title: WT2_rep2

description: GEO_processed excel file_YHLLAB.xlsx


title: HFO 4

status: Public on Dec 17 2020

type: RNA

city: Hannover

first: Robert

last: Zweigerdt

address: Carl-Neuberg-Str. 1

country: Germany

institute: Hannover Medical School

hyb_protocol: cRNA fragmentation, hybridization and washing steps were carried-out as recommended in the ‘One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling Protocol V6.7’, except that 2500ng of labeled cRNA were used for hybridization.

scan_protocol: Slides were scanned on the Agilent Micro Array Scanner G2565CA (pixel resolution 3 µm, bit depth 20).

data_processing: Data extraction was performed with the ‘Feature Extraction Software V10.7.3.1’ by using the recommended default extraction protocol file ‘GE1_107_Sep09.xml’.  Measurements of on-chip replicates (quintuplicates) were averaged using the geometric mean of processed intensity values of the green channel, ‘gProcessedSignal’ (gPS) to retrieve one resulting value per unique non-control probe. Single Features were excluded from averaging, if they i) were manually flagged, ii) were identified as Outliers by the Feature Extraction Software, iii) lay outside the interval of ‘1.42 x interquartile range‘ regarding the normalized gPS distribution of the respective on-chip replicate population, or, iv) showed a coefficient of variation of pixel intensities per Feature that exceeded 0.5.   Averaged gPS values were normalized by global linear scaling: All gPS values of one sample were multiplied by an array-specific scaling factor. This factor was calculated by dividing a ‘reference 75th Percentile value’ (set as 1500 for the whole series) by the 75th Percentile value of the particular Microarray to be normalized (‘Array I’ in the formula shown below). Accordingly, normalized gPS values for all samples (microarray data sets) were calculated by the following formula:   normalized gPSArray i = gPSArray i  x (1500 / 75th PercentileArray i)  After normalization, a lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed at 10 normalized gPS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 10.  Finally, normalized processed data were imported into Omics Explorer software v3.5 (Qlucore) under default import settings for Agilent One Color mRNA Microarrays, except that any (further) normalization option during import was deselected. Accordingly, data processing steps during Omics Explorer import were: 1) removal of control measurements, 2) log base 2 transformation, 3) baseline transformation to the median.

molecule: total RNA

organism: Homo sapiens

source_name: HES3 NKX2.5-eGFP-derived organoid

label_protocol: 110ng of total RNA were used as input. Synthesis of Cy3-labeled cRNA was performed in ¾ reaction volumes with the ‘Low Input Quick Amp Labeling Kit One-Color’ (#5190-2305, Agilent Technologies) according to the manufacturer’s recommendations.

extract_protocol: Prior to RNA isolation, single HFOs were incubated in 20 uL Collagenase II (Worthington) for 30 min at 37C for dissociation, whereby every 5 - 10 min, HFOs were disrupted by pipetting up and down. 330 uL RLT buffer (Qiagen) containing 1% β-Mercaptoethanol (Thermo Fisher Scientific) was added on top of the dissociated HFOs and vortexed for 15 s. Total RNA was prepared using the RNeasy Micro Kit (Qiagen) according to manufacturer's instructions. For RNA isolation from human embryonic hearts, cardiomyocytes from 2D differentiation, and hPSCs, samples were put into Trizol (Thermo Fisher Scientific) and mechanically disrupted by vortexing and pipetting. RNA isolation was performed using the NucleoSpin RNA Kit (Macherey-Nagel) according to manufacturer's instructions.

tag: developmental stage


title: KO2_rep2

type: SRA

city: Seoul

first: CheolJun

last: Choi

address: 1, Gwanak-ro, Gwanak-gu, 29-310

country: South Korea

institute: Seoul National University

description: GEO_processed excel file_YHLLAB.xlsx

library_source: transcriptomic

data_processing:
bcl2fastq2-v2.20.0 package was used for basecalling.
Sequenced reads were trimmed for adaptor sequence and masked for low-complexity or low-quality sequence with Trimmomatic0.38. (Trimmomatic option : ILLUMINACLIP:[TruSeq3-PE-2.fa]:2:30:10:5:true ,LEADING:3 , TRAILING:3 , SLIDINGWINDOW:4:15, MINLEN:36)
To estimate expression levels, the RNA-Seq reads were mapped to Mus musculus genome (mm10) using HISAT2 version 2.1.0, which is capable of reporting split-read alignments across splice junctions and determined using StringTie version 1.3.4d software in default options.
The read count data of filtered genes was normalized by RLE (Relative Log Expression) normalization with DESeq2. The nbinomWaldTest function in DESeq2 was used to test for differential expression. The output prints foldChange, p-value and FDR values which corrected by the Benjamin-Hochberg procedure.  DEGs was determined by |fold change|>=2 and p-value <0.05. For DEGs, hierarchical clustering analysis was performed with complete linkage method and Euclidean distance as a measure of similarity.
Gene-enrichment analysis and KEGG pathway analysis for DEGs were also performed based on Gene Ontology (http://geneontology.org/) and KEGG pathway (https://www.genome.jp/kegg/) database respectively. All statistical analysis and visualization of differentially expressed genes was conducted using R.3.5.1 (www.r-project.org).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include baseMean values for each Comparison

organism: Mus musculus

source_name: inguinal white adipose tissue

growth_protocol: Mice were fed a standard chow diet and were kept on a 12-h light/12-h dark cycle with free access to food and water at 22±1 °C.

extract_protocol:
RNA was isolated using Trizol reagent (Invitrogen) and processed as per manufacturer’s instructions.
Libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit, according to the manufacturer’s

tag: strain

tag: genotype

tag: treatment

value: untreated


title: RNAseq: E13.5 Sox2hi epithelial cells-2

city: Tokyo

first: Yasuhiro

last: Yamada

address: 7-3-1 Hongo, Bunkyo-ku

department: Department of Molecular Pathology

country: Japan

institute: University of Tokyo

data_processing:
After trimming adaptor sequences and low-quality bases by cutadapt-1.16, the sequenced reads were mapped to the mouse reference genome (mm10) plus EGFP and tdTomato reporter sequences using HISAT2-2.1.0 with the GENCODE version M17 annotation gtf file. The uniquely mapped reads with high mapping quality were used for further analyses. The expression level of each gene (protein coding) was calculated as fragments per kilobase per million mapped reads (FPKM) by cufflinks-2.2.1
Genome_build: mm10
Supplementary_files_format_and_content: processed_file.xlsx (cufflinks.FPKM)

source_name: stomach epithelial cells

extract_protocol:
RNAeasy plus Micro Kit (QIAGEN) for RNA.
To generate RNA-seq libraries, Truseq Stranded mRNA LT sample prep kit (Illumina) was used according to the supplier’s instruction.

value: Sox2-EGFP; Gata4-td Tomato

tag: tissue

value: stomach epithelium

tag: cell type

value: epithelial cells


title: RNAseq: E13.5 Sox2hi epithelial cells-1


title: WT1_rep1

description: GEO_processed excel file_YHLLAB.xlsx

value: Wild-type


title: SNPC13ChIP_SNPC13flag-SNPC4aid-TIR1_48h_input_rep1

city: Baltimore

first: Margaret

middle: R

last: Starostik

state: MD

address: 3400 Charles Street

department: Department of Biology

country: USA

institute: Johns Hopkins University

description:
snpc-1.3::3xflag; snpc-4::aid; TIR1 L4 (48h) SNPC-1.3 ChIP-seq input replicate 1
SNPC13ChIP_SNPC13flag-SNPC4aid-TIR1_48h_rep1_SignificantBins.txt

library_source: genomic

data_processing:
Raw small RNA-seq reads were trimmed for Illumina adapters and quality (SLIDING WINDOW: 4:25) using Trimmomatic 0.39 (Bolger et al., 2014).
Trimmed reads were then filtered using bbmap 38.23 (http://jgi.doe.gov/data-and-tools/bb-tools) to retain reads that were 15-30 nucleotides in length.
These filtered reads were aligned to the C. elegans WBcel235 (Cunningham et al., 2018) reference genome using Bowtie 1.1.1 (Langmead et al., 2009) with parameters -v 0 -k 5 --best --strata --tryhard.
Mapped reads were assigned to genomic features using featureCounts from Subread 1.6.3 (Liao et al., 2014), taking into account overlapping and multi-mapping reads (-O -M).
Raw counts were normalized within DESeq2 1.26.0 (Love et al., 2014).
For SNPC-1.3 and TRA-1 ChIP seq data, reads were trimmed for adapters and filtered for sequencing quality score > Q25 using Trim Galore! 0.5.0 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/).
Filtered reads were aligned to C. elegans reference genome WBcel235 (Cunningham et al., 2018) using Bowtie2 2.3.4.2 (Langmead and Salzberg, 2012) with default parameters.
Post-alignment filtering was then performed to remove PCR duplicates using the MarkDuplicates utility within Picard 2.22.1 (http://broadinstitute.github.io/picard/).
In addition, SAMtools 1.9 was applied to remove unmapped reads and reads that mapped with MAPQ 30 but were not of primary alignment or failed sequence platform quality checks (SAMtools -F 1804 -q 30) (Li et al., 2009).
To identify DNA binding sites for SNPC-1.3, mapped and filtered SNPC-1.3 ChIP-seq reads were extended to 200 base pairs to account for the average length of ChIP fragments.
We then partitioned the genome into consecutive, non-overlapping 1,000 base pair bins and calculated read coverage, normalized by sequencing depth of each library, based on the total read count in each bin.
Bins with read coverages in the IP sample that fell below the median read coverage of piRNA-depleted regions on chromosome IV in the relevant input control were excluded from further analysis.
TRA-1 ChIP-seq peaks were called by callpeak within MACS 2.1.2 (Zhang et al., 2008) (--pvalue 0.05) with filtered TRA-1 ChIP-seq reads and relevant input controls.
Genome_build: WBcel235

molecule: genomic DNA

organism: Caenorhabditis elegans

source_name: whole worms, L4 (48 h, 20°C )

growth_protocol: Worms were grown in liquid culture at 20°C and fed E. coli strain HB101.

