 kinase active mutant DD36 rep2


title: Ribo-seq cpc-3 kinase active mutant DD24 rep2


title: Ribo-seq cpc-3 kinase active mutant DD20 rep2


title: Ribo-seq cpc-3 kinase active mutant DD32 rep2


title: RNA-Seq wild-type DD24 rep2


title: TU0064_Mtb_Lysate

value: Mtb lysate


title: RNA-Seq wild-type DD20 rep1


title: Ribo-seq cpc-3 kinase active mutant DD28 rep2

value: cpc-3 kinase active mutant


title: Ribo-seq cpc-3 kinase active mutant DD16 rep2

value: mycelia


title: RNA-Seq wild-type DD12 rep1


title: BL77 (Cancer Detection)

tag: training/validation group in colorectal diagnostic model


title: Ribo-seq cpc-3 kinase active mutant DD44 rep2


title: TU0063_MAC_Lysate


title: Ribo-seq cpc-3 kinase active mutant DD12 rep2


title: Ribo-seq cpc-3 kinase active mutant DD48 rep1


title: BL72 (Cancer Detection)


title: TU0063_aCD3_CD28


title: GIST_48, NT#2shRNA-NT

tag: cell line

tag: phenotype

tag: treatment

value: NTshRNA

tag: sample type


title: Ribo-seq cpc-3 kinase active mutant DD44 rep1

tag: molecule subtype


title: BL68 (Cancer Detection)

value: Control

tag: training/validation group in kidney diagnostic model

tag: training/validation group in lung diagnostic model

tag: training/validation group in prostate diagnostic model

tag: training/validation group in pan-cancer diagnostic model


title: BL64 (Cancer Detection)


title: TU0057_MTB300

value: IGRA-HC


title: BL61 (Cancer Detection)


title: TU0057_Negative

value: PBMC


title: TU0057_Mtb_Lysate


title: Ribo-seq cpc-3 kinase active mutant DD40 rep1

tag: genotype

tag: time point

tag: replicate

tag: tissue


title: Ribo-seq cpc-3 kinase active mutant DD36 rep1


title: Ribo-seq cpc-3 kinase active mutant DD32 rep1


title: TU0057_MAC_Lysate

tag: disease group

tag: stimulation

tag: cell type


title: BL69 (Cancer Detection)

tag: diagnosis

tag: training/validation group in bladder diagnostic model

tag: training/validation group in breast diagnostic model

value: Validation

tag: training/validation group in colorectal diagnostic model

value: Training


title: BL60 (Cancer Detection)


title: BL55 (Cancer Detection)


title: TU0057_aCD3_CD28


title: Ribo-seq cpc-3 kinase active mutant DD24 rep1


title: Ribo-seq cpc-3 kinase active mutant DD28 rep1


title: BL53 (Cancer Detection)


title: TU0063_Mtb_Lysate


title: BL52 (Cancer Detection)


title: TU0056_MTB300


title: Ribo-seq cpc-3 kinase active mutant DD16 rep1


title: TU0056_Mtb_Lysate


title: BL50 (Cancer Detection)


title: Ribo-seq cpc-3 kinase active mutant DD12 rep1


title: BL5 (Cancer Detection)


title: TU0056_Negative

value: Unstimulated


title: Ribo-seq cpc-3 kinase active mutant DD20 rep1


title: TU0056_aCD3_CD28


title: RNA-Seq cpc-3 kinase active mutant DD48 rep2


title: BL45 (Cancer Detection)


title: RNA-Seq cpc-3 kinase active mutant DD44 rep2


title: TU0056_MAC_Lysate


title: TU0055_Negative


title: SEPT2-deficient PMs, HSV-1 infection_2

city: Chongqing

first: Beibei

last: Fu

address: 174th, Shazheng Street, Shapingba District

country: China

institute: Chongqing University

data_processing:
The raw paired-end reads were trimmed and quality controlled by SeqPrep and Sickle with default parameters.
Then clean reads were separately aligned to Mus_musculus GRCm39 reference genome with orientation mode using HISAT2 software.
The mapped reads of each sample were assembled by StringTie.
Differential expression analysis and functional enrichment was performed using the online platform of Majorbio Cloud Platform.
Assembly: Mus_musculus GRCm39 reference genome
Supplementary files format and content: tab-delimited text file includes gene count matrix and annotation for each sample

organism: Mus musculus

source_name: peritoneal macrophages

growth_protocol: Wild-type and SEPT2-deficient PMs were obtained from WT and SEPT2 conditional knockout mice, respectively. PMs were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37°C.

extract_protocol:
RNA was harvested using TRIZOL. RNA-seq transcriptome library was prepared following TruSeq RNA sample preparation Kit from Illumina (San Diego, CA) using 1 μg of total RNA.
Double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol.
Libraries were size selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (New England Biolabs) for 15 PCR cycles.
After quantified by TBS380, paired-end RNA-seq library was sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2×150 bp read length).

treatment_protocol: Wild-type and SEPT2-deficient PMs were infected with VSV (MOI of 1) or HSV-1 (MOI of 5) for 12 hours.

tag: strain

value: peritoneal macrophages

value: SEPT2 knockout

tag: treatment

value: HSV-1 infection for 12 hours


title: SEPT2-deficient PMs, HSV-1 infection_3


title: RNA-Seq cpc-3 kinase active mutant DD40 rep2


title: TU0055_MTB300


title: SEPT2-deficient PMs, HSV-1 infection_1


title: BL44 (Cancer Detection)

tag: training/validation group in kidney diagnostic model


title: BL43 (Cancer Detection)

tag: training/validation group in lung diagnostic model


title: RNA-Seq cpc-3 kinase active mutant DD36 rep2


title: BL41 (Cancer Detection)

tag: training/validation group in prostate diagnostic model


title: Wild-type PMs, HSV-1 infection_3

value: WT


title: mouse-epidermis-frozen-F

type: genomic

city: Copenhagen

first: Olivia

last: Luxford Meyer

address: Frederik V's Vej 11

department: Department of Forensic Medicine

country: Denmark

institute: University of Copenhagen

hyb_protocol: Standard Illumina Protocol

scan_protocol: Standard Illumina Protocol

data_processing: Data processed with the R package SeSAMe (Sensible Step-wise Analysis of DNA Methylation BeadChips) v. 1.14.2 using default parameters

source_name: mouse epidermis, frozen, no treatment

label_protocol: Standard Illumina Protocol

extract_protocol: Geonomic DNA extracted with DNeasy Blood and Tissue kit from Qiagen

treatment_protocol: Mice were euthanized in CO2-atmosphere. The skin of the back of each mice was removed and placed in sodium chloride solution (58.44 g/L) at room temperature for 24 hours. Afterwards, the epidermis was gently removed from dermis using a tweezer.

value: C3.Cg-HRhr/Tac hairless (Taconic)

tag: Sex

value: female

value: epidermis


title: mouse-epidermis-frozen-E


title: BL40 (Cancer Detection)

tag: training/validation group in pan-cancer diagnostic model


title: TU0055_Mtb_Lysate

value: IGRA+HC

value: Mtb lysate


title: Wild-type PMs, HSV-1 infection_2

tag: genotype


title: RNA-Seq cpc-3 kinase active mutant DD28 rep2

value: cpc-3 kinase active mutant

tag: time point

tag: replicate

value: mycelia


title: RNA-Seq cpc-3 kinase active mutant DD32 rep2

tag: tissue


title: TU0055_MAC_Lysate

tag: disease group

tag: stimulation

value: Mav lysate


title: TU0055_aCD3_CD28

tag: cell type


title: Wild-type PMs, HSV-1 infection_1


title: BL39 (Cancer Detection)

tag: diagnosis

value: Control

tag: training/validation group in bladder diagnostic model


title: mouse-epidermis-frozen-B

description:
Mouse was UV treated with three standard erythema doses UVR 3x pr. week for four weeks
T2_frozen

source_name: mouse epidermis, frozen, UV treated

value: UV 4 weeks


title: mouse-epidermis-fresh-E

source_name: mouse epidermis, fresh, no treatment


title: mouse-epidermis-frozen-A

description:
Mouse was UV treated with three standard erythema doses UVR 3x pr. week for four weeks
T1_frozen


title: RNA-Seq cpc-3 kinase active mutant DD24 rep2


title: SEPT2-deficient PMs, VSV infection_3

value: VSV infection for 12 hours


title: BL38 (Cancer Detection)

tag: training/validation group in breast diagnostic model

tag: training/validation group in colorectal diagnostic model

value: Validation


title: RNA-Seq cpc-3 kinase active mutant DD24 rep1


title: RNA-Seq cpc-3 kinase active mutant DD20 rep2


title: mouse-epidermis-frozen-D

description:
Mouse was UV treated with three standard erythema doses UVR 3x pr. week for eight weeks
T2-8_frozen

tag: strain

tag: treatment

value: UV 8 weeks


title: SEPT2-deficient PMs, VSV infection_2

value: peritoneal macrophages


title: TU0053_MTB300

value: IGRA-HC

value: PBMC


title: mouse-epidermis-fresh-B

description:
Mouse was UV treated with three standard erythema doses UVR 3x pr. week for four weeks
T2_fresh

source_name: mouse epidermis, fresh, UV treated


title: RNA-Seq cpc-3 kinase active mutant DD16 rep2


title: BL34 (Cancer Detection)

tag: training/validation group in kidney diagnostic model

tag: training/validation group in lung diagnostic model


title: RNA-Seq cpc-3 kinase active mutant DD12 rep2


title: SEPT2-deficient PMs, VSV infection_1

value: SEPT2 knockout


title: TU0053_MAC_Lysate


title: mouse-epidermis-fresh-D

description:
Mouse was UV treated with three standard erythema doses UVR 3x pr. week for eight weeks
T2-8_fresh

value: C3.Cg-HRhr/Tac hairless (Taconic)

tag: Sex


title: BL21 (Cancer Detection)

tag: training/validation group in prostate diagnostic model

tag: training/validation group in pan-cancer diagnostic model


title: Wild-type PMs, VSV infection_3

tag: genotype

value: WT


title: TU0053_Mtb_Lysate


title: BL37 (Cancer Detection)

value: Training


title: mouse-epidermis-fresh-A

description:
Mouse was UV treated with three standard erythema doses UVR 3x pr. week for four weeks
T1_fresh

value: female

value: epidermis


title: RNA-Seq cpc-3 kinase active mutant DD48 rep1

value: cpc-3 kinase active mutant

tag: time point

tag: replicate


title: Wild-type PMs, VSV infection_2


title: mouse-epidermis-fresh-C

description:
Mouse was UV treated with three standard erythema doses UVR 3x pr. week for eight weeks
T1-8_fresh


title: mouse-epidermis-fresh-F


title: RNA-Seq cpc-3 kinase active mutant DD44 rep1

tag: tissue

value: mycelia


title: mouse-epidermis-frozen-C

description:
Mouse was UV treated with three standard erythema doses UVR 3x pr. week for eight weeks
T1-8_frozen


title: TU0053_aCD3_CD28

tag: disease group

tag: stimulation


title: TU0053_Negative

value: Unstimulated

tag: cell type


title: BL20 (Cancer Detection)

tag: diagnosis

value: Control

tag: training/validation group in bladder diagnostic model


title: BL16 (Cancer Detection)

tag: training/validation group in breast diagnostic model

tag: training/validation group in colorectal diagnostic model


title: TU0050_MTB300


title: Wild-type PMs, VSV infection_1

tag: strain

tag: treatment


title: BL12 (Cancer Detection)

tag: training/validation group in kidney diagnostic model

tag: training/validation group in lung diagnostic model


title: N2 + cco-1 RNAi + atfs-1 RNAi_ rep3 [N3]

city: Lausanne

first: Terytty Yang

address: EPFL SV IBI-SV LISP AI 1145

country: Switzerland

institute: EPFL

data_processing:
After sequencing on the BGIseq-500 platform, the raw reads were filtered. Data filtering includes removing adaptor sequences, contamination and low-quality (phred quality < 20) reads from raw reads.
Sequenced reads were mapped to the WBcel235 assembly of the worm genome using ensembl release 89 gene annotation with STAR aligner version 2.6.0a
Reads were counted using htseq-count version 0.10.0 using these flags: -f bam -r pos -s no -m union -t exon -i gene_id
Genome_build: Caenorhabditis_elegans.WBcel235.89
Supplementary_files_format_and_content: xlsx file with counts per gene per sample

organism: Caenorhabditis elegans

source_name: N2 + cco-1 RNAi + atfs-1 RNAi_ rep3

growth_protocol: Worms were synchronized by bleaching. Synchronized worm eggs were plated in NGM plates under the described conditions and raised at 20 ̊C. Worms were harvested after 2 days (at L4/young adult stage) and washed with M9 buffer for three times to remove the bacteria, then snap frozen in liquid nitrogen.

extract_protocol:
On the day of the extraction, 1 ml of TriPure Isolation Reagent (Cat. 11667165001, Roche) was added to each tube. The samples were then frozen and thawed quickly 8 times in liquid nitrogen to rupture cell membranes. RNA was then extracted by using a column-based kit from Macherey-Nagel (Cat. 740955.250).
RNA libraries were prepared for sequencing using standard BGISeq500 protocol with polyA enrichment

treatment_protocol: RNAi treatment was started from the egg phase and treated until worms reached L4/young adult phase and then collected in 1.5 mL tubes

tag: RNAi clone

value: E. coli HT115, cco-1 RNAi, atfs-1 RNAi

value: Wild-type

tag: stage at harvesting

value: L4/young adult


title: BL108 (Cancer Detection)

tag: training/validation group in prostate diagnostic model

tag: training/validation group in pan-cancer diagnostic model

value: Validation


title: N2 + cco-1 RNAi + atfs-1 RNAi_ rep2 [N2]

source_name: N2 + cco-1 RNAi + atfs-1 RNAi_ rep2

tag: genotype


title: TU0050_Mtb_Lysate

value: Mtb lysate


title: N2 + cco-1 RNAi + atfs-1 RNAi_ rep1 [N1]

source_name: N2 + cco-1 RNAi + atfs-1 RNAi_ rep1


title: TU0050_MAC_Lysate

value: IGRA-HC

value: Mav lysate


title: N2 + cco-1 RNAi + vha-19  RNAi_rep3 [M3]

source_name: N2 + cco-1 RNAi + vha-19  RNAi_rep3

value: E. coli HT115, cco-1 RNAi, vha-19 RNAi


title: BL100 (Cancer Detection)


title: TU0050_aCD3_CD28

value: PBMC


title: 3D gastruloids Control_rep1 [A1]

city: Baronissi

first: Giovanna

middle: Maria

last: Ventola

state: Salerno

address: Via S. Allende, 43/L

country: Italy

data_processing:
Illumina bcl2fastq software used for basecalling.
Sequenced reads were trimmed for adaptor sequence and masked for low-complexity or low-quality sequence by using cutadapt software (version 2.5).
The sample was mapped on m27 - GrCm39 (latest version of GenCode) using the bioinformatics tool STAR (version 2.7.5c) [4], with the standard parameters for paired reads.
The quantification of transcripts expressed for each sequenced samples was performed using featureCount algorithm (version 2.0.
The Bioconductor package DESeq2 was used to normalize the data, using the median of ratio, to perform the differential expression analysis. The counts were divided by sample-specific size factors determined by median ratio of gene counts relative to geometric mean per gene.
Assembly: m27 - GrCm39
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample
Supplementary files format and content: tab-delimited text files include normalized values (median of ratio of gene counts) for each Sample.

source_name: 3D ESC aggregates

extract_protocol:
Total RNA was extracted using the RNAeasy kit.
RNA libraries were prepared for sequencing using standard Illumina protocols. The quality of RNA were evaluated by NanoDrop One spectrophotometer (NanoDrop) and by TapeStation 4200 (Agilent Technologies  ), respectively. Indexed libraries were prepared from 1 µg/ea purified RNA with TruSeq Stranded mRNA (Illumina) Li-brary Prep Kit according to the manufacturer’s instructions.

tag: esc line

value: Untreated


title: N2 + cco-1 RNAi + vha-19  RNAi_rep2 [M2]

source_name: N2 + cco-1 RNAi + vha-19  RNAi_rep2


title: MMI+T3-3

city: Shanghai

first: ying

last: yan

address: 320 Yueyang Road, Shanghai

institute: Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences

data_processing:
Libraries were prepared according to the Illumina TruSeq protocol
Sequenced reads pooled pooled  for deep sequencing by using Illumina Hiseq Xten (2 x 150) platforms
Adaptor sequences and read sequences on both ends with  Phred quality scores below 30 were trimmed. Trimmed reads were then mapped with the Hisat2  algorithm (Hisat2 v2.1.0) to target sequences
Gene expression levels were quantified by the software  package HTSeq (v0.6.1p1).
Genome_build: GRCm38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: MMI+T3, liver

growth_protocol: C57 mice were rendered hypothyroid (MMI group) by addition of 0.1% methimazole in their drinking water for 20 days.

extract_protocol:
Total RNA was isolated by using Trizol reagent from liver
Libraries were prepared according to the Illumina TruSeq protocol and pooled for deep sequencing by using Illumina Hiseq Xten (2 x150) platforms

treatment_protocol: MMI mice received 5 daily T3 injection were euthanized 24 hours after last T3 injection (MMI+T3-5d mice)

tag: tissue

value: liver

tag: gender

value: male


title: TU0035_Negative

tag: disease group

value: IGRA+HC

tag: stimulation

tag: cell type


title: N2 + cco-1 RNAi + vha-19  RNAi_rep1 [M1]

source_name: N2 + cco-1 RNAi + vha-19  RNAi_rep1


title: MMI-3

source_name: MMI, liver


title: TU0035_MTB300

source_name: blood - PBMC


title: N2 + cco-1 RNAi + vha-16  RNAi_rep2 [L2]

source_name: N2 + cco-1 RNAi + vha-16  RNAi_rep2

value: E. coli HT115, cco-1 RNAi, vha-16 RNAi


title: TU0035_MAC_Lysate


title: MMI-2


title: MMI+T3-2


title: MMI+T3-1


title: TU0035_Mtb_Lysate


title: TU0035_aCD3_CD28


title: N2 + cco-1 RNAi + vha-16  RNAi_rep1 [L1]

source_name: N2 + cco-1 RNAi + vha-16  RNAi_rep1


title: N2 + cco-1 RNAi + vha-4  RNAi_rep3 [J3]

source_name: N2 + cco-1 RNAi + vha-4  RNAi_rep3

value: E. coli HT115, cco-1 RNAi, vha-4 RNAi


title: TU0026_MTB300


title: TU0026_Negative

value: Unstimulated


title: TU0026_Mtb_Lysate


title: N2 + cco-1 RNAi + vha-4  RNAi_rep2 [J2]

source_name: N2 + cco-1 RNAi + vha-4  RNAi_rep2


title: N2 + cco-1 RNAi + vha-4  RNAi_rep1 [J1]

source_name: N2 + cco-1 RNAi + vha-4  RNAi_rep1


title: N2 + cco-1 RNAi + vha-1  RNAi_rep3 [I3]

source_name: N2 + cco-1 RNAi + vha-1  RNAi_rep3

value: E. coli HT115, cco-1 RNAi, vha-1 RNAi


title: TU0026_MAC_Lysate


title: N2 + cco-1 RNAi + vha-1  RNAi_rep2 [I2]

source_name: N2 + cco-1 RNAi + vha-1  RNAi_rep2


title: N2 + cco-1 RNAi + vha-16  RNAi_rep3 [L3]

source_name: N2 + cco-1 RNAi + vha-16  RNAi_rep3


title: N2 + cco-1 RNAi + vha-1  RNAi_rep1 [I1]

source_name: N2 + cco-1 RNAi + vha-1  RNAi_rep1


title: TU0026_aCD3_CD28


title: TU0025_Negative


title: N2 + cco-1 RNAi_ rep1 [H3]

source_name: N2 + cco-1 RNAi_ rep1

value: E. coli HT115, cco-1 RNAi


title: TU0025_MTB300


title: TU0025_Mtb_Lysate


title: TU0025_MAC_Lysate


title: N2 + atfs-1 RNAi_ rep3 [G3]