extract_protocol:
 After washing, the gut was cleared for 15 min by nutation in M9, followed by three washes in M9. Worms were live-crosslinked in 2.6% formaldehyde in water for 30 min at room temperature, nutating. Crosslinking was quenched with a final concentration of 125 mM glycine for 5 min with nutation. After three washes with water, worms were flash-frozen in liquid nitrogen. Frozen worms were ground into powder using a mixer mill MM400 (Retsch) for 2 rounds of 1 min at 30-1. Powder was resuspended in 1x RIPA buffer (1xPBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) for 10 min at 4°C. Crosslinked chromatin was sonicated using a Diagenode Bioruptor Pico for three 3-min cycles, 30 sec on/off. 10 µg chromatin was nutated overnight at 4°C with 2 µg of Flag antibody (Sigma-Aldrich, F1804) and then for 1.5 h with 50 µL mouse IgG Dynabeads (Invitrogen). Input amount was 10% of IP. Chromatin was decrosslinked and extracted as described previously (Weiser et al., 2017).
Libraries were prepared and multiplexed using the Ovation Ultralow Library Systems v2 (NuGEN Technologies) according to the manufacturer’s protocol.  The Illumina HiSeq 4000 platform was used to generate 50 bp single-end reads.

treatment_protocol: 250 uM auxin was added 4 hours before collection

value: whole worm

tag: age

value: snpc-4(xk23) I ; unc-11(ed3) III; ieSi38 IV

tag: replicate


title: Frameshifting-region RNA 25mM 5NIA In Vitro

city: Chapel Hill

first: Mark

middle: A

last: Boerneke

state: NC

address: Campus Box 3290

department: Chemistry

institute: University of North Carolina

data_processing:
Library strategy: RNP-MaP
Adduct locations identified by Shapemapper2 software
RNA structure models were generated from SHAPE data using SuperFold software
RNP-MaP analysis was generated with in-house software from shapemapper outputs

organism: Severe acute respiratory syndrome coronavirus 2

source_name: Reconstituted solution

extract_protocol: In vitro transcribed RNA was treated with 5NIA or DMSO (control) for SHAPE-MaP RNA structure probing experiments.  The binding of recombinantly expressed and purified SARS-CoV-2 N-protein at 160x, 80x, 20x, or 0x (control) molar excess concentrations to in vitro transcribed RNA was chemically probed using the bivalent chemical crosslinker SDA for RNP-MaP experiments. Chemically modified RNA samples from SHAPE-MaP and RNP-MaP experiments were subjected to MaP reverse transcription, which encodes the position of chemical adducts as non-templated nucleotides or deletions in the product cDNA. Sequencing libraries were generated using the Nextera XT workflow and sequenced on an Illumin MiSeq instrument.

value: isolate 2019-nCoV WHU01

value: 25mM 5NIA

tag: target

value: Frameshifting-region RNA


title: SNPC13ChIP_N2_48h_IP_rep1

description:
N2 L4 (48h) SNPC-1.3 ChIP-seq IP replicate 1
SNPC13ChIP_N2_48h_rep1_SignificantBins.txt


title: KO-Thymus

city: New Brunswick

first: Daniel

last: Herranz

state: New Jersey

address: 195 Little Albany Street

institute: Rutgers Cancer Institute of New Jersey

description: Filtered features-barcode UMI count matrix for 7283 barcoded cells and 31053 features, with associated features and barcode labels (.tsv) files

data_processing:
CellRanger pipeline of 10X Genomics was used to prepare the Filtered feature-barcode matrices (.mtx files) - these matrices contain only detected cellular barcodes. The matrix elements are the number of UMIs associated with the respective features (rows) and barcodes (columns), respectively.
The filtered feature-barcode matrices are stored in the Market Exchange Format (MEX) for sparse matrices.
Genome_build: mm10
Supplementary_files_format_and_content: Filtered feature-barcode UMI counts matrix in MEX format

source_name: Thymus

extract_protocol:
Normal thymus from ~6 week-old littermate mice were removed. Single-cell suspensions were obtained by pressing them through a 40mm filter, and subsequently subjected to red blood cell lysis. Part of the single-cell suspensions of thymocytes were subjected to enrichment in immature thymocytes (CD4-CD3-) by staining with biotinylated antibodies against CD3 (#13-0031-85, ThermoFisher) and CD4 (13-0041-85, ThermoFisher), followed by magnetic depletion of antibody-labeled cells using streptavidin microbeads (Miltenyi Biotec 130-048-101). CD4−CD3−- enriched thymocytes were then mixed 1:1 with single-cell suspensions from total unenriched thymocytes
single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3′ v3 Reagent Kit (10x Genomics PN-1000092)

tag: strain background

value: Pten-enhancer deleted

value: normal thymus


title: RNAseq: E13.5 Gata4hi epithelial cells-1


title: SNPC13ChIP_SNPC13flag_48h_input_rep1

description:
snpc-1.3::3xflag L4 (48h) SNPC-1.3 ChIP-seq input replicate 1
SNPC13ChIP_SNPC13flag_48h_rep1_SignificantBins.txt

value: snpc-1.3(xk32)[snpc-1.3a::3xflag] V


title: 5′-End RNA DMSO In Vitro

value: DMSO

value: 5′-End RNA


title: RNA-seq : E11.5 Gata4hi epithelial cells


title: 5′-End RNA 25mM 5NIA In Vitro


title: WT4_rep4

description: GEO_processed excel file_YHLLAB.xlsx


title: SNPC13ChIP_N2_48h_input_rep1

description:
N2 L4 (48h) SNPC-1.3 ChIP-seq input replicate 1
SNPC13ChIP_N2_48h_rep1_SignificantBins.txt


title: RNAseq: E11.5 Sox2hi epithelial cells


title: WT-Thymus

description: Filtered features-barcode UMI count matrix for 8931 barcoded cells and 31053 features, with associated features and barcode labels (.tsv) files

value: Control


title: ArCre_E17-5_UGS

city: Duarte

first: Zijie

last: Sun

state: CA

address: 1500 E. Duarte Rd.

department: Cancer Biology

institute: City of Hope

description: mRNA from single cells

data_processing:
The library purity and size was validated by capillary electrophoresis using 2,100 Bioanalyzer (Agilent Technologies)
The library quantity was measured fluorometrically using Qubit dsDNA HS Assay Kit from Invitrogen
The libraries were sequenced with a NovaSeq 6000 instrument (Illumina) to a depth of ~130k reads per cell. Raw sequencing data were processed using the 10x Genomics’ Cell Ranger pipeline (version 3.1.0) to generate FASTQ files and were aligned to the mm10 genome with an added mGFP sequence to generate gene expression counts
Genome_build: mm10 reference genome with added EGFP sequence
Supplementary_files_format_and_content: Txt file with counts for each cell corresponding to each gene

source_name: E17.5 Male Urogenital Sinus

extract_protocol:
UGS tissue was trimmed and dissociated to single cells using collagenase and TrypLE (Gibco)
The library was prepared using 10x Genomics Chromium Single Cell 3' Solution with v3 chemistry following manufacturer protocols (10x Genomics)
scRNAseq

value: Urogenital Sinus

tag: condition

value: Normal

tag: gender

value: male


title: multiple myeloma bone marrow 9 [mm9]

status: Public on Dec 16 2020

city: Atlanta

first: Samuel

middle: Spence

last: McCachren

state: GA

address: 1760 Haygood Dr. NE

institute: Emory University

description:
mm9
BM single cell RNA seq

data_processing:
Reads were aligned, filtered, de-duplicated, and converted into a digital count matrix using Cell Ranger 1.2 (10X Genomics)
Additional quality control (QC) was performed by visual inspection of QC plots using Seurat [Satija et al. Nat Biotech 33, 495-502 (2015)]. Outlier cells were identified and filtered based on (1) anomalously high mitochondrial gene expression (cells with >10% mitochondrial content were removed) and (2) potential doublets/multiplets, as identified by comparing the number of expressed genes detected by per cell versus the number of unique molecular identifiers (UMIs) detected per cell (cells with greater than 7000 expressed genes were removed). In addition, we removed clusters of plasma (cells expressing CD138) and erythroid cells (cells expressing hemoglobin).
To co-analyse all samples, an aggregate digital expression matrix was produced using Seurat, and then the above steps were carried out.
The processed files are combined digital expression matrix divided in three files for each of the sample. The matrix.mtx file contains the values, barcodes.tsv contains the cell barcodes detected, and genes.tsv contains the list of genes detected. The three files for each sample are copied in one folder, and this folder was read-in using the Seurat package.
For Seurat to work correctly the suffix for each file has to be removed.
Genome_build: hg38
Supplementary_files_format_and_content: The processed files are combined digital expression matrix divided in three files for each of the sample. The matrix.mtx file contains the values, barcodes.tsv contains the cell barcodes detected, and genes.tsv contains the list of genes detected. The three files for each sample are copied in one folder, and this folder was read-in using the Seurat package.

source_name: multiple myeloma bone marrow

growth_protocol: Bone marrow sample from a healthy donor, MGUS patient, or myeloma patient was thawed; myeloma samples were sorted to remove CD138+ malignant plasma cells.