source_name: N2 + atfs-1 RNAi_ rep3

value: E. coli HT115, atfs-1 RNAi

value: Wild-type


title: TU0025_aCD3_CD28


title: N2 + cco-1 RNAi_ rep1 [H1]

value: L4/young adult


title: N2 + atfs-1 RNAi_ rep1 [G1]

source_name: N2 + atfs-1 RNAi_ rep1


title: N2 + atfs-1 RNAi_ rep2 [G2]

source_name: N2 + atfs-1 RNAi_ rep2


title: TU0021_Negative


title: TU0021_MTB300


title: N2 + vha-19 RNAi_rep2 [F2]

source_name: N2 + vha-19 RNAi_rep2

value: E. coli HT115, vha-19 RNAi


title: TU0021_MAC_Lysate


title: N2 + vha-19 RNAi_rep1 [F1]

source_name: N2 + vha-19 RNAi_rep1


title: N2 + cco-1 RNAi_ rep1 [H2]


title: LOXL2 KO  rep 3

city: Wenzhou

first: Xufeng

last: Lu

state: Zhejiang

address: Caolong Road

institute: Wenzhou Medical University

description: Gene expression in LOXL2 d/d mice uterus tissue rep 3

hyb_protocol: 1.65 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55ul containing 25x fragmentation buffer and 10x blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55ul of 2xGE hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Gene Expression Microarray 4×44K for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 and 1 minute with at room temperature GE Wash buffer 2 , then dried immediately by brief centrifugation.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR Lo 10% ).

data_processing: The scanned images were analyzed with Agilent Feature Extraction Software using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. The algorithm used to normalize data: Threshold raw signals to 1.0. Normalization algorithm 75% Percentile shift. Baseline to median of all samples.

source_name: LOXL2 KO

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 1ug RNA using the One-Color One-Color Quick Amp Labering kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop 2000 Spectrophotometer.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions. And RNA was prepared using the RNeasy mini kit (Qiagen) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-2000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies).

value: uterus tissue

value: female

value: LOXL2 d/d mice


title: LOXL2 WT  rep 3

description: Gene expression in LOXL2 f/f mice uterus tissue rep 3

source_name: LOXL2 WT

value: LOXL2 f/f mice


title: MMI-1

source_name: MMI, liver


title: N2 + vha-16 RNAi_rep3 [E3]

source_name: N2 + vha-16 RNAi_rep3

value: E. coli HT115, vha-16 RNAi


title: LOXL2 KO  rep 2

description: Gene expression in LOXL2 d/d mice uterus tissue rep 2


title: LOXL2 WT  rep 2

description: Gene expression in LOXL2 f/f mice uterus tissue rep 2


title: TU0013_Negative

source_name: blood - PBMC

value: PBMC


title: TU0021_Mtb_Lysate

value: Mtb lysate


title: N2 + vha-19 RNAi_rep3 [F3]

source_name: N2 + vha-19 RNAi_rep3


title: LOXL2 KO  rep 1

description: Gene expression in LOXL2 d/d mice uterus tissue rep 1


title: N2 + vha-16 RNAi_rep2 [E2]

source_name: N2 + vha-16 RNAi_rep2


title: TU0013_MTB300

value: IGRA+HC


title: LOXL2 WT  rep 1

description: Gene expression in LOXL2 f/f mice uterus tissue rep 1


title: N2 + vha-16 RNAi_rep1 [E1]

source_name: N2 + vha-16 RNAi_rep1


title: feeding_rep1

description: Gene expression in ad libitum-fed mouse liver

hyb_protocol: Hybridization was performed in Agilent’s SureHyb Hybridization Chambers.

scan_protocol: After washing, slides were scanned with the Agilent DNA Microarray Scanner.

data_processing:
Data was extracted using Agilent Feature Extraction software. Normalization was performed using Agilent GeneSpring GX 11.5.
Raw signal intensities were normalized in quantile method by GeneSpring GX v11.5, and low intensity genes were filtered.

source_name: liver,feeding

label_protocol: Agilent Quick Amp Labeling Kit was used for sample labeling.

growth_protocol: All the mice were housed in a specific-pathogen-free (SPF) environment with controlled conditions, a temperature of 23±3°C and a humidity of 35±5% under a12-h dark/light cycle

extract_protocol: Total RNA was isolated by using Trizol reagent from liver

treatment_protocol: Fasting group were fast overnight about 14-16h,  feeding group were given a nutrition ingestion after fast.

value: liver

value: male


title: N2 + vha-4 RNAi_rep3 [C3]

source_name: N2 + vha-4 RNAi_rep3

value: E. coli HT115, vha-4 RNAi


title: TU0013_Mtb_Lysate


title: TU0013_MAC_Lysate

value: Mav lysate


title: fasting_rep1

description: Gene expression after 16hr fasting in mouse liver

source_name: liver,fasting


title: N2 + vha-4 RNAi_rep2 [C2]

source_name: N2 + vha-4 RNAi_rep2


title: TU0021_aCD3_CD28


title: LTBI8

city: San Antonio

first: Dhiraj

middle: Kumar

last: Singh

address: 8715 West Military Drive

department: Southwest National Primate Research Center

institute: Texas Biomedical Research Institute

description: LTBI

hyb_protocol: manufacturers instructions/reagents were used (Agilent: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90050_GeneExpression_Two_Color_v6.5.pdf). Arrays were hybridized overnight in a SciGene rotating oven at 65oC, washed according to manufacturer’s protocols and scanned using GenePix Pro.

scan_protocol: GenePix 4000B scanner was used at nm settings between 600-660

data_processing: Data was processed as described earlier (Dutta NK et al, PLoS ONE 2012, Sestak K et al, PLoS ONE 2011, Mehra S et al, PLoS ONE 2010). Raw data files generated by the scanner were uploaded into a licenced version of Spotfire DecisionSite for Microarray Analysis running in conjunction with SPLUS. Unreliable datapoints were removed based on bad flags as well as low 'SNR ' values. The remaining data were then normalized using the built in Loess script. Differential genes were identified  based on fold-changes. In order to identify gene-ontologies significantly overrepresented in our data, we employed the functional analysis tool of DAVID (Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov/).

organism: Macaca mulatta

source_name: Lung

label_protocol: manufacturers instructions/reagents were used (Agilent: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90050_GeneExpression_Two_Color_v6.5.pdf). Briefly, RNA was labeled with Cy3/Cy5 fluorescent dyes using the Agilent Low RNA Input Linear Amplification Kit (LRILAK) (Agilent Technologies), and hybridized to Rhesus macaque microarrays in a 4x44k format (Agilent Technologies).

extract_protocol: RNA was extracted from lungs from different group of animals  described above using RNeasy lipid tissue mini kit, following the manufacturer’s instructions (Qiagen Inc., Valencia, CA)

tag: disease status

value: Naïve subject

value: Latent TB


title: LTBI11

description: LTBI


title: TU0050_Negative

value: IGRA-HC

value: Unstimulated


title: TU0010_Negative


title: LTBI7

description: LTBI


title: MtbSIV7

description: MtbSIV

value: SIV infected + active TB


title: N2 + vha-1 RNAi_rep2 [B2]

source_name: N2 + vha-1 RNAi_rep2

value: E. coli HT115, vha-1 RNAi

value: Wild-type

value: L4/young adult


title: TU0010_MTB300


title: MtbSIV1

description: MtbSIV


title: MtbSIV5

description: MtbSIV


title: LTBI3

description: LTBI


title: N2 + vha-1 RNAi_rep1 [B1]

source_name: N2 + vha-1 RNAi_rep1


title: TU0010_Mtb_Lysate


title: N2 + ev_rep3 [A3]

source_name: N2 + ev_rep3

value: E. coli HT115


title: TU0010_MAC_Lysate


title: LTBI5

description: LTBI


title: TU0010_aCD3_CD28


title: TU0013_aCD3_CD28


title: N2 + ev_rep1 [A1]

source_name: N2 + ev_rep1


title: ATB13

description: ATB

value: Active TB


title: N2 + ev_rep2 [A2]

source_name: N2 + ev_rep2


title: TU0005_MTB300

value: PBMC


title: TU0005_Negative


title: N2 + vha-1 RNAi_rep3 [B3]

source_name: N2 + vha-1 RNAi_rep3


title: 1575242_JKB_K7

data_processing:
The raw reads were trimmed by Trim-Galore (version 0.60), and  mapped to mouse referenece genome mm10  by STAR (v2.7).
Gene level expression values were quantified (HTseq v1.31) based on GENCODE annotation (vM16) .
Genome_build: mm10
Supplementary_files_format_and_content: gene-level Htseq raw count txt files

source_name: Cerebral cortex

extract_protocol:
Total RNA was isolated by using mirVana microRNA Isolation Kit (Life Technologies, Carlsbad, CA)
Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Kit according to the manufacturer’s instructions (Illumina)

value: Brain

tag: age

value: 2 month old


title: TU0005_Mtb_Lysate

value: IGRA+HC

value: Mtb lysate


title: ATB5

description: ATB


title: LTBI2

description: LTBI


title: ATB7

description: ATB


title: ATB6

description: ATB


title: TU0005_aCD3_CD28


title: 1575240_JKB_K5

tag: strain


title: 1575239_JKB_W4

value: wild type


title: 1575238_JKB_W3


title: ATB3

description: ATB


title: TP0044_Negative


title: Normal,replicate 2, scRNAseq

city: Guangzhou

first: Bin

last: Zhang

state: Guangdong

address: 107 Yanjiang West Road

department: Department of Cardiovascular Surgery

institute: Sun Yat-sen Memorial Hospital, Sun Yat-sen University

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Ascending aortic wall tissue