extract_protocol:
To prepare the libraries for sequencing, the Chromium Gel Bead and Library Kit (10X Genomics) and Chromium instrument (10X Genomics) were used following manufacturer’s protocol. Further, the barcoded libraries were sequenced using HiSeq2500.
10X Genomics manufacturer protocol

tag: disease state

value: multiple myeloma

value: Bone Marrow Immune Cells


title: multiple myeloma bone marrow 8 [mm8]

description:
mm8
BM single cell RNA seq


title: multiple myeloma bone marrow 5 [mm5]

description:
mm5
BM single cell RNA seq


title: multiple myeloma bone marrow 10 [mm10]

description:
mm10
BM single cell RNA seq


title: multiple myeloma bone marrow 11 [mm11]

description:
mm11
BM single cell RNA seq


title: multiple myeloma bone marrow 7 [mm7]

description:
mm7
BM single cell RNA seq


title: multiple myeloma bone marrow 6 [mm6]

description:
mm6
BM single cell RNA seq


title: multiple myeloma bone marrow 2 [mm2]

description:
mm2
BM single cell RNA seq


title: multiple myeloma bone marrow 3 [mm3]

description:
mm3
BM single cell RNA seq


title: multiple myeloma bone marrow 1 [mm1]

description:
mm1
BM single cell RNA seq


title: multiple myeloma bone marrow 4 [mm4]

description:
mm4
BM single cell RNA seq


title: MGUS bone marrow 14 [mgus14]

description:
mgus14
BM single cell RNA seq

source_name: MGUS bone marrow

value: monoclonal gammopathy of unkown significance (MGUS)


title: MGUS bone marrow 13 [mgus13]

description:
mgus13
BM single cell RNA seq


title: MGUS bone marrow 4 [mgus4]

description:
mgus4
BM single cell RNA seq


title: MGUS bone marrow 6 [mgus6]

description:
mgus6
BM single cell RNA seq


title: MGUS bone marrow 9 [mgus9]

description:
mgus9
BM single cell RNA seq


title: MGUS bone marrow 11 [mgus11]

description:
mgus11
BM single cell RNA seq


title: MGUS bone marrow 12 [mgus12]

description:
mgus12
BM single cell RNA seq


title: MGUS bone marrow 5 [mgus5]

description:
mgus5
BM single cell RNA seq


title: MGUS bone marrow 8 [mgus8]

description:
mgus8
BM single cell RNA seq


title: MGUS bone marrow 2 [mgus2]

description:
mgus2
BM single cell RNA seq


title: MGUS bone marrow 10 [mgus10]

description:
mgus10
BM single cell RNA seq


title: healthy donor bone marrow 6 [hd6]

description:
hd6
BM single cell RNA seq

source_name: healthy donor bone marrow

value: healthy


title: healthy donor bone marrow 5 [hd5]

description:
hd5
BM single cell RNA seq


title: MGUS bone marrow 1 [mgus1]

description:
mgus1
BM single cell RNA seq


title: healthy donor bone marrow 8 [hd8]

description:
hd8
BM single cell RNA seq


title: healthy donor bone marrow 7 [hd7]

description:
hd7
BM single cell RNA seq


title: healthy donor bone marrow 3 [hd3]

description:
hd3
BM single cell RNA seq


title: healthy donor bone marrow 2 [hd2]

description:
hd2
BM single cell RNA seq


title: healthy donor bone marrow 4 [hd4]

description:
hd4
BM single cell RNA seq


title: RF-AvrRpt2 Rep2 (R)

city: Durham

first: Xinnian

last: Dong

address: 124 Science Drive

department: Biology

institute: Duke University

description:
rps2_RPKM.txt
RFA_R2

data_processing:
Library strategy: Ribo-Seq
Basecalls performed using CASAVA version 1.8.2
Illumina adapter removal and size filter by FastX
removal of reads aligned to tRNA and rRNA sequences by Bowtie 2.0, default parameters
align reads to genome by Bowtie 2.0, default parameters
assign reads to genes,(exon,CDS), by HT-seq, default parameters
Genome_build: TAIR10
Supplementary_files_format_and_content: Col_RPKM.txt (tab delimited file of RPKM assigned to genes by exons)
Supplementary_files_format_and_content: rps2_RPKM.txt (tab delimited file of RPKM assigned to genes by CDS)

organism: Arabidopsis thaliana

source_name: RF-AvrRpt2 (R)

growth_protocol: Plants were grown on soil (Metro Mix 360) at 22 °C under a 12/12-h light/dark cycle

extract_protocol:
Leaves from ~24 three-week-old plants (2 leaves/plant; ~1.0 g) were collected. Tissue was fast frozen and ground in liquid nitrogen. 5 ml cold polysome extraction buffer [PEB; 200 mM Tris pH 9.0, 200 mM KCl, 35 mM MgCl2, 25 mM EGTA, 5 mM DTT, 1 mM phenylmethanesulfonylfluoride (PMSF), 50 μg/ml cycloheximide, 50 μg/ml chloramphenicol, 1% (vol/vol) Brij-35, 1% (vol/vol) Igepal CA630, 1% (vol/vol) Tween 20, 1% (vol/vol) Triton X-100, 1% Sodium deoxycholate (DOC), 1% (vol/vol) polyoxyethylene 10 tridecyl ether (PTE)] was added. After thawing on ice for 10 min, lysate was centrifuged at 4 °C/16,000 g for 2 min. Supernatant was transferred to 40 μm filter falcon tube and centrifuged at 4 °C/7,000 g for 1 min. Supernatant was then transferred into a 2 ml tube and centrifuged at 4 °C/16,000 g for 15 min and this step was repeated once. 0.25 ml lysate was saved for total RNA extraction for making the RNA-seq library. Another 1 ml lysate was layered on top of 0.9 ml sucrose cushion [400 mM Tris•HCl (pH 9.0), 200 mM KCl, 35 mM MgCl2, 1.75 M sucrose, 5 mM DTT, 50 μg/ml chloramphenicol, 50 μg/ml cycloheximide] in an ultracentrifuge tube (#349623, Beckman). The samples were then centrifuged at 4 °C/70 k rpm for 4 h. The pellet was washed twice with cold water, resuspended in 300 μl RNase I digestion buffer [RNID; 20 mM Tris•HCl (pH 7.4), 140 mM KCl, 35 mM MgCl2, 50 μg/mL cycloheximide, 50 μg/ml chloramphenicol]11 and then transferred to a new tube for brief centrifugation. The supernatant was then transferred to another new tube where 10 μl RNase I (100 U/μl) was added for 60 min incubation at 25 °C/300 rpm. 15 μl SUPERase-In (20 U/μl) was then added to stop reaction.
For RF library, ribosome recovery, footprint fragment purification, PNK treatment and linker ligation were performed according to the public protocol (Ingolia et al.,, 2012, Nature protocol). 2.5 μl 5’ deadenylase (NEB) was then added to the ligation system and incubated at 30 °C for 1 h. 2.5 μl RecJf exonuclease (NEB) was subsequently added for 1 h incubation at 37 °C. The enzymes were inactivated at 70 °C for 20 min and 10 μl of the samples were taken as template for reverse transcription. The rest of steps for the library construction were performedaccording to the public protocol (Ingolia et al., 2012, Nature protocol) except biotinylated oligos, rRNA1 and rRNA2, for Arabidopsis according to another reported method Juntawong et al., 2014, PNAS). For RNA-seq library, 0.75 ml TRIzol® LS (Ambion) was added to the 0.25 ml lysate saved from the Ribo-seq library construction. Total RNA was extracted, quantified and qualified using Nanodrop (Thermo Fisher Scientific Inc). 50-75 μg total RNA was used for mRNA purification with Dynabeads® Oligo (dT)25 (Invitrogen). 20 μl of the purified poly (A) mRNA was mixed with 20 μl 2× fragmentation buffer (2 mM EDTA, 10 mM Na2CO3, 90 mM NaHCO3) and incubated for 40 min at 95 °C before cooling on ice. 500 μl of cold water, 1.5 μl of GlycoBlue and 60 μl of cold 3 M sodium acetate were then added to the samples and mixed. Subsequently, 600 μl isopropanol was added before precipitation at -80 °C for at least 30 min. Samples were then centrifuged at 4°C/12,000 rpm for 30 min to remove all liquid and air dried for 10 min before resuspension in 5 μl of 10 mM Tris (pH 8). The rest of the steps were the same as Ribo-seq library preparation from footprint fragment purification.

treatment_protocol: 4-week-old Arabidopsis plants were infiltrated with either 0.01 AvrRpt2 or 10 mM MgCl2 as Mock

tag: ecotype

tag: transgene

value: AvrRpt2 inoculation

value: leaf

tag: rna fraction

value: ribosomal associated mRNA

value: rps2 mutant background


title: healthy donor bone marrow 1 [hd1]

description:
hd1
BM single cell RNA seq


title: MGUS bone marrow 7 [mgus7]

description:
mgus7
BM single cell RNA seq


title: RS-Mock Rep2 (R)

description:
rps2_RPKM.txt
RSM_R2

data_processing:
Basecalls performed using CASAVA version 1.8.2
Illumina adapter removal and size filter by FastX
removal of reads aligned to tRNA and rRNA sequences by Bowtie 2.0, default parameters
align reads to genome by Bowtie 2.0, default parameters
assign reads to genes,(exon,CDS), by HT-seq, default parameters
Genome_build: TAIR10
Supplementary_files_format_and_content: Col_RPKM.txt (tab delimited file of RPKM assigned to genes by exons)
Supplementary_files_format_and_content: rps2_RPKM.txt (tab delimited file of RPKM assigned to genes by CDS)

source_name: RS-Mock (R)

value: Mock

value: total mRNA


title: RS-AvrRpt2 Rep1 (R)

description:
rps2_RPKM.txt
RSA_R1

source_name: RS-AvrRpt2 (R)


title: RF-Mock Rep1 (R)

description:
rps2_RPKM.txt
RFM_R1

source_name: RF-Mock (R)


title: RF-AvrRpt2 Rep3 (C)

description:
Col_RPKM.txt
RFA_C3

source_name: RF-AvrRpt2 (C)

value: WT background


title: MGUS bone marrow 3 [mgus3]

description:
mgus3
BM single cell RNA seq


title: RS-Mock Rep1 (R)

description:
rps2_RPKM.txt
RSM_R1


title: RS-AvrRpt2 Rep2 (R)

description:
rps2_RPKM.txt
RSA_R2


title: RF-AvrRpt2 Rep2 (C)

description:
Col_RPKM.txt
RFA_C2


title: RF-AvrRpt2 Rep1 (C)

description:
Col_RPKM.txt
RFA_C1


title: RS-AvrRpt2 Rep2 (C)

description:
Col_RPKM.txt
RSA_C2

source_name: RS-AvrRpt2 (C)


title: RF-Mock Rep2 (C)

description:
Col_RPKM.txt
RFM_C2

source_name: RF-Mock (C)