extract_protocol:
The fresh ascending aortic wall tissues were washed at least 3 times with the Hanks Balanced Salt Solution (HBSS) (14025092, Thermo Fisher Scientific, Waltham, MA, USA) to wash away the blood. The adipose tissue attached to the adventitia was removed by blunt separation using the forceps. The single cell preparation was performed on ice to preserve cell viability. The aortic tissues were cut into smaller pieces (1–2 mm3) and digested in a 15-ml centrifuge tube with a digestive enzyme cocktail that included 3 mg/ml Type I collagenase (LS004176, Worthington Biochemical Corp., Lakewood, NJ, USA), 0.156 mg/ml Type XI collagenase (H3506, Sigma Corp., Kanagawa, Japan), 0.25 mg/ml soybean trypsin inhibitor (LS003571, Worthington), 0.1875 mg/ml lyophilized elastase (LS002292, Worthington), 0.24 mg/ml Type I hyaluronidase (H3506, Sigma) and 60 U/ml DNase I (10104159001, Roche, Shanghai, China) as previously described. The samples were shaken slowly for 2-3h in a 37°C water bath to facilitate complete digestion. The digested supernatant was separated from the tissue remnants. The di-gestive reaction was stopped by adding Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal bovine serum. The remaining tissue was further digested with a fresh enzyme cocktail. The digestion process was repeated 3 times to ensure that all the cells from the aortic tissues were isolated. The cells were pelleted by centrifugation at 300 xg and 4℃ for 5min and resuspended in 1X PBS to generate a single cell suspension. The cells were counted using the TC20 automated cell counter (Singleron). The live cell counts were obtained by staining with trypan blue (302643, Sigma). The samples with >80% cell viability and 1×106 cells / ml were further processed for sequencing.
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: Ascending aortic wall tissue

tag: cell line

value: RNA

tag: group

value: Normal


title: Normal,replicate 1, scRNAseq

description: 10x Genomics


title: DCR1-Tun-REP_3

city: Koln

first: Jorge

last: Boucas

address: Joseph-Stelzmann-Str. 9b

department: Bioinformatics

country: Germany

institute: Max Planck Institute for Biology of Ageing

data_processing:
Quality check: FastQC 0.11.7
Alignment: Hisat version 2.1.0
Guided assembly: StringTie 1.3.4d; -p 18 -G ORIGINAL_GTF -f 0.99
Merging assemblies: Cuffmerge 2.2.1; --min-isoform-fraction 1.0
Quantification: Cuffquant 2.2.1
Differential analysis: Cuffdiff 2.2.1; --dispersion-method per-condition
Supplementary_files_format_and_content: tab-separated values or excel table of gene expression
Genome_build: GRCm38_RELEASE_91

source_name: AGO2 +/+ treated with tunicamycin

growth_protocol: We passaged and plated AGO2 -/- and AGO-2 +/+ MEF cells into 150-cm2 flasks for collection of samples. After a 3-d growth (at around 70% confluency), we  induced ER stress by adding 500 nM thapsigargin

extract_protocol:
1 ml of Trizol was added to cells and cells were frozen at -80°C for at least 1h. Subsequently cells were thawed at 37°C. RNA release was performed using silica beads and using the Precellys 24- Dual cell homogenizer (Peq-Lab) twice for 20 s at 5000 rpm. Samples were incubated for 15 min at room temperature. Subsequently, 100 µl 1-bromo-3-chloropropane was added to the samples and centrifuged for 15 min at 8000 rcf at 4°C to separate aqueous and organic phase. The aqueous phase was used to isolate total RNA with the RNeasy mini kit (Qiagen) following manufacturer instructions.
RNA libraries were prepared using the RNAeasy mini kit (Qiagen)

treatment_protocol: induction of ER stress by adding 500 nM thapsigargin

value: embryonic fibroblasts

tag: treatment

value: treated with tunicamycin


title: AD,replicate 6, scRNAseq

description: 10x Genomics

value: sporadic type A aortic dissection


title: DCR1-Tha-REP_3

source_name: AGO2 +/+ treated with thapsigargin

value: treated with thapsigargin


title: ATB1

description: ATB

value: Naïve subject


title: TP0044_MTB300

source_name: blood - PBMC

value: IGRA-HC


title: TP0044_MAC_Lysate

value: Mav lysate


title: TP0044_Mtb_Lysate


title: TP0044_aCD3_CD28


title: TU0005_MAC_Lysate


title: 1575241_JKB_K6


title: AD,replicate 4, scRNAseq

description: 10x Genomics


title: AD,replicate 5, scRNAseq

description: 10x Genomics


title: DCR1-dms-REP_3

source_name: AGO2 +/+ treated with DMSO

value: treated with DMSO


title: Normal,replicate 3, scRNAseq

description: 10x Genomics


title: 1575236_JKB_W1


title: TP0043_MTB300

value: PBMC


title: AGO2-Tun-REP_3

source_name: AGO2 -/- treated with tunicamycyin


title: TP0043_Negative

value: Unstimulated


title: TP0043_Mtb_Lysate


title: AD,replicate 1, scRNAseq

description: 10x Genomics


title: AGO2-dms-REP_3

source_name: AGO2 -/- treated with DMSO


title: AD,replicate 2, scRNAseq

description: 10x Genomics


title: DCR1-Tun-REP_2


title: TP0043_aCD3_CD28


title: TP0043_MAC_Lysate


title: DCR1-dms-REP_2


title: DCR1-Tha-REP_2

source_name: AGO2 -/- treated with thapsigargin


title: Day21_organoid_Day7RSPO1

city: Parkville

first: Sean

last: Wilson

state: Victoria

address: 50 Flemington Road

country: Australia

institute: MCRI

data_processing:
10x library demultiplexing: CellRanger 3.1.0
The 10x Genomics Cell Ranger pipeline (v3.1.0) was used to align, filter and de-duplicate reads before counting those overlapping genes to produce count matrices.
Genome_build: GRCh38
Supplementary_files_format_and_content: Cell Ranger output files

source_name: Kidney organoid

growth_protocol: Briefly, undifferentiated iPSCs were dissociated using TrypLE- select enzyme (Cat# 12563029; ThermoFisher Scientific) and seeded onto Matrigel-coated (Cat#FAL354277; Corning) vessels at the cell density of 192 K cells/cm2 in Essential 8TM medium with 5 μM Y27632 for 24 hours. The next day, cells were incubated with 4 to 6 μM CHIR99021 (Cat# 4423; Tocris Bioscience) in APEL 2 (Cat# 5275; StemCell Technologies) for 2 days. Then the medium was aspirated, and cells were treated with 200ng/mL FGF9 (Cat# 273-F9; R&D Systems), 1ug/mL heparin (Cat# H4784; Sigma Aldrich), 0.2uM ATRA (Cat#R2625; Sigma Aldrich), 0.2uM cis-RA (Cat#BML-GR101; Sapphire Biosciences), 100ng/ml GDNF (Cat#212-GD; R&D Systems) and 0.1uM LDN-193189 (Cat#04-0074; Stemgent) for five days, refreshing the media every two days. After seven days of differentiation, the cells were detached using Trypsin EDTA (0.05%) at 37°C for 3 minutes to make organoids according to Takasato et al.29, 54. Aggregates were made by centrifuging three times at 400xg for 3min and were gently picked up using a wide-bore pipette tip and were placed onto a transwell 0.4μm pore polyester membrane (Corning) in media containing 200ng/mL FGF9, 1ug/mL heparin, 0.2uM ATRA, 0.2uM cis-RA, 100ng/ml GDNF, 0.1uM LDN-193189 and 300ng/ml R-Spondin 1 (Cat#4645-RS, R&D Systems), refreshing the medium every two days until harvested.

extract_protocol:
Organoids were harvested by pooling 3 organoids, cutting into small pieces with a scalpel blade and dissociating in 1mL Accutase at 37 degrees Celcius, pipetting every 3 minutes. The incubation was stopped after 15 minutes by placing on ice. The cell solution was passed through a 40um cell strainer into a FACS tube and then washed with 2mL E6 (room temperature). FACS tubes were spun (1500 rpm, 3 minutes) and resuspended in 500uL of E6 media. Samples were cell counted and tested for viability and diluted to the required density for the sequencing protocol (300-1000 cells per uL) in E6 media. The cells were then transported to the sequencing facility on ice (30 minutes total).
Chromium Single Cell Library kit V2
Single cell RNA seq, sequenced using Illumina Novoseq.

treatment_protocol: For Day 21 samples only, RSPO1 at 300ng/ml was added either at Day 7 or Day 14 as specified

value: iPSC-derived kidney organoid


title: AD,replicate 3, scRNAseq

description: 10x Genomics


title: AGO2-Tha-REP_2


title: AGO2-Tun-REP_2


title: AGO2-Tha-REP_3


title: retina, P28, NRL, 4

city: Bethesda

first: Matthew

middle: J

last: Brooks

state: MD

address: 6 Center Dr Bldg 6, Rm 303

department: NEI

institute: NIH

description: postnatal day 28 retina probed with anti-NRL anitbody replicate 4

data_processing:
Library strategy: CUT&RUN
Sequencing reads were trimmed using Trimmomatic v0.38 for library adapters, 6-base tail cropping, end-wise PHRED 20 quality score, and requiring a minimum length of 25 bases for further analysis.
Trimmed sequencing reads were aligned to a chimeric index using bowtie2 v2.3.5.1 with the following settings: --end-to-end --dovetail -I 10 -X 700 --very-sensitive --no-unal --no-mixed --no-discordant -q --phred33. A chimera bowtie2 index was created by concatenating genomic DNA fasta files of Mus musculus GRCm38, Saccharomyces cerevisiae S288C, and Escherichia coli ASM584v2 prior to running the bowtie2 index function.
Quality alignments were kept if MAPQ scores were greater than 20 using samtools v1.9 (Li, Handsaker et al. 2009). Duplicate aligned reads were removed using picard v2.20.8 (https://broadinstitute.github.io/picard/) MarkDuplicates algorithm. Peaks were identified using MACS2 v2.2.5 (Zhang, Liu et al. 2008). Called peaks overlapping ENCODE blacklist regions (Amemiya, Kundaje et al. 2019) were removed using Bedtools v2.29.0 (Quinlan and Hall 2010).
The Irreproducible Discovery Rate approach was used to determine consensus peaks at each timepoint using IDR v2.0.3 with an IDR threshold of 0.05.
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: bigwig, peaks.  peak_quantitation.tsv:  represents the quantitation for the individual samples used in the IDR peak analysis at each timepoint (2 replicates x 4 timepoints; for P2, P4, and P28 replicates 3 and 4 were used, for P10 replicates 1 and 4 were used). Take the mean at each timepoint to get the final figure data.