title: RS-AvrRpt2 Rep3 (C)

description:
Col_RPKM.txt
RSA_C3


title: RF-Mock Rep3 (C)

description:
Col_RPKM.txt
RFM_C3


title: RF-Mock Rep1 (C)

description:
Col_RPKM.txt
RFM_C1


title: RS-AvrRpt2 Rep1 (C)

description:
Col_RPKM.txt
RSA_C1


title: RF-AvrRpt2 Rep1 (R)

description:
rps2_RPKM.txt
RFA_R1


title: RF-Mock Rep2 (R)

description:
rps2_RPKM.txt
RFM_R2


title: RS-Mock Rep3 (C)

description:
Col_RPKM.txt
RSM_C3

source_name: RS-Mock (C)


title: RS-Mock Rep2 (C)

description:
Col_RPKM.txt
RSM_C2


title: RS-Mock Rep1 (C)

description:
Col_RPKM.txt
RSM_C1


title: Gilteritinib+MI3454_MOLM13_3

city: Indianapolis

first: James

last: Ropa

state: IN

address: 950 W Walnut St

institute: Indiana University

data_processing:
Quality trimming using Cutadapt version 2.1 with arguments: -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCA -A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT --nextseq-trim=20 --minimum-length 50
Indices generated using STAR v2.7.1a with options: --sjdbOverhang 99 --genomeSAsparseD 2
Sequences aligned using STAR v2.7.1a with options: --outSAMtype BAM SortedByCoordinate --outSAMunmapped Within
Aligned reads counted using htseq-count from HTSeq v0.11.2 with options: -s reverse -r pos -f bam
Differential expression analysis using DESeq2 alpha < 0.05 lfcThreshold = log2(1.5)
Genome_build: GENCODE release 33 (GRCh38) primary assembly
Supplementary_files_format_and_content: tab-delimited files containing unnormalized count data

source_name: Acute monocytic leukemia cell line

growth_protocol: MOLM-13 cells were cultured in RPMI 1640 medium with 10% heat inactivated FBS and 1% pen/strep antibiotics.

extract_protocol:
Rneasy Mini Kit (Qiagen 74106)
Ribo depleted RNA was used to generate Illumina compatible TruSeq libraries

treatment_protocol: Cells were seeded at 3x10^5 cells/mL in triplicates and treated with Gilteritinib, MI3454, Gilteritinib+MI3454, or DMSO control.

tag: cells

value: Gilteritinib + MI3456


title: 21_BM

city: Pittsburgh

first: Timothy

last: Billiar

state: Pennsylvania

address: 200 Lothrop Street

department: Surgery

institute: University of Pittsburgh

description:
19, 21 and 23 (3 samples of PBMC and 3 samples of BMMC, total 6 samples) were processed in the same batch.
Mouse18-23_aggr.txt.gz

data_processing:
To minimize the potential batch effect, we analyzed each batch of mouse data separately. For each batch, raw sequencing data was processed using the 10x CellRanger pipeline, cellranger count followed by cellranger aggr (--normalize=mapped, mouse: cell ranger v2.1.0, mapped to mm10) to generate a UMI count matrix and then further processed using the Seurat (v2.3.4). Genes expressed in ≥ 3 cells were retained. Cells with gene counts less than 200 or more than 5000 were filtered out. The UMI count matrixes, which pass the above preliminary filtering and contain the data collected from all the samples from the same batch (01-04 or 11-14) or paralel batches (18-23) , are provided as the processed data.
Based on the mouse experiments, biological replicates prepared in parallel were highly reproducible. Thus, similar to the mouse analysis, different time points sampled from the same patients with the matched healthy control were processed by cellranger count/aggr (human: cell ranger v3.0.0, mapped to GRCh38, aggr: --normalize=mapped) and then by Seurat (v3.0.2) for quality control and pre-processing. Genes expressed in ≥ 3 cells were retained. Cells with gene counts less than 200 or more than 5000 or ≥ 20% mitochondria genes were filtered out. The UMI count matrixes, which passed the above preliminary filtering and contained the data collected from 3 time points and corresponding control for each patient, are provided as the processed data. To overcome human heterogeneity and to identify the same cell type or functional state in population, the data from different individuals were integrated by Seurat integration standard workflow.
Genome_build: Mouse: mm10. Human: GRCh38.
Supplementary_files_format_and_content: For the samples processed in the same batch (samples for each patient, mouse 01-04 or mouse 11-14) or parallel batches (mouse 18-23), the UMI count matrixes which were generated by cellranger aggr (--normalize=mapped) and passed Seurat preliminary filtering are provided.

source_name: BMMC

extract_protocol:
Ficoll centrifugation to isolate PBMC or BMMC.
RNA libraries were prepared for sequencing using 10x Genomic single cell gene expression procotol: mouse (v2) and human (v3).

value: BMMC

tag: group

value: Ctrl


title: 22_BM

description:
18, 20 and 22 (3 samples of PBMC and 3 samples of BMMC, total 6 samples) were processed in the same batch.
Mouse18-23_aggr.txt.gz

tag: strain


title: 13_BM

description:
11-14, 4 samples of PBMC and 4 samples of BMMC (total 8 samples) were processed in the same batch.
Mouse11-14_aggr.txt.gz


title: 23_PB

source_name: PBMC

value: PBMC


title: 20_PB


title: 20_BM


title: 11_BM


title: 22_PB


title: 21_PB


title: 19_BM


title: 18_PB


title: 18_BM


title: 14_PB


title: 23_BM


title: 13_PB


title: 04_PB

description:
01-04, 4 samples of PBMC were processed in the same batch.
Mouse01-04_aggr.txt.gz


title: 19_PB


title: 03_PB


title: 02_PB


title: 14_BM


title: 01_PB


title: H1299_control vs. H1299_miR-7_overexpression

city: Daejeon

first: Seong-Lan

last: Yu

address: Gasuwon-dong

department: Myunggok Medical Research Institute

institute: Konyang university

description: H1299_LN vs H1299_L7

hyb_protocol: Each 825ng of cyanine 3-labeled and cyanine 5-labeled cRNA target was mixed and the cRNA fragmentation was performed by adding a 10X blocking agent and 25X fragmentation buffer and incubating at 60 oC for 30min.

scan_protocol: The hybridization images were analyzed by an Agilent DNA Microarray Scanner (Agilent Technology) and data quantification was performed using Agilent Feature Extraction software 9.3.2.1 (Agilent Technology).

data_processing: All data normalization and the selection of fold-changed genes were performed using GeneSpringGX 7.3.1.

source_name: H1299 cells

label_protocol: The transcription master mix was prepared according to the manufacturer’s protocol (4X Transcription buffer, 0.1M DTT, NTP mix, 50% PEG, RNase-Out, inorganic pyrophosphatase, T7-RNA polymerase, and cyanine 3/5-CTP). Transcription of the dsDNA was performed by adding the transcription master mix to the dsDNA reaction samples and incubating at 40 oC for 2 hours.

growth_protocol: The H1299 cells were cultured with RPMI1640 supplemented with 10% FBS and 1% penicillin/streptomycin solution.

extract_protocol: Total RNA extracted using Trizol following manufacturer's instructions.

treatment_protocol: The H1299 cells were transfected with hsa-miR7-5p mimic using Lipofectamine RNAiMAX reagent.

tag: cell line

value: Non-small cell lung cancer cell line

tag: overexpression

value: control

source_name: hsa-miR7 overexpressing H1299 cells


title: 12_BM

tag: tissue


title: KOinWT_Day21_rep2 [ATAC-Seq]

city: Geneva

first: Sylvain

last: LEMEILLE

address: 1 rue Michel-Servet

department: Department of Pathology and Immunology

country: Switzerland

institute: University of Geneva Medical School

data_processing:
Basecalls performed using CASAVA version 1.8.2
Adaptors were trimming using cutadapt 1.9.1
Reads were mapped to the reference genome using bowtie2 2.2.26
Duplicated reads were marked with picard-tools-1.126 and removed with samtools 1.2
Reads were shifted by +4bp on the forward strand and -5bp on the reverse strand
Peaks were called using macs2 v2.1.2 with -p 0.01 –to-large –nomodel and shiftsize set to 75
Reproducible peaks were obtained by IDR procedure with -p 0.01
Reproducible peaks overlapping blacklist (https://www.nature.com/articles/s41598-019-45839-z) were removed
Genome_build: GRCm38.89
Supplementary_files_format_and_content: bigwig
Supplementary_files_format_and_content: peaks

source_name: P14 CD8+ T cells

extract_protocol:
To perform ATAC-seq, we FACS-sorted brain-infiltrating P14 cells (Tox +/+ and Tox -/-) from WT and MOG-GP mice at different time points after intracranial LCMV-GP infection. Each biological replicate (n = 2) represents 5x10E4 sorted P14 cells sorted in FACS buffer.
Cells were spun down at 500 g for 20 min at 4 °C. Nuclei were isolated in 50 μl of lysing solution composed of 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAL CA-630. The nuclear pellet was resuspended into 50 μl of tagmentation buffer containing the Tn5 transposase (Illumina) and incubated at 37 °C for 30 min. The tagmented DNA was purified using MiniElute PCR Purification Kit (Qiagen). Libraries were preamplified for 5 cycles using indexed primer from Nextera kit (Illumina) and NEBNext High Fidelity PCR master mix (New England Biolabs). Preamplified libraries served as input to determine the number of remaining cycles needed for the second round of PCR amplification. Libraries were cleaned by Ampure Beads (Agencourt) at a 1.5× ratio.