source_name: retina

extract_protocol:
Retinas were dissected at P2, P4, P10, and P28 and cells were dissociated using dissociation solution (30 U/mL papain, HBSS pH 7.4, 1 mg/mL glucose, 10 mM Hepes, 100 U/mL DNase I, 5 µg/mL superoxide dismutase, 5 µg/mL catalase, 10 µg/mL D-alpha-tocopherol acetate, 1 mg/mL glucoscysteine-HCl, 5 µg/mL superoxide dismutase, 50 µg/mL gentamycin). Cells were pelleted at 200g for 5 min, washed and re-suspended in Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM Spermidine) with Complete Protease Inhibitor tablet (MilliporeSigma, Burlington, MA, USA). Concanavalin A–coated beads (Polysciences, Warrington, PA, USA) were incubated with the cell suspension containing 250,000 cells for 10 min, washed to remove unbound cells, and incubated with NRL antibody (1:200) (Cat#ab-137193, Abcam, Cambridge, UK) in Antibody Buffer (20 mM EDTA and 0.02% digitonin in Wash Buffer) for overnight. Next day beads were washed with Wash Buffer containing 0.02% digitonin and incubated with 700 ng/ml pA-MNase for 1 hr at 4 °C. Beads were washed, re-suspended in 100 ul of Wash Buffer containing 0.02% digitonin and incubated with 2 mM CaCl2 at 0 °C for 30 min. Cleavage reaction was quenched by adding 100 ul 2X Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 25 μl RNase A, 0.05 mg glycogen and 10 pg heterologous spike-in DNA). DNA fragments were released from insoluble nuclear chromatin by incubation at 37°C for 10 min and extracted by spin column (Qiagen, Hilden, Germany).
Libraries were prepared with ThruPLEX DNA-seq kit (Takara Bio USA, Inc, Mountain View, USA).

value: retina

value: postnatal day 28 (P28)

tag: antibody

value: anti-NRL

tag: replicate


title: Day14_organoid


title: DCR1-Tun-REP_1


title: HASMCs, siBAF60c_3

city: Ann Arbor

first: Guizhen

last: Zhao

state: MI

address: 2800 Plymouth Rd

department: Cardiovascular Center

institute: University of Michigan

data_processing:
The quality control of the sequencing reads from each sample was assessed with FastQC (Babraham Bioinformatics).
The human reference cDNA sequences were downloaded from the Ensembl database (GRCh38 ftp://ftp.ensembl.org/pub/release-97/fasta/homo_sapiens/cdna/).
The reference was indexed with Salmon v 1.2.0, and gene expression quantification was performed using Salmon in its nonalignment-based mode.
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab-delimited text files include count and TPM value for each sample

source_name: Human aortic smooth muscle cells

growth_protocol: HASMCs (CC-2571) were purchased from Lonza (Walkersville, MD). HASMCs were cultured in Smooth Muscle Cell Growth Medium 2 (Promo Cell, Germany) containing 5% fetal bovine serum at 37°C/5% CO2 humidified incubator.

extract_protocol:
Total RNA was extracted from cells using RNeasy Mini Kit (74106, QIAGEN, Hilden, Germany)
RNA library preparation and sequencing were performed by the DNA sequencing core at the University of Michigan. Briefly, the RNA library was prepared with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) and sequenced on a NovaSeq 6000 platform (Illumina) to generate pair-end 51 bp reads according to the manufacturer’s recommendation. In total, ~360 million reads were generated with an average of 30 million paired-reads per sample.

treatment_protocol: HASMCs were transfected with 30 nM siBAF60c (ThermoFisher Scientific, #252172) or siControl (SilencerTM select negative control siRNA, ThermoFisher Scientific, #4390843) using Lipofectamine RNAiMAX Reagent (Invitrogen, #13778150) for 48h, and then serum-starved for 24h.

value: primay vascular smooth muscle cells

tag: passage

value: transfected with BAF60c siRNA

value: BAF60c knockdown


title: DCR1-Tha-REP_1

value: embryonic fibroblasts


title: AGO2-dms-REP_2


title: HASMCs, siBAF60c_1


title: HASMCs, siBAF60c_2


title: HASMCs, siControl_3

value: transfected with control siRNA

value: control


title: retina, P28, NRL, 3

description: postnatal day 28 retina probed with anti-NRL anitbody replicate 3


title: retina, P28, NRL, 1

description: postnatal day 28 retina probed with anti-NRL anitbody replicate 1


title: retina, P28, NRL, 2

description: postnatal day 28 retina probed with anti-NRL anitbody replicate 2


title: DCR1-dms-REP_1


title: Day21_organoid_Day14RSPO1


title: HASMCs, siControl_2


title: AGO2-Tun-REP_1

value: treated with tunicamycin


title: AGO2-Tha-REP_1

value: treated with thapsigargin


title: HASMCs, siControl_1


title: retina, P10, NRL, 4

description: postnatal day 10 retina probed with anti-NRL anitbody replicate 4

value: postnatal day 10 (P10)


title: HASMCs, siBAF60c_DSG_BRG1

data_processing:
ChIP-seq reads were aligned to the human genome (NCBI GRCh19) using bowtie2 version 2.3.5 with default settings.
Peaks were called using MACS2 software and the reads from input served as a control with parameters: -f BAMPE -g hs -B --SPMR -q 0.05.
Samples were normalized to adjust for sequencing depth.
Genome_build: GRCh19
Supplementary_files_format_and_content: bigwig files were generated with UCCS tools 'bedGraphToBigWig' from the MACS2 .bdg file

extract_protocol:
Chromatin immunoprecipitation was performed with the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling, #9003), according to the manufacturer's recommendation. Histone-DNA complexes were isolated with antibodies against H3K9ac (Cell Signaling, #9649, 1:100 dilution), H3K27ac (Cell Signaling, #8173, 1:100 dilution), H3K9me2 (Cell Signaling, #4658, 1:100 dilution), H3K27me3 (Cell Signaling, #9733, 1:100 dilution) or BRG1 (Abcam, ab110641, 1:100 dilution). The sample for BRG1 Chip-seq was crosslinked using 2mM disuccinimidyl glutarate (DSG, ThermoFisher Scientific, #20593) at room temperature for 45min before paraformaldehyde crosslinking.
The DNA library preparation and sequencing were performed by the DNA sequencing core at the University of Michigan. Briefly, the DNA library was prepared with NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) and sequenced on a NovaSeq 6000 S1 flowcell (Illumina) with100bp pair-end reads.

value: Primary vascular smooth muscle cells

tag: passages

tag: chip antibody

value: BRG1 (Abcam, ab110641)


title: AGO2-dms-REP_1

tag: genotype

value: treated with DMSO


title: retina, P10, NRL, 3

description: postnatal day 10 retina probed with anti-NRL anitbody replicate 3

tag: tissue


title: HASMCs, siBAF60c_DSG_input


title: retina, P4, NRL, 4

description: postnatal day 4 retina probed with anti-NRL anitbody replicate 4

value: postnatal day 4 (P4)


title: retina, P10, NRL, 1

description: postnatal day 10 retina probed with anti-NRL anitbody replicate 1


title: HASMCs, siBAF60c_2_H3K27me3

value: H3K27me3 (Cell Signaling, #9733)


title: HASMCs, siBAF60c_2_H3K9me2

value: H3K9me2 (Cell Signaling, #4658)


title: HASMCs, siBAF60c_H3K27ac

value: H3K27ac (Cell Signaling, #8173)


title: HASMCs, siBAF60c_2_input


title: HASMCs, siBAF60c_H3K9ac

value: H3K9ac (Cell Signaling, #9649)


title: retina, P4, NRL, 2

description: postnatal day 4 retina probed with anti-NRL anitbody replicate 2

value: retina

value: anti-NRL


title: retina, P4, NRL, 3

description: postnatal day 4 retina probed with anti-NRL anitbody replicate 3


title: retina, P4, NRL, 1

description: postnatal day 4 retina probed with anti-NRL anitbody replicate 1


title: retina, P2, NRL, 4

description: postnatal day 2 retina probed with anti-NRL anitbody replicate 4

value: postnatal day 2 (P2)


title: HASMCs, siBAF60c_input

value: transfected with BAF60c siRNA

value: BAF60c knockdown


title: CD74-tdTomato Negative

city: Baltimore

middle: D

last: Smith

address: 600 N Wolfe Street

department: Neurology

institute: Johns Hopkins University

description: 10x Genomics GEX 3' v3

data_processing:
Raw sequencing data was demultiplexed, barcodes were processed, and reads aligned and quantified using Cell Ranger (v6.1.0) from 10X Genomics
Assembly: mm10 (Gencode vM23) transcriptome modified to contain a tdTomato gene sequence (symbol TDT)
Supplementary files format and content: tab-seperated values files with cell-associated barcodes, detected features, and feature X cell expression matrix in MEX format

source_name: Brain

extract_protocol:
Mice were perfused with HBSS without cations containing 5ug/ml actinomycin and 10uM triptolide. Brains were dissected out of skull, chopped into small pieces, then dissociated in EBSS buffer with papain (20U/ml), Dnase (100U/ml), actinomycin (5ug/ml), triptolide (10uM), anisomycin (27ug/ml), 22.5mM D-glucose, 0.5mM EDTA, 2.2g/L NaHCO3, and 5.5mM L-cysteine  at pH 7.4. Dissociations were done at 37c for 30 minutes with triturations every 10 minutes. Dissociated tissue was passed over 70um filters and myelin was removed with 2 rounds of debris removal solution (Miltenyi) following manufacturer’s recommendation. Cells were labeled with Zombie Violet for dead cell exclusion then sorted on Aria IIu (VBR) into viable;tdTomato+ and viable;tdTomato- fractions.
Libraries were constructed following manufacturer's instructions (Gene Expression 3' v3 chemistry, 10x genomics). For all 3 samples, cells were loaded at concentrations aiming for capture of 10,000 cells.