tag: cell type

value: CD8+ T cells

tag: mouse recipient

value: WT

tag: weeks post-infection

tag: genotype of p14

value: Tox -/-


title: KOinGP_Day21_rep1 [ATAC-Seq]

value: MOG-GP


title: KOinGP_Day21_rep2 [ATAC-Seq]


title: WTinGP_Day21_rep2 [ATAC-Seq]

value: wild type


title: KOinWT_Day21_rep1 [ATAC-Seq]


title: 12_PB


title: 11_PB


title: WTinGP_Day7_rep1 [ATAC-Seq]


title: WTinWT_Day7_rep2 [ATAC-Seq]


title: KOinWT_day63_rep1

data_processing:
Basecalls performed using CASAVA version 1.8.2
Reads were mapped to the reference genome using STAR 2.4 with --outFilterMultimapNmax 1
Raw count measurements were obtained with featureCounts 1.4.6 keeping only unambiguous reads
Genome_build: GRCm38.89
Supplementary_files_format_and_content: Raw count measurements

source_name: P14 CD8+ T cells_KOinWT_day63

extract_protocol:
To perform RNA-seq, we FACS-sorted brain-infiltrating P14 cells (Tox +/+ and Tox -/-) from WT and MOG-GP mice at different time points after intracranial LCMV-GP infection. Each biological replicate (n = 3) represents 10E4 sorted P14 cells sorted in buffer RLT with 1% ß-mercaptoethanol and processed with RNAeasy Micro Kit (Qiagen) following manufacturer’s instructions.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: strain background


title: WTinWT_day63_rep2

source_name: P14 CD8+ T cells_WTinWT_day63


title: KOinWT_day63_rep3


title: WTinWT_Day7_rep1 [ATAC-Seq]


title: WTinWT_Day21_rep2 [ATAC-Seq]


title: KOinWT_day63_rep2


title: KOinGP_day21_rep3

source_name: P14 CD8+ T cells_KOinGP_day21


title: KOinGP_day21_rep2


title: KOinWT_day21_rep3

source_name: P14 CD8+ T cells_KOinWT_day21


title: KOinWT_day21_rep1


title: KOinWT_day21_rep2


title: WTinGP_day21_rep2

source_name: P14 CD8+ T cells_WTinGP_day21


title: WTinGP_day21_rep3


title: WTinWT_day21_rep3

source_name: P14 CD8+ T cells_WTinWT_day21


title: WTinWT_Day21_rep1 [ATAC-Seq]


title: WTinWT_day63_rep1


title: WTinGP_day21_rep1


title: WTinWT_day21_rep2


title: AL1679T

city: New York

first: Tiphaine

middle: Christiane

last: Martin

address: 1470 Madison Ave

department: Oncological Sciences

institute: Icahn School of Medicine at Mount Sinai, Tisch Cancer Institute

description: RNA-seq

data_processing:
We first assessed the quality of paired-end reads with FASTQC  (version 0.11.8). Next, we filtered reads with BBDUK from BBTOOLS  (version 37.53) to remove adapters, known artefacts and then quality trimmed (PHRED quality score < 10). Reads that became too short after trimming (N < 60 bp) were discarded. We did not retain singleton reads (i.e., reads whose mate has been discarded). We estimated the transcript-level quantification of cleaned RNA-seq data using SALMON  (version 1.0.0) by a quasi-mapping on Mus musculus GRCm38. We estimated the gene-level quantification using tximport  library and converted mouse gene expression to human gene expression using biomaRt  library.
Genome_build: GRCm38
Genome_build: Mus_musculus.GRCm38.cds.all.fa.gz (ENSEMBL)

molecule: polyA RNA

source_name: Liver tumors

growth_protocol: hydrodynamic tail vein delivery

extract_protocol:
Trizol® (Invitrogen) followed by digestion with DNase I (Roche) and purification with the RNeasy Kit (Qiagen).
TruSeq RNA Sample Preparation

treatment_protocol: no treatment

tag: protocol

value: Hydrodynamic tail-vein injection method

tag: mutations

value: MYC;Tert

value: Tumor

tag: tumor stage

value: advanced tumors


title: AL1678T

description: RNA-seq


title: PM440T

description: RNA-seq


title: WTinWT_day21_rep1


title: AL416T_S4

description: RNA-seq

value: MYC;sg-Pten


title: AL407T_S6

description: RNA-seq


title: WTinGP_Day21_rep1 [ATAC-Seq]


title: WTinGP_Day7_rep2 [ATAC-Seq]


title: AL396T

description: RNA-seq


title: pm-117-t_S17

description: RNA-seq


title: pm-118-t_S18

description: RNA-seq


title: AL382T

description: RNA-seq


title: AL419T_S1

description: RNA-seq


title: AL380T

description: RNA-seq


title: AL417T_S2

description: RNA-seq


title: AL569T

description: RNA-seq


title: K11-3

city: Guangzhou

first: Hongjian

last: Li

address: No.6 Dafeng Road, Tianhe District

department: Department of tea resources and breeding

country: China

institute: Tea Research Institute, Guangdong Academy of Agricultural Sciences

data_processing:
Reads obtained from the sequencing machines included raw reads containing adapters or low quality bases which would affect the following assembly and analysis. Thus, to get high quality clean reads, reads were further filtered according to the following rules: 1) Removing reads containing adapters; 2) Removing reads consisting of all A bases; 3) Removing reads containing more than 10% of unknown nucleotides (N); 4) Removing low quality reads containing more than 50% of low quality (Q-value≤20) bases
Short reads alignment tool Bowtie2 (2.2.8) was used for mapping reads to ribosome RNA (rRNA) database. The rRNA mapped reads were then removed. The remaining reads were further used in assembly and analysis of transcriptome.
The rRNA removed reads of each sample were then mapped to reference genome by TopHat2 (version 2.1.1), respectively. The alignment parameters were as follows: 1) Maximum read mismatch is 2 2) Disables the coverage based search for junctions 3) The distance between mate-pair reads is 50bp 4) The standard deviation for the distribution on inner distance between mate-pair reads is 80bp
After aligned with reference genome, unmapped reads (or mapped very poorly) were then re-aligned with Bowtie2, the enriched unmapped reads were split into smaller segments which were then used to find potential splice sites. The section and the section position of these short segments were predicted as well. A set of splice sites was built with initial unmapped reads by TopHat2 without relying on the known genes annotation. Not only be used for identifying expressed genes and their quantitative expression, the sequence alignment will also be helpful to find alternative splicing and new transcripts.
Genome_build: Camellia sinensis (http://pcsb.ahau.edu.cn:8080/CSS/)
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

organism: Camellia sinensis

source_name: leaf

growth_protocol: All the materials were grown in Germplasm Bank of Tea Plant in Guangdong Province (Yingde, Guangdong, China, 24°18′N，113°23′E), and details on agro-techniques (fertilization and irrigation) are available upon request.

extract_protocol:
Retinas were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
After total RNA was mixed, rRNAs were removed to retain mRNAs and ncRNAs. The enriched mRNAs and ncRNAs were fragmented into short fragments by using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP (dUTP instead of dTTP) and buffer. Next, the cDNA fragments were purified with QiaQuick PCR extraction kit, end repaired, poly(A) added, and ligated to Illumina sequencing adapters.

value: leaf

tag: genotype


title: AL1467T

description: RNA-seq


title: K11-2


title: al-421-t

description: RNA-seq


title: AL612T

description: RNA-seq


title: AL608T

description: RNA-seq


title: K11-1


title: K6-3


title: K6-2


title: K6-1


title: QX1-3


title: AL422T_S9

description: RNA-seq


title: QX1-2


title: QX1-1


title: YH-3


title: AL384T

description: RNA-seq


title: JAX214-myc-lucOS-bcat

description: RNA-seq

value: myc-lucOS-bcat


title: AL1466T

description: RNA-seq


title: JAX192-myc-lucOS-bcat

description: RNA-seq


title: JAX193-myc-lucOS-bcat

description: RNA-seq


title: JAX208-myc-luc-bcat

description: RNA-seq

value: myc-luc-bcat


title: YH-2


title: RNA-seq mut06-2

city: TAMPA

first: Marco

last: Davila

state: Florida

address: 12902 USF MAGNOLIA DRIVE, MRC2

institute: Moffitt Cancer Center

description: featureCounts.txt [JB21]

data_processing:
For RNA-seq: Description of Analysis Steps  1. Read Mapping: RNA-sequencing performed by Active Motif generates 100-nt sequence reads using Illumina HiSeq 4000. The reads are mapped to the genome using the STAR algorithm with default settings. Alignment information for each read is stored in the BAM format.  2. Fragment Assignment: This process step is to count the number of reads (for single-end library) or fragments (for paired-end library) overlapping predefined genomic features of interest (e.g. genes). Only read pairs that have both ends aligned were counted. Read pairs that have their two ends mapping to different chromosomes or mapping to same chromosome but on different strands were discarded. We also require that at least 25 bp overlapping bases in a fragment for read assignment. The gene annotations we used were obtained from Subread package [2]. These annotations were originally from NCBI RefSeq database and then adapted by merging overlapping exons from the same gene to form a set of disjoint exons for each gene. Genes with the same Entrez gene identifiers were also merged into one gene [2].  3. Differential Analysis: After obtaining the gene table containing the fragment (or read) counts of genes, we performed differential analysis to identify statistically significant differential genes using DESeq2 [3]. The following lists the pre-processing steps before differential calling.  a. Data Normalization: DESeq2 expects un-normalized count matrix of sequencing reads (for single-end RNA-seq) or fragments (for paired-end RNA-seq) for DESeq2’s statistical model to hold. The DESeq2 model internally corrects for library size using their median-of-ratios method [3]. The gene table obtained from Analysis Step 2) was used as input to perform DESeq2.  b. Filtering: DESeq2 by default performs independent filtering which is a statistical technique to filter out the statistical tests (i.e. genes) that have no or little chance of being significant. The filtering criteria is the mean of normalized counts. The idea is to increase detection power (i.e. maximizing the number of significantly differential genes detected) by filtering out genes not likely to show expression difference (e.g. genes with low counts) as these genes have an influence on the multiple testing adjustment.  8 of 8  Active Motif, Inc. • 1914 Palomar Oaks Way, Suite 150 • Carlsbad, CA 92008 • toll free 877.222.9543 • fax  760.431.1351  www.activemotif.com  c. Differential Calling: When replication data is available, differential genes were detected by DESeq2 at 10% adjusted P-value level. Otherwise, differential genes were defined using shrunkenLog2FC (default cutoff is 0.3) when there is no replication data.  4. Gene Set Enrichment Analysis (GSEA): Using a gene list ranked by shrunkenLog2FC obtained from DESeq2, we performed GSEA with default setting to determine whether members of a priori defined gene set based on biological knowledge (e.g. genes sharing the same GO category) tend to occur toward the top (or bottom) of the gene list. Please refer to GSEA’s User Guide (section IV) for more information.
For ATAC-seq: Description of Analysis Steps.  1. Sequence Analysis: The paired-end 42 bp sequencing reads generated by Illumina sequencing  (using NextSeq 500) are mapped to the genome using the BWA algorithm with default settings.  Alignment information for each read is stored in the BAM format. Only reads that pass Illumina’s  purity filter, align with no more than 2 mismatches, and map uniquely to the genome are used in the  subsequent analysis. In addition, unless stated otherwise, duplicate reads (“PCR duplicates”) are  removed.  2 of 7  Active Motif, Inc. • 1914 Palomar Oaks Way, Suite 150 • Carlsbad, CA 92008 • toll free 877.222.9543 • fax 760.431.1351  www.activemotif.com  2. “Peak Finding”: Since both reads (tags) from paired-end sequencing represent transposition  events, both reads are used for peak-calling. Unlike ChIP-seq, where in-silico extension is  performed to represent the length of the fragment bound by the protein of interest, ATAC-Seq aims  to identify enrichment of transposome accessibility, thus no in-silico extension is performed. Rather,  the 42 bp length of the reads is used for peak-calling. The generic term “Interval” is used to  describe genomic regions with local enrichments in tag numbers. Intervals are defined by the  chromosome number and a start and end coordinate. The peak caller used for ATAC-Seq at Active  Motif is MACS2 (Zhang et al., Genome Biology 2008, 9:R137), using both PE reads from each  aligned fragment.  3. Determination of Fragment Density: To identify the density of transposition events along the  genome, the genome is divided into 32 bp bins and the number of fragments in each bin is  determined. For this purpose, reads are extended to 200 bp in order to smooth the data. This  information (“signal map”; histogram of fragment densities) is stored in a bigWig file, which can be  visualized in genome browsers (see Section V.). BigWig files are also the basis of the peak metrics  in the Active Motif analysis program described below. “Interval” or “Merged Region” intensity values  are calculated based on the number of fragments within 32 bp bins that overlap with these features.  4. Normalization: In the default analysis, the tag number of all samples is reduced (by random  sampling) to the number of tags present in the smallest sample. When samples are not directly  comparable or in cases of projects with outlier samples, this strategy is either forgone or can  potentially be customized to fit the experimental design.  5. Merged Region Analysis: To compare peak metrics between 2 or more samples, overlapping  Intervals are grouped into “Merged Regions”, which are defined by the start coordinate of the most  upstream Interval and the end coordinate of the most downstream Interval (= union of overlapping  Intervals). In locations where only one sample has an Interval, this Interval defines the Merged  Region. The use of Merged Regions is necessary because the locations and lengths of Intervals  are rarely exactly the same when comparing different samples.  6. Annotations: After defining the Intervals and Merged Regions, their genomic locations along with  their proximities to gene annotations and other genomic features are determined and presented in  Excel spreadsheets. In addition, average and peak (i.e. at “Summit”) fragment densities within  Intervals and Merged Regions are compiled.
Supplementary_files_format_and_content: featureCounts.txt, tab delimited text file (.txt), matrix of gene counts
Supplementary_files_format_and_content: 3774Moffitt_ATAC_ucsctracks.bed, tab delimited text file, BED formatted feature tracks

source_name: CAR T cell

growth_protocol: 6 week old C57BL6 mice were purchased from Jackson Labs and housed in the Moffitt animal facility.  Spleens were collected at 8 weeks

extract_protocol:
After stimulation live CAR positive cells were sorted using a FACSAriaII cell sorter then pelleted and snap frozen in liquid nitrogen. See library construction protocol section for RNA-seq and ATAC-seq processing.
RNA-seq: Total RNA was isolated from the cells using the Qiagen RNeasy Mini Kit (Qiagen, cat# 74104). For each sample, 2 ug of total RNA was then used in Illumina’s TruSeq Stranded mRNA Library kit (Cat# 20020594). Libraries were sequenced on Illumina NextSeq 500 as paired-end 42-nt reads. Sequence reads were analyzed with the STAR alignment – DESeq2 software pipeline described in the Data Explanation document. ATAC-seq: Cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the Nextera Library Prep Kit (Illumina). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter).  Resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems), and sequenced with PE42 sequencing on the NextSeq 500 sequencer (Illumina).

treatment_protocol: T cells were isolated from splenocytes and transduced with CARs by retro-gamma viral spin transduction.  Transduction efficiency was then measured by flow cytometry.  CAR T cells were then stimulated by 3T3-mCD19 target cells at a 10:1 effector:target ratio for 24hrs.

tag: sorted cells


title: YH-1


title: JAX204-myc-luc-bcat

description: RNA-seq


title: RNA-seq mut06-1

description: featureCounts.txt [JB20]


title: PM149-T

description: RNA-seq


title: AL1589T

description: RNA-seq


title: PM148-T

description: RNA-seq


title: RNA-seq m19hBBz-2

description: featureCounts.txt [JB19]


title: RNA-seq m1928z-2

description: featureCounts.txt [JB15]


title: RNA-seq m1928z-1

description: featureCounts.txt [JB14]


title: ATAC-seq mut06-1


title: al-456-t

description: RNA-seq


title: AL423T_S11

description: RNA-seq


title: al-458-t

description: RNA-seq


title: AL453T

description: RNA-seq


title: ATAC-seq m19hBBz-2


title: PM510-Healthyliver

description: RNA-seq

source_name: Liver

value: normal liver

tag: age

value: 10-14 weeks


title: PM509-Healthyliver

description: RNA-seq


title: ATAC-seq m19hBBz-1


title: JAX210-myc-luc-bcat

description: RNA-seq


title: ATAC-seq m1928z-2


title: PM508

description: RNA-seq


title: RNA-seq m19hBBz-1

description: featureCounts.txt [JB17]


title: ATAC-seq m1928z-1


title: DD044P_1

first: Kenichi

last: Okuda

address: 125 Mason Farm Road

department: Department of Medicine, Cystic Fibrosis/Pulmonary Research and Treatment Center

institute: University of North Carolina at Chapel Hill

description: Sample 7

data_processing:
read 1 contains both a cell barcode and a molecular barcode (also known as a UMI); read 2 is aligned to the reference genome
The first 12 bases of the barcoded read contain the cell barcode; the next 8 bases containing the molecular barcode.
Raw sequences from the same library (sample) in separate lanes but sequenced in the same batch were pooled at mapping step, while raw reads from the same library sequenced at different batches were processed separately and combined during secondary analysis; merged after QC filtering during analysis
Drop-seq_tools-2.3.0
Genome_build: GRCh38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: Bronchiole

extract_protocol:
Freshly brush-biopsied human bronchial samples and micro-dissected bronchioles were incubated with Accutase solution for 30 min at 37°C. The Accutase-treated cells were centrifuged (600 g, 2 min, 4°C) and then incubated with 10 mL HBSS (Ca+, Mg+) buffer containing DNase I (0.1 mg/ml) and collagenase IV (1 mg/ml) for 10 min at 37°C. After incubation, collagenase IV was inactivated by adding 500 μL fetal bovine serum. Dissociated cells were filtered through a 40-μm cell strainer, centrifuged (600 g, 2 min, 4°C) and resuspended in PBS, adjusted to 105 cells/ml. Cell viability was examined by trypan blue dye exclusion.
single cell-based RNA libraries were prepared for sequencing using standard Drop-Seq library preparation protocols (Macosko EZ, et al., Cell 2015; 161: 1202-1214). The libraries were sequenced using HiSeq X with 150-bp paired-end sequencing.

value: Bronchiole

tag: collection

value: Microdissection

tag: age of donor

tag: Sex

value: Male

value: freshly isolated human cells


title: DD044P_2

description: Sample 7


title: ATAC-seq mut06-2

tag: strain


title: DD040P

description: Sample 6

value: Female


title: KOinGP_day21_rep1

value: CD8+ T cells

value: MOG-GP

value: Tox -/-


title: AL416CL_S5

description: RNA-seq

value: Hydrodynamic tail-vein injection method

value: Cell line

tag: passage

value: 8-10 passages


title: DD036P

description: Sample 5

tag: tissue


title: pm322

description: RNA-seq


title: pm221

description: RNA-seq


title: AL407CL_S8

description: RNA-seq

value: MYC;sg-Pten


title: 5282019_2

description: Sample 4

source_name: Bronchi

value: Bronchi

value: Bronchial brushing

tag: cell type


title: pm173-t_S20

description: RNA-seq


title: 5282019_1

description: Sample 4


title: AL396CL-resub

description: RNA-seq


title: pm118a-cl_S16

description: RNA-seq


title: N2a_Gep94_KD_rep3 [Affy_Grp94_7]

city: Niigata

first: Tomoyuki

last: Yamanaka

address: 1-757 Asahimachidori, Chuo-ku

department: Brain Research Institute

institute: Niigata University

hyb_protocol: Hybridization was performed according to the Affymetrix standard protocol.

scan_protocol: Arrays were scanned according to the Affymetrix standard protocol.

data_processing: Obtained microarray data were analyzed using an Agilent GeneSpring GX software to identify differentially expressed genes (DEGs) by gene knockdown. Genes with low expressions (less than 20th percentile) were excluded to reduce false positives.

label: biotin

label_protocol: Labeled cDNAs were prepared by Ambion WT Expression Kit and Affymetrix WT Terminal Labeling Kit according to the manufacture's protocol.

extract_protocol: Total RNAs were prepared from transfected neuro2a cells by Qiagen RNeasy kit according to the manufacture's protocol.