treatment_protocol: Mice were immunized sub-cutaneously with 150ul of emulsion made by mixing MOG 35-55 (2mg/ml) 1:1 with CFA (8mg/ml) and given 250ng of pertussis toxin via IP injection on day of immunization and 2 days later.

value: Brain

value: 4 pooled female mice sac'd on DPI 14

value: tdTomato negative

value: MOG 35-55 EAE


title: B2m-tdTomato Positive

description: 10x Genomics GEX 3' v3

value: 3 pooled male mice sacrified at DPI 13

value: tdTomato positive


title: HFSCs, control, Day70

first: Mou

last: Wen

state: TX

address: 6670 Bertner Avenue

institute: Houston Methodist Research Institute

data_processing:
The sequencing data was filtered with SOAPnuke.
(1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 15) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format.
The subsequent analysis and data mining were performed on Dr. Tom Multi-omics Data mining system (https://biosys.bgi.com).
Assembly: mm10
Supplementary files format and content: tab-delimited text files include FPKM, TPM and read count values for each Sample

source_name: skin

growth_protocol: All mice were maintained in a specific pathogen-free conditions. HFSCs were isolated from dorsal skin through cell sorting and matintained in DMEM supplemented with 10% fetal bovine serum (FBS).

extract_protocol:
HFSCs were sorted, spun down and dissolved in TRIzol (Life Technology), and total RNA was harvested by means of the Direct-zol RNA MicroPrep Kit (Genesee Scientific). Total RNA quality was determined with a spectrophotometer (Thermo scientific). Avove 800 mg of total RNA of each sample was used for the construction of sequencing libraries.
The corresponding library quality control protocol will be selected depending upon product requirements.

value: skin

value: primary cell

value: Hair follicle stem cells


title: HFSCs, CKO, Day70


title: A549 Fra-2 rep 2

city: Columbus

first: Carlo

middle: M.

last: Croce

state: Ohio

address: 460 West 12th Avenue

department: Department of Cancer Biology and Genetics

institute: The Ohio State University

hyb_protocol: Following fragmentation, 2.3 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Hybridization Oven 645. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.

data_processing: The analysis for Clariom™ S Human Affymetrix panel of ~20K genes was carried out by Transcriptome Analysis Console software (v4 - Thermo Fisher Scientific) and it consists of three main steps: 1) data quality control; 2) normalization (signal space transformation robust multiple-array average); 3) differentially expression analysis employing the eBayes method from limma R package.

label: biotin

source_name: Lung Cancer Cell Line

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5.5 ug total RNA (GeneChip WT PLUS Reagent Kit, 2013, Affymetrix).

growth_protocol: A549 cell lines were transfected with pCMV6-Entry-Fra2 (Myc-DDK-tagged) and pCMV6-Entry as control (ORIGENE, Rockville, MD) and, after 48 hours, RNA was collected and isolated.

extract_protocol: RNA was collected and isolated with Trizol and RNA clean-up and concentration kit Norgen. Total RNA was treated with RNase-free DNase I Kit by Norgen, in order to avoid DNA contamination.

value: transfected with Fra-2 overexpressing vector


title: A549 Fra-2 rep 1


title: retina, P2, NRL, 3

description: postnatal day 2 retina probed with anti-NRL anitbody replicate 3


title: retina, P10, NRL, 2

description: postnatal day 10 retina probed with anti-NRL anitbody replicate 2

value: postnatal day 10 (P10)


title: A549 empty rep 3

value: transfected with empty vector


title: A549 empty rep 2


title: retina, P2, NRL, 1

description: postnatal day 2 retina probed with anti-NRL anitbody replicate 1

value: retina


title: HFSCs, CKO, Day26


title: A549 Fra-2 rep 3


title: HASMCs, siCtrl_2_H3K27me3

value: Primary vascular smooth muscle cells

value: transfected with control siRNA

value: control

value: H3K27me3 (Cell Signaling, #9733)


title: CD74-tdTomato Positive

description: 10x Genomics GEX 3' v3

value: Brain


title: HASMCs, siCtrl_DSG_BRG1

value: BRG1 (Abcam, ab110641)


title: HASMCs, siCtrl_DSG_input


title: HFSCs, control, Day26


title: rod photoreceptors, Jun_KD_3

description: postnatal day 10 flow-sorted rod photoreceptors electroporated with Jun shRNA plasmid replicate 3

data_processing:
Illumina adapter, polyA, and polyT sequence trimming was performed with Trimmomatic v0.39. Transcript level quantitation was performed using Kallisto v0.44.0 (Bray, Pimentel et al. 2016) employing a merged transcript cDNA and ncRNA FASTA file downloaded from Ensembl v94 (http://oct2018.archive.ensembl.org/index.html).
Gene level quantification performed in R (https://www.r-project.org/) was generated by summarization of the transcript level quantitation using tximport v1.14.0 (Soneson, Love et al. 2015) with the option “countFromAbundance=lengthScaledTPM”.
The gene level count values were TMM normalized in edgeR v3.28.1 package (Robinson, McCarthy et al. 2010).
Genome_build: Ensembl v94
Supplementary_files_format_and_content: Gene_counts.tsv, Gene_normalized-CPM.tsv, Transcript_counts.tsv

source_name: flow-sorted rod photoreceptors

growth_protocol: shRNA-mediated knockdown of mouse Jun was performed with shRNA-expressing vector (TRC Clone ID TRCN0000042695, MilliporeSigma, Burlington, MA, USA) and control pLKO.1-Scrambled vector (Addgene, Watertown, MA, USA). Nrlp-EGFP vector was adopted from (Kautzmann, Kim et al. 2011). Neonatal mice at P0 were used for in vivo electroporation as previously described (Matsuda and Cepko 2004) with shRNA vectors (2.5 μg/μl, 0.4 μl) and the Nrlp-EGFP vector (1.5 μg/μl, 0.4 μl).

extract_protocol: Full length cDNA were generated from purified cells using SMART-seq v4 Ultra Low Input RNA Kit (Takara, Kusatsu, Shiga, Japan). Library generation from cDNA were prepared using Nextera XT DNA Library Kit (Illumina, San Diego, CA, USA).

treatment_protocol: EGFP positive rods electroporated with control and Jun shRNA constructs were flow-sorted at P10 as previously described (Kim, Yang et al. 2016).

value: rod photoreceptors

tag: plasmid

value: Jun shRNA

value: Jun knockdown

value: postnatal day 10


title: KO2

first: Junwen

last: Qu

address: No.639 Zhizaoju Road

institute: 9th people's hospital

data_processing:
The raw reads were aligned to the mouse reference genome (mm10) using Tophat2 RNASeq alignment software Hisat2.
The raw reads alignment files were processed using Hisat SAMtools and HTSeq to generate read counts for every gene.
The read counts were then normalized using R package DESeq2. The normalized read counts were then centered and scaled for each gene, generating z-scores. The z-scores basically represent how many standard deviations one particular count is from the average count for that gene. P-values obtained from multiple tests were adjusted using Benjamini-Hochberg correction.
Assembly: mm10
Supplementary files format and content: excel file containing read count for each gene

source_name: bone marrow derived DCs

extract_protocol:
Total RNA was harvested using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: BMDCs isolated from Hif2αfl/fl and CD11c-Cre;Hif2αfl/fl mice were cultured for 4 hours with α-GalCer following 24 hour DMOG incubation

value: bone marrow

value: BMDCs cultured for 4 hours with α-GalCer following 24 hour-DMOG


title: rod photoreceptors, Jun_KD_2

description: postnatal day 10 flow-sorted rod photoreceptors electroporated with Jun shRNA plasmid replicate 2


title: KO3


title: rod photoreceptors, Jun_KD_1

description: postnatal day 10 flow-sorted rod photoreceptors electroporated with Jun shRNA plasmid replicate 1


title: HFSCs, CKO, Day20

value: skin

value: primary cell

value: Hair follicle stem cells


title: A549 empty rep 1

value: transfected with empty vector


title: HASMCs, siCtrl_2_input


title: KO1


title: rod photoreceptors, cntl_3

description: postnatal day 10 flow-sorted rod photoreceptors electroporated with control plasmid replicate 3


title: HASMCs, siCtrl_H3K27ac

value: H3K27ac (Cell Signaling, #8173)


title: N2 + vha-4 RNAi_rep1 [C1]

data_processing:
After sequencing on the BGIseq-500 platform, the raw reads were filtered. Data filtering includes removing adaptor sequences, contamination and low-quality (phred quality < 20) reads from raw reads.
Sequenced reads were mapped to the WBcel235 assembly of the worm genome using ensembl release 89 gene annotation with STAR aligner version 2.6.0a
Reads were counted using htseq-count version 0.10.0 using these flags: -f bam -r pos -s no -m union -t exon -i gene_id
Genome_build: Caenorhabditis_elegans.WBcel235.89
Supplementary_files_format_and_content: xlsx file with counts per gene per sample

source_name: N2 + vha-4 RNAi_rep1

extract_protocol:
On the day of the extraction, 1 ml of TriPure Isolation Reagent (Cat. 11667165001, Roche) was added to each tube. The samples were then frozen and thawed quickly 8 times in liquid nitrogen to rupture cell membranes. RNA was then extracted by using a column-based kit from Macherey-Nagel (Cat. 740955.250).
RNA libraries were prepared for sequencing using standard BGISeq500 protocol with polyA enrichment

tag: RNAi clone

value: E. coli HT115, vha-4 RNAi

value: Wild-type

tag: stage at harvesting

value: L4/young adult


title: HFSCs, control, Day20

tag: cell type


title: WT3


title: WT2


title: rod photoreceptors, cntl_2

description: postnatal day 10 flow-sorted rod photoreceptors electroporated with control plasmid replicate 2

value: control


title: HASMCs, siCtrl_H3K9ac

value: Primary vascular smooth muscle cells

value: transfected with control siRNA

value: H3K9ac (Cell Signaling, #9649)


title: HASMCs, siCtrl_input


title: rod photoreceptors, cntl_1

description: postnatal day 10 flow-sorted rod photoreceptors electroporated with control plasmid replicate 1

value: rod photoreceptors


title: WT1


title: TgS1 cells in DMEM

city: Kurashiki

first: Tatsufumi

last: Murakami

address: 577 Matsushima

country: Japan

institute: Kawasaki Medical School

data_processing:
Trim Galore! 0.06
Trimmomatic 0.39
Cutadapt 3.1
STAR 2.7.9a
featureCounts 2.0.1
Assembly: GRCm38.p6 (mm10) + human TTR gene (NM_000371.4)
Supplementary files format and content: tab-delimited text files include FPKM values for each sample

extract_protocol:
Total RNA was extracted from TgS1 cells using an Purelink™ RNA Mini kit
Double-stranded cDNA libraries (RNA-seq libraries) were prepared using SMARTer Stranded Total RNA Sample Prep Kit – HI Mammalian and DNBSEQ MGIEasy Universal Library Conversion Kit according to the manufacturer’s instruction.

value: Schwann cells

value: TTR V30M tg

value: Long-term culture  in DMEM


title: PBS control fetal mouse brain, E18.5 (M19B7)

status: Public on Sep 22 2022

city: East Lansing

first: Jonathan

last: Hardy

state: Michigan

address: 775 Woodlot Drive

department: Microbiology and Molecular Genetics

institute: Michigan State University

data_processing:
low quality bases and sequencing adopter were trimmed using Trimmomatic
Single end RNA seq reads were mapped to the mouse reference genome (mm10) using Spliced Transcriptions Alignment to a Reference)
Mapped reads were assinged to genes with FeatureCounts
DESeq2 was performed for differential gene expression analysis
Assembly: mm10
Supplementary files format and content: read counts for each sample (excel file)

source_name: whole fetal mouse brain

extract_protocol:
Total RNA was isolated using the phenol/guanidine based QIAzol lysis reagen
Illumina TruSeq Standard mRNA Library Preparation Kit with IDT for Illumina Unique Dual Index

treatment_protocol: CD-1 pregnant mice were injected with PBS on gestation day 14.5

value: whole fetal mouse brain

value: Control


title: PBS control fetal mouse brain, E18.5 (M19B3)


title: HASMCs, siCtrl_2_H3K9me2

value: H3K9me2 (Cell Signaling, #4658)


title: PBS control fetal mouse brain, E18.5 (M9B3)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M18B11)

treatment_protocol: CD-1 pregnant mice were injected with 2x10^5 CFU L. monocytogenes on gestation day 14.5

value: Exposed


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M18B10)


title: PBS control fetal mouse brain, E18.5 (M9B2)


title: PBS control fetal mouse brain, E18.5 (M10B3)


title: TgS1 cells in SCM

value: Long-term culture in SCM


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M18B7)


title: PBS control fetal mouse brain, E18.5 (M10B2)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M18B8)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M17B11)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M18B6)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M17B3)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M17B2)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M17B9)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M3B5)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M3B1)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M1B4)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M3B6)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M1B2)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M1B3)


title: Satb1_cKO_rep2_RNAseq

city: Tampa

first: Tomas

last: Zelenka

state: Florida

address: 12902 Magnolia Drive

department: Immunology

institute: H. Lee Moffitt Cancer Center & Research Institute

data_processing:
Raw reads were mapped to the mm10 mouse genome using HISAT2 (Version 2.1.0; Kim et al., 2019) with parameters: --rna-strandness RF --fr --phred33. Only mapped, paired reads with a map quality >20 were retained after Bamtools (version 2.4.1) filtering and sorting.
Transcripts were assembled with StringTie (version 1.3.4d; Pertea et al., 2015) using an evidence-based Ensembl-Havana annotation file (version M18, Ensembl 93) with parameters: --rf -G -C -f 0.15 -m 200 -a 10 –j. A unified set of transcripts (isoforms) across multiple RNA-Seq samples was generated by merging the assembled transcripts with the reference Ensembl-Havana annotation file using stringtie --merge with parameters: -G -m 50 -c 0 -F 1.0 -T 1.0 -f 0.01 -g 250. Transcripts and separately also genes were summarized using featureCounts (version 1.6.2; Liao et al., 2014) with parameters: -s 2 -C -a -t exon -g transcript_id AND/OR gene_id. The output in a form of Gene-ID "\t" read-count was used for statistical evaluation of differential expression using DESeq2 (1.18.1; Love et al., 2014). Output of the differential analysis as well as DESeq2 output of normalized values is also attached here. Note that for an intra-sample transcript comparison, DESeq2 normalized values need to be further normalized to the gene length.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include results from DESeq2 differential analysis and matrices of DESeq2 normalized counts; files are provided separately for gene_ID and transcript_ID attributes
Supplementary_files_format_and_content: gtf file contains a complete annotation of genes and transcripts based on reference-based transcript assembly

source_name: thymus

extract_protocol:
Freshly isolated thymocytes from female animals were resuspended in 1 ml of TRIzol Reagent (Invitrogen, 15596026) and RNA was isolated according to manufacturer’s protocol. The aqueous phase with RNA was transferred into a tube and combined with 10 μg of Linear Acrylamide (Ambion, AM9520), 1/10 of sample volume of 3M CH3COONa (pH 5.2), 2.5 volumes of 100% Ethanol and tubes were mixed by flipping. Samples were incubated at –80°C for 40 minutes. Samples were centrifuged at 16,000 g, at 4°C for 30 minutes. The supernatant was removed and the pellet was washed twice with 75% Ethanol. The air-dried pellets were resuspended in 40 μl RNase-free water and incubated at 55°C for 15 minutes to dissolve RNA. To remove any residual DNA contamination, RNase-free DNase Buffer was added to the samples until 1x final concentration together with 20 units of DNase I (NEB, M0303L) and incubated at 37°C for 20 minutes. Samples were then purified using RNeasy Mini Kit (Qiagen, 74104) according to the manufacturer’s protocol. RNA quality was evaluated using Agilent 2100 Bioanalyzer with Agilent RNA 6000 Nano Kit (Agilent Technologies, 5067-1511).
Libraries were prepared using an Illumina TruSeq Stranded Total RNA kit with ribosomal depletion by Ribo-Zero Gold solution from Illumina according to the manufacturer’s protocol and sequenced on an Illumina HiSeq 4000 (2x 75 bp).

value: thymocytes

value: 4-8 weeks


title: Satb1_cKO_rep1_RNAseq


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M18B9)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M17B12)


title: WT_rep1_RNAseq

value: wild type


title: WT_rep3_RNAseq


title: WT_rep2_RNAseq


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M17B1)


title: Listeria monocytogenes exposed fetal mouse brain, E18.5 (M3B2)


title: shPODXL#2_1

city: Nankang

first: Jean

state: Taipei

address: 128 Academia Road, Section 2, IBMS, N401F

department: Genomics Research Center

country: Taiwan

institute: Academia Sinica

data_processing:
RNA-seq samples were pseudoaligned to the Ensembl GRCh38 human transcriptome release 90 using Kallisto, version 0.46.2.
Transcript abundance was quantified with 100 bootstraps using default k-mer length 31, mean fragment length 250, and sd 50.
Assembly: GRCh38
Supplementary files format and content: xlsx files include TPM values for each sample

extract_protocol:
Total RNA were extracted with TRIzol™ Reagent and then RNAs were cleaned and concentrated with kit (Zymo research, R1015).
RNA libraries for RNA-seq were prepared using Stranded RNA (Poly-A) method.

treatment_protocol: HUES6 were transucted with shRNA lentivirus two times with 10 M.O.I. and selected with puromycin for 4 days.

value: hESCs

value: Normal 46XX


title: shPODXL#2_2


title: WT_rep4_SATB1_HiChIP_just_for_peak_calling

data_processing:
Raw reads were mapped with bowtie2 (version 2.3.4.3; Langmead and Salzberg, 2012) to the mm10 genome and fully processed using the HiC-Pro pipeline (version 2.11.1; Servant et al., 2015) with default parameters. Biological replicates were processed individually to assess their quality and then combined for downstream analyses and visualization. The Hi-C and HiChIP matrices are provided as individual replicates and also as a single file representing merged biological replicates. All matrices are in hic format (Juicer Tools, version 1.8.9; Durand et al., 2016) and include multiple resolutions from 5 kbp to 2 mbp as well as DpnII restriction fragment based resolutions. They were produced by the following command utilizing a HiC-Pro script: bash hicpro2juicebox.sh -i xxx.allValidPairs -g chrom_mm10.sizes -j juicer_tools.1.8.9_jcuda.0.8.jar -o -r DpnII_resfrag_mm10.bed.
The HiChIP datasets were used to identify binding sites of the targeted factors. The SATB1 binding sites were extracted from an unsuccessful HiChIP experiment with >60% Dangling End Pairs (~280 million reads; here referred to as rep3 and rep4), using PeakInferHiChIP.sh script from FitHiChIP (version 6.0; Bhattacharyya et al., 2019), with MACS2 (version 2.1.2) parameters: --nomodel --extsize 147; filter peaks with >2.5 enrichment. The peaks were called separately for two biological replicates and then merged. Raw data of the unsuccessful HiChIP as well as called peaks are provided. The H3K27ac peaks were derived similarly but from the HiChIP datasets that were also used for the chromatin interaction analysis. The CTCF peaks were also derived the same way and motif analysis with MEME (Bailey and Elkan, 1994) validated the high enrichment of the CTCF binding motif in the HiChIP derived peaks, confirming its specificity. However, due to a relatively low number of HiChIP-derived CTCF binding sites, for FitHiChIP loop calling we employed a CTCF ChIP-seq dataset from the ENCODE project (ENCFF714WDP; Dunham et al., 2012).
We used the described peaks and the allValidPairs HiChIP output from the HiC-Pro pipeline (biological replicates were combined) to identify HiChIP loops by FitHiChIP (Bhattacharyya et al., 2019) with the following parameters: 5,000 kbp resolution, 20000-2000000 distance threshold, FDR 0.01, coverage specific bias correction, merged nearby peak to all interactions. Resulting HiChIP loops are provided here.  For the differential analysis of H3K27ac WT and Satb1 cKO HiChIP datasets, we utilized the differential analysis pipeline from FitHiChIP (Bhattacharyya et al., 2019) and only utilized loops that were classified as differential in 3D but not in 1D. These were loops that showed differences in interaction counts but no significant differences in H3K27ac occupancy at loop anchors. We divided the loops into two categories depending if they evinced decreased (referred to as underinteracting loops) or increased interaction in Satb1 cKO (referred to as overinteracting loops). Loops from both categories are provided in the excel file. Some regions contained H3K27ac loops from both under- and over-interacting categories, hence we further calculated a difference between the number of under- and over-interacting loops and this score including the affected genes are also provided.
Genome_build: mm10
Supplementary_files_format_and_content: hic files are interaction matrices produced by Juicer Tools
Supplementary_files_format_and_content: narrowPeak files contain HiChIP-inferred binding sites (peaks) of targeted factors in HiChIP experiments
Supplementary_files_format_and_content: bed files contain coordinates of chromatin loops identified from the HiChIP experiments
Supplementary_files_format_and_content: xlsx is a Microsoft Excel file containing results of the differential analysis of H3K27ac loops – showing H3K27ac loops that are over- and underinteracting in Satb1 cKO and also the genes affected by these differential loops