tag: cell line

value: N2a mouse neuroblastoma

tag: treatment

value: Grp94 knockdown


title: N2a_Gep94_KD_rep2 [Affy_Grp94_6]


title: N2a_Gep94_KD_rep4 [Affy_Grp94_8]


title: HuCCT1_shBEX2-2_2

city: Natori, Miyagi

first: Keiichi

last: Tamai

address: 1-47, Nodayama, Shiote, Medeshima

institute: Miyagi Cancer Center

description: BEX2-knock down cells

hyb_protocol: 600 ng of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65Â°C in a rotating Agilent hybridization oven.  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37Â°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (GE1_107_Sep09) to obtain background subtracted and spatially detrended Processed Signal intensities.  Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

source_name: HuCCT1 cells, transfected shBEX2-2 vector

label_protocol: 100 ng of total RNA was labeled using Low Input Quick Amp Labeling Kit

extract_protocol: RNA from HuCCT1 cells was purified using RNeasy Mini Kits (Qiagen, Canada)

tag: shRNA

value: BEX2 knock down


title: N2a_Gep94_KD_rep1 [Affy_Grp94_5]


title: N2a_Vector_KD_rep4 [Affy_Grp94_4]

value: Vector knockdown


title: HuCCT1_shBEX2-1_2

description: BEX2-knock down cells

source_name: HuCCT1 cells, transfected shBEX2-1 vector


title: HuCCT1_shBEX2-1_1

description: BEX2-knock down cells


title: HuCCT1_shBEX2-2_1

description: BEX2-knock down cells


title: AL609-T

description: RNA-seq

value: Tumor

value: advanced tumors


title: pm117a-cl_S15

description: RNA-seq


title: N2a_Vector_KD_rep3 [Affy_Grp94_3]


title: HuCCT1_shNontarget_1

description: control cells

source_name: HuCCT1 cells, transfected shNontarget vector

value: control


title: pm172-t_S19

description: RNA-seq

value: normal liver

value: 10-14 weeks


title: HuCCT1_shNontarget_2

description: control cells


title: N2a_Vector_KD_rep2 [Affy_Grp94_2]


title: HuCCT1_shControl_1

description: control cells

source_name: HuCCT1 cells, transfected shControl vector


title: 2252019_2

description: Sample 2

value: Male

value: freshly isolated human cells


title: AL417CL_S3

description: RNA-seq

value: Hydrodynamic tail-vein injection method


title: WTinWT_day63_rep3

tag: strain background

value: CD8+ T cells

tag: mouse recipient

value: WT

tag: weeks post-infection

tag: genotype of p14

value: wild type


title: 2252019_1

description: Sample 2

value: Bronchi


title: al-419b-cl-1_S7

description: RNA-seq

tag: protocol

tag: mutations

value: Cell line

value: 8-10 passages


title: N2a_Vector_KD_rep1 [Affy_Grp94_1]

value: N2a mouse neuroblastoma


title: 4292019

description: Sample 3

value: Bronchial brushing


title: 2042019_2

description: Sample 1

value: Female


title: 2252019_3

description:
Sample 2
processed data file for this sample is on GSM4876743.


title: AL382CL-resub-2

description: RNA-seq


title: al-421-c

description: RNA-seq


title: 2042019_1

description: Sample 1


title: AL614CL-resub-2

description: RNA-seq


title: RD cells, KnockDown_G9a3

city: Singapore

first: Reshma

last: Taneja

state: Singapore

address: 2 Medical Drive, MD9, National University of Singapore

department: Department of Physiology

country: Singapore

institute: National University of Singapore

data_processing:
the raw data ( FASTQ files) were filtered and mapped to Homo sapiens Hg19 using STAR software .Raw image data file from highthroughput sequencing (like illumina) was transformed to Sequence Reads by CASAVA base.
Differential expression analysis of two conditions/groups was performed using the DESeq2 R package.And the resulting P values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate.
Genome_build: Homo sapiens hg19
Supplementary_files_format_and_content: xlx and gtf file containing FPKM values, read count and log2 fold change for each sample is provided

source_name: RD ERMS cell line derived from biopsy specimen of a 7year old female with pelvic RMS

growth_protocol: 1.5X10^6 RD cells/well were plated in 6-well tissue cutlture dishes and placed in a 5% CO2 37 degree celcius incubator for 12 hours.

extract_protocol:
RNA was isolated using Trizol and checked for RNA purity(260/280 and 260/230 >1.8) and integrty(using agarose gel electrophoresis and Agilent 2100)
Then first strand cDNA is synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis is subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA is purified using AMPure XP beads. Remaining overhangs of the purified double- stranded cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization(1). In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments are purified with AMPure XP system (Beckman Coulter, Beverly, USA). Finally, the final library is gotten by PCR amplification and purification of PCR products by AMPure XP beads.

treatment_protocol: 12 hours after seeding , cells were transfected using lipofectamine RNAimax with control siRNA for the control group and siG9a for the other group.

value: RD ERMS

tag: passages

tag: genotype

value: G9a knockdown


title: AL609-CL

description: RNA-seq


title: RD cells, KnockDown_G9a2


title: RD cells, KnockDown_G9a1


title: N2a_YAYC_KD_rep2 [Affy_YAYC_4]

value: NFYA and NFYC knockdown


title: N2a_YAYC_KD_rep3 [Affy_YAYC_5]


title: N2a_Vector_KD_rep2 [Affy_YAYC_2]

value: Vector knockdown


title: RD cells, G9acontrol2

value: control


title: AL423B-CL_S14

description: RNA-seq


title: PAO1-2

city: seoul

first: Kyung Bae

last: Min

address: yonsei-ro 50-1

department: college of medicine

institute: Yonsei University

data_processing:
RNA quality was assessed by an Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using an ND-2000 Spectrophotometer (Thermo Inc., DE, USA). For control and testing RNAs, rRNA was removed using a Ribo-Zero Magnetic kit (Epicentre Inc., USA) from each 5 ug of total RNA. The construction of a library was performed using a SMARTer Stranded RNA-Seq Kit (Clontech Lab Inc., CA, USA) according to the manufacturer’s instructions. The rRNA depleted RNAs were used for the cDNA synthesis and shearing, following manufacturer’s instructions. Indexing was performed using the Illumina indexes 1–12. The enrichment step was carried out using PCR.
libraries were checked using the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the library quantification kit using a StepOne Real-Time PCR System (Life Technologies Inc., USA). High throughput sequencing was performed as paired-end 100 sequencing using HiSeq 2500 (Illumina Inc., USA). Bacterial-Seq reads were mapped using a Bowtie2 software tool to obtain the alignment file.
Differentially expressed genes were determined based on counts from unique and multiple alignments using EdgeR within R (R development Core Team, 2016) using Bioconductor (45). The alignment file also was used for assembling transcripts. The quantile normalization method was used for comparisons between samples. Gene classification was based on searches done by DAVID (http://david.abcc.ncifcrf.gov/).

organism: Pseudomonas aeruginosa

source_name: PAO_bacterial total RNA

growth_protocol: P. aeruginosa PAO1 was incubated for 2 h in 5 ml of LB with vigorous shaking at 37°C. Then, additional incubation was conducted for 1 h under the same condition with 150 𝜇M of Dkstatin-1 or Dkstatin-2 supplement.

extract_protocol:
Total RNA samples were extracted using an RNeasy mini kit (Qiagen), and total DNA was eliminated using RNase-free DNase set (Qiagen) following the manufacturer’s instructions. The extraction was performed as an experimental duplicate.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: none (control)


title: RD cells, G9acontrol3


title: PAO1-1


title: DKST-2-2

source_name: DKST-2_bacterial total RNA

value: 150 µM of Dkstatin-2 supplement


title: DKST-1-2

source_name: DKST-1_bacterial total RNA

value: 150 µM of Dkstatin-1 supplement


title: DKST-2-1


title: AL422CL_S10

description: RNA-seq

value: Hydrodynamic tail-vein injection method


title: MX26

city: Pullman

first: Michael

middle: K

last: Skinner

state: WA

address: Abelson 507

department: SBS

institute: WSU

description: prostate.dmr.results.csv.gz

data_processing:
Basic read quality was verified using summaries produced by FastQC. The raw reads were trimmed and filtered using Trimmomatic. The reads for each MeDIP sample were mapped to the Rnor 6.0 rat genome using Bowtie2 with default parameter options. The mapped read files were then converted to sorted BAM files using SAMtools.
To identify DMRs, the reference genome was broken into 1000 bp windows. Genomic windows with a mean of less than 10 mapped reads per sample were removed prior to further analysis. The MEDIPS R package was then used to calculate differential coverage between disease and non-disease sample groups. The edgeR p-value was used to determine the relative difference between the two groups for each genomic window.
Windows with an edgeR p-value less than an arbitrarily selected threshold were considered DMRs. The DMR edges were extended until no genomic window with a p-value less than 0.1 remained within 1000 bp of the DMR.
CpG density and other information was then calculated for the DMR based on the reference genome.
Genome_build: Rnor_6.0
Supplementary_files_format_and_content: The results of the edgeR analysis in CSV format. This includes raw read counts for each genomic window as well as the calculated p-value and other summary values.

organism: Rattus norvegicus

source_name: Sperm

growth_protocol: Female and male rats of an outbred strain Hsd:Sprague Dawley SD (Harlan) at 70 to 100 days of age were fed ad lib with a standard rat diet and ad lib tap water. The gestating female rats treated were designated as the F0 generation. F1- F3 generation control and  methoxychlor lineages were housed in the same room and racks with lighting, food and water. All experimental protocols for the procedures with rats were pre-approved by the Washington State University Animal Care and Use Committee (protocol IACUC # 6252).