extract_protocol:
Both Hi-C and HiChIP experiments were performed identically until the chromatin immunoprecipitation step. Aliquots of 10 million isolated thymocytes resuspended in 1X PBS were fixed by adding 1/10th sample volume of fixation butter [11% methanol-free formaldehyde (Pierce, 28908), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes pH 8.0] and rocking at RT for 10 minutes. To quench the reaction, glycine was added to 0.125 M final concentration and incubated at RT for 5 minutes, while rocking. After two washes with 1X PBS, cell pellet was resuspended in 500 μl of ice-cold Hi-C Lysis Buffer (10 mM Tris-HCl – pH 8, 10 mM NaCl, 0.2% NP40, 0.5 mM PMSF) and rotated at 4°C for 1.5 hours. Cells were centrifuged at 2,500 g, at 4°C for 5 minutes and the supernatant was discarded. The cell pellet was washed once with 500 μl of ice-cold Hi-C Lysis Buffer and then resuspended in 100 μl of 0.5% SDS. Cells were incubated at 62 °C for 10 minutes and then combined with 296 μl of H2O and 50 μl of 20% Triton X-100. Samples were incubated at 37°C for 15 minutes and then combined with 50 μl of 10X DpnII Buffer and 200 U of DpnII restriction enzyme (NEB, R0543M) and digested for additional 16 hours at 37 °C while shaking (160 rpm). The restriction enzyme was inactivated at 62°C for 20 minutes and the nuclei were centrifuged at 2,500 g, at 4 °C for 6 minutes. The supernatant was discarded and the nuclei were resuspended in 300 μl Fill-in Buffer [30 μl Klenow Buffer 10X (NEB, M0210L), 238.5 μl water, 15 μl 1 mM Biotin-16-dCTP (Jena Bioscience, NU-809-BIO16-L), 1.5 μl of each 10 mM dATP, dGTP, dTTP (Promega, U1240), 12 μl 5 U/μl DNA Polymerase I, Klenow Fragment (NEB, M0210L)].  The biotinylation mixture was incubated at 37°C for 30 minutes with rotation. SDS was added to a final concentration of 0.5% to inactivate the Klenow enzyme. Triton X-100 was added to 1% final concentration and samples were incubated at 37°C for 5 minutes. Samples were centrifuged at 2,500 g, at 4 °C for 10 minutes and the supernatant was discarded. The nuclei pellet was resuspended in the Ligation Buffer [120 μl 10X NEB T4 DNA Ligase Buffer with 10 mM ATP (NEB, B0202), 60 μl 20% Triton X-100 (1% final), 6 μl 2% (20 mg/ml) BSA, 40 μl 30% PEG 6,000 (1% final), 5 μl 400 U/μl T4 DNA Ligase (NEB, M0202L), 969 μl water]. Samples were incubated for 6 hours at RT with rotation. Nuclei were centrifuged at 2,400 g, at RT for 15 minutes and the supernatant was discarded. The pellet was resuspended in 60 μl Lysis Buffer (1% SDS, 50 mM Tris-HCl – pH 8, 20 mM EDTA, 1X protease inhibitors) and incubated at RT for 15 minutes. The lysate was diluted to 600 μl using ice cold TE Buffer supplemented with protease inhibitors and then sonicated with a Labsonic M – Tip sonicator for 3 cycles (30 seconds ON/OFF, 40% power). The sonicated material was centrifuged at 16,000 g, at RT for 15 minutes and the supernatant was collected into a new tube. Samples from the same genotype were merged and then split again: separately 100 μl for Hi-C and 450 μl for HiChIP.  Hi-C samples were combined with two volumes of Hi-C Elution Buffer (10 mM Tris-HCl – pH 8, 5 mM EDTA, 300 mM NaCl, 1% SDS) and incubated at 65°C overnight. De-crosslinked material was diluted to 500 μl with TE Buffer and treated with RNase A and Proteinase K. DNA was purified using a ChIP DNA Clean & Concentrator kit following the manufacturer’s instructions (Zymo Research, D5205). Purified DNA was quantified using Qubit dsDNA BS Assay Kit (Invitrogen, Q32853) checked on an agarose gel for shearing efficiency and 100 μg were used for library construction.  HiChIP samples were combined with Triton X-100 to 1% final concentration and samples were incubated at 37°C for 15 minutes. Samples were combined with an equal volume of 2X ChIP Binding Buffer (20 mM Tris-HCl – pH 8, 2 mM EDTA, 0.2 % sodium deoxycholate, 2X protease inhibitors). Chromatin preclearing and antibody binding to magnetic beads were performed as described in the ChIP protocol. The following antibodies were used for the immunoprecipitation step: SATB1 long isoform (8 μg, custom-made from Davids Biotechnologies), CTCF (7 µg, Abcam, ab70303), H3K27ac (2 µg, Abcam, ab4729). Antibody-coupled beads were incubated with chromatin at 4°C with rotation for 16 hours. Beads were washed five times with ice cold RIPA Buffer (50 mM Hepes pH 8, 1% NP-40, 0.70% Na-Deoxycholate, 0.5 M LiCl, 1 mM EDTA, protease inhibitors) and then twice with TE Buffer. After the first wash with TE Buffer, resuspended beads were transferred into a new tube. Immune complexes bound to beads were eluted in 125 μl Hi-C Elution Buffer (10 mM Tris-HCl – pH 8, 5 mM EDTA, 300 mM NaCl, 1% SDS) at 65°C for 16 hours. De-crosslinked material was diluted to 250 μl with TE Buffer and treated with RNase A and Proteinase K as previously described. DNA was purified using a ChIP DNA Clean & Concentrator kit following the manufacturer’s instructions (Zymo Research, D5205). Purified DNA was quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Q32854) and used for library construction.
Samples were brought to 25 μl with water. 5 μl and 20 μl for HiChIP / Hi-C samples, respectively, of Dynabeads MyOne Streptavidin C1 beads (Invitrogen, 65001) were washed with 500 μl Tween Wash Buffer (5 mM Tris-HCl – pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20). Beads were resuspended in 25 μl of 2X Biotin Binding Buffer (10 mM Tris-HCl – pH 7.5, 1 mM EDTA, 2M NaCl) and combined with samples. Samples were incubated at RT for 20 minutes. Beads were separated on a magnet and washed twice with 400 μl of Tween Wash Buffer. Beads were washed by 100 μl of 1X TD Buffer (10 mM Tris-HCl – pH 7.5, 5 mM MgCl2, 10% Dimethylformamide). Beads were resuspended in 25 μl of 2X TD Buffer and combined with Tn5 enzyme from the Nextera DNA Sample Preparation Kit (Illumina, FC-121-1030) and water to final volume 50 μl. The amount of Tn5 enzyme was adjusted according to the input DNA amount: 4.5 μl for Hi-C libraries, 1.5 μl for SATB1 HiChIP and 1 μl for other HiChIP libraries. The reaction was incubated at 55°C for 10 minutes. The beads were collected with a magnet and the supernatant was discarded. Beads were resuspended in 300 μl of Strip Buffer (0.15% SDS, 10 mM Tris-HCl – pH 8, 50 mM EDTA) and incubated for 5 minutes at RT to strip off and deactivate Tn5. Beads were washed once with 400 μl of Tween Wash Buffer and once with 500 μl of 10 mM Tris-HCl (pH 8.0).  The beads were resuspended in 50 μl of the following PCR master mix with indexed primers from the Nextera DNA Sample Preparation Index Kit (Illumina, FC-121-1011): 25 μl Phusion HF 2X (NEB, M0531L), 1 μl (1.5 for Hi-C) Nextera Index 1 N7XX, 1 μl (1.5 for Hi-C) Nextera Index 2 N5XX, 23 μl (22 for Hi-C) water. The PCR reaction was performed according to the following program: initial incubation at 72°C for 5 min and 98°C for 1 min, then x cycles of 98°C for 15 s, 63°C for 35 s and 72°C for 60 s. The number of PCR cycles was estimated based on post-ChIP quantification and amplification was 6 cycles for Hi-C libraries, 11 cycles for SATB1 HiChIP and 13 cycles for the other HiChIP libraries. DNA libraries were purified and size-selected using AMPure XP beads, quantified by Qubit and analyzed on a Bioanalyzer. The DNA Libraries were sequenced on an Illumina® HiSeq 4000 2x 75 bp platform.

value: extra exon of long SATB1 isoform (Davids Biotechnologies, custom-made)


title: shPODXL#1_2


title: WT_rep3_SATB1_HiChIP_just_for_peak_calling


title: Satb1_cKO_rep3_RNAseq


title: WT_rep1_HiC


title: Satb1_cKO_rep2_HiC


title: shPODXL#1_1


title: Satb1_cKO_rep1_HiC


title: WT_rep2_HiC


title: Satb1_cKO_rep2_H3K27ac_HiChIP

value: H3K27ac (Abcam, ab4729)


title: shRFP_2

value: control


title: Satb1_cKO_rep1_H3K27ac_HiChIP


title: shRFP_1


title: WT_rep2_H3K27ac_HiChIP


title: WT_rep2_CTCF_HiChIP

value: CTCF (Abcam, ab70303)


title: WT_rep1_H3K27ac_HiChIP


title: WT_rep1_CTCF_HiChIP