extract_protocol:
For each sample, the epididymis was dissected free of fat and connective tissue, then, after cutting open the cauda, placed into 6 ml of phosphate buffer saline (PBS) for 20 minutes at room temperature. Further incubation at 4ºC will immobilize the sperm.  The tissue was then minced, the released sperm pelleted at 4ºC 3,000 x g for 10 min, then resuspended in NIM buffer and stored at -80ºC for further processing.   DNA protocol: While sperm are thawing, turn on the centrifuge to 4ºC.   1. Re-suspend sperm pellet with pestle (and pestle motor if available).   o If there is some liquid left with the pellet, there is no need to add any additional buffer  o If not add 100 ?l 1xPBS or NIM buffer  2. Take 25 to 50 ?l of the suspension (depending on how big the pellet is) and transfer it to a microfuge tube with 200 ?l 1xPBS.   o Mix  3. Sonicate suspension at 25% with probe sonicator for 5 seconds.  4. Spin at 12,000 rpm for 10 minutes at 4?C.  o While waiting, turn on heat plate to 65?C   5. Discard supernatant. Be careful – pellet is loose.  6. Wash with 200 ?l 1xPBS and spin as above.  o While waiting, retrieve DTT from freezer (make sure there is enough)    o Turn on the incubator  o Vortex DTT once it has thawed a bit  7. Resuspend pellet in 100 ?l 1xPBS, then continue with protocol below.  General:  1. Re-suspend tissue in 100 ?l PBS or NIM buffer, then   o add 820 ?l DNA extraction buffer   o add 50 ?l 0.1M DTT   o vortex briefly  o incubate at 65?C for 15 minutes   o Retrieve ProteinaseK from freezer  2. Add 50 ?l proteinase K (20 mg/ml) and   o incubate at 55?C for 2-3 hours under constant rotation  o use Eppendorf SafeLock tubes and wrap the tops with Parafilm  3. After incubation add 300 ?l of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A)  o mix (vortex) thoroughly and make sure that the solutions are mixed   o then incubate for 20 min on ice (or longer if needed)  4. Centrifuge at 13,500 g for 30 minutes at 4?C.  o Prepare 2 mL tubes  o Take glycoblue out of the freezer  5. Transfer 1 ml of the supernatant to a new 2 ml tube.   o add 2 ?l of glycoblue   o add 1 ml of cold 100 % isopropanol  6. Mix well by inverting tubes several times.  7. Incubate for 60 min at -20?C.  8. Centrifuge for 20 min at 13,500 g at 4?C.   9. Take off and discard supernatant without disturbing (blue) pellet.   10. Wash pellet 2-3 times with 70% cold ethanol.   o Add 500?l of 70% ethanol to each pellet and   o return to freezer for 20 minutes  11. Centrifuge for 10 min at 4?C at maximum speed (18,200 g)   o discard supernatant  12. Spin again briefly to collect residual ethanol at bottom of tube and remove as much liquid as possible with gel loading tip.  13. Air-dry at RT until pellet looks dry, but be careful not to over-dry.   o This will take about 5 minutes  14. Re-suspend in 100 ?l of nuclease free water or TE.  15. Measure DNA concentration using Nanodrop.   Methylated DNA immunoprecipitation protocol: Shear & Add Antibody to DNA  1. Sonicate purified genomic DNA using Covaris:  a. Dilute 6?g genomic DNA into 50 or 130 ?l TE buffer in the appropriate Covaris tube.  b. Set Covaris to desired program (300 or 500 bp program works well).  c. Run program for each tube  • While waiting, turn on the dry bath to 95ºC.   2. Run 10 ?l sonicated DNA & 5 ?l DNA ladder on 2% agarose gel to verify fragment size.  • While waiting for gel, clean up Covaris.   3. Measure volume of sonicated DNA after gel run.  4. Dilute sonicated DNA with TE buffer to 400 ?l (400 – 130 + 10 = 280 µL)  5. Heat-denature for 10min at 95?C, and   • immediately cool on (watery) ice for 5min  6. To the denatured sonicated DNA:  a. Add 100?l of cold 5X IP buffer   b. Add 5?g (volume depending on concentration) of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006). ALWAYS check antibody concentration. Diagenode sometimes changes it.  c. Incubate the DNA-antibody mixture overnight on a rotator at 4?C.  Rotate at a low enough speed to prevent significant foaming.  Bind Beads to DNA: Antibody Mixture  1. Pre-wash Magnetic Beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) as follows:  • Resuspend the beads in the vial (pipet up and down or rotate, do not vortex)  • Transfer desired volume (50 ?l per sample) to a centrifuge tube, add the same volume of Washing Buffer (at least 1 mL), and resuspend.  • Place the tube in a magnet for 1-2 minutes and discard supernatant.   • Resuspend beads in 1 ml of washing buffer and incubate for 1 minute on bench. Put tube on magnet for 1-2 minutes and discard supernatant.  • Remove the tube from the magnet and resuspend the washed beads in the same volume of 1xIP buffer as the initial volume of beads.  2. Add 50?l of beads to the 500?l of DNA-antibody mixture from step 2  3. Incubate 2h on a rotating platform at 4?C.  Wash DNA: Antibody: Beads Mixture & Add Proteinase K Mix  1. Wash beads three times with 1X IP buffer as follows:  • Place tube in magnet for 1-2 minutes and discard supernatant  • Remove tube from magnet  • Add 1ml cold 1X IP buffer  • Mix by inverting tube or gently vortexing (set Vortex to low).  • Incubate tube(s) for 1 minute on bench.  • Place tube in magnet for 1-2 minutes and discard supernatant  • Repeat twice for a total of 3 washes  2. Resuspend the beads in 250?l digestion buffer.  3. Add 3.5?l Proteinase K Mix (20mg/ml) to the resuspended beads.  4. Incubate 2-3 hours on a rotating platform at 55?C (make sure to seal lids so they don’t leak). Use USA Scientific SealRite tubes or Eppendorf Safe-Lock tubes and seal top with Parafilm.  Purify DNA  • Retrieve Glycoblue from freezer.   1. Remove Parafilm and add 250?l Phenol-Chloroform-Isoamylalcohol to each tube.    • Vortex for 30 sec and centrifuge at 14,000g for 5min at RT.  • Remove the aqueous supernatant and transfer to a fresh microcentrifuge tube.  2. Add 250?l chloroform to the supernatant from step 1.    • Vortex for 30sec and centrifuge at 14,000g for 5min at RT.   • Remove the aqueous supernatant and transfer to a fresh microcentrifuge tube.  3. Add 500 ?l ethanol  • Then add 20?l 5M NaCl   • Then add 2?l glyco-blue (20mg/ml)   • Vortex well   4. Precipitate in -20?C freezer for 1 hour to overnight.  5. Centrifuge at 14,000g for 20min at 4?C.  Carefully remove the supernatant, while not disturbing the pellet.  6. Wash the pellet with 500?l cold 75% ethanol. Leave in -20?C freezer for 15 min.  7. Centrifuge at 14,000g for 5min at 4?C.  Carefully remove the supernatant.  8. Completely dry samples.  9. Resuspend in 25?l H2O.    10. Measure the DNA concentration in Qubit with ssDNA kit and matching buffer.
MeDIP DNA was used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra DNA Library following the manufacturer’s protocol  and indexing each sample individually with NEBNext Multiplex Oligos for Illumina. NEBNext Ultra II Library Prep Kit for Illumina: ssDNA  • Start with 20 µl in PCR strips.   o ~40 ng of DNA  o Complete the volume to 20 µl   • Add 2.5 µL of diluted (1:10) random primer to each tube.   o for 10 samples, 27 µL 0.1x TE and 3 µL primers  o for 12 samples 36 µL 0.1x TE and 4 µL primers  o for 22 samples, 54 µL 0.1x TE and 6 µL primers  • Place strips in the thermocycler and use the Denaturation program. Immediately remove (as soon as time runs out on the program) and cool on watery ice following denaturation. Let tubes rest on watery ice for 5 minutes, then transfer to refrigerated block.   I. Second Strand cDNA Synthesis  Assemble the second strand cDNA synthesis reaction on ice by adding the following components to the 20 µl DNA.   Second Strand Synthesis Reaction  DNA          20 µL  ORANGE NEBNet 2nd Strand Synthesis Reaction Buffer  8 µL  ORANGE NEBNext 2nd Strand Synthesis Enzyme Mix 4 µL  Nuclease-free water      45.5 µL  Total Volume        ~70 µL  1. Keeping the tube on ice, mix thoroughly by pipetting t

treatment_protocol: Outbred Sprague Dawley gestating female rats (F0 generation) were administered an intraperitoneal dose of 200 mg/kg body weight of methoxychlor. These doses were administered at 90 days of age, during embryonic days 8-14 (E8-E14) of fetal gonadal sex determination. The F1 generation offspring was directly exposed as a fetus and F2 generation grand-offspring exposed as the germline in the F1 generation. These were each bred at 90 days of age within the lineage. The F3 generation great-grand-offspring is required to establish the transgenerational inheritance generation of ancestral exposure.

tag: measurement

value: MeDIP

tag: disease

value: prostate

tag: lineage

value: methoxychlor


title: N2a_Vector_KD_rep1 [Affy_YAYC_1]


title: PM149-CL

description: RNA-seq

tag: strain

value: Cell line

tag: passage


title: N2a_YAYC_KD_rep1 [Affy_YAYC_3]

tag: cell type


title: MX25

description: prostate.dmr.results.csv.gz


title: RD cells, G9acontrol1

tag: cell line


title: PM148-CL

description: RNA-seq

tag: tissue

value: 8-10 passages


title: AL1589CL

description: RNA-seq


title: MX24


title: MX29

description: kidney.dmr.results.csv.gz


title: HuCCT1_shControl_2

description: control cells

tag: shRNA


title: MX27

description: obesity.dmr.results.csv.gz

value: obesity


title: MX28

description: multiple.dmr.results.csv.gz

value: multiple disease


title: MX21

description:
multiple.dmr.results.csv.gz
kidney.dmr.results.csv.gz
prostate.dmr.results.csv.gz
obesity.dmr.results.csv.gz

value: no disease


title: MX22

value: testis


title: Input_rep3

city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

description:
spike normalized ChIP-Sequencing (ChIP-rx)
Input_H3_H3S10_rep3
Input_H3p3_rep3

data_processing:
Base calling perfomed using Illumina's FASTQ Generation software v1.0.0
ChIPseq reads were aligned with Bowtie1 to hg19 and to mm10 for the spike-in
human reads were normalized to the number of spike-in reads
metagenes were established with ngs.plot.r v2.63
read distribution was determined with betdools intersect v2.26.0, either over specified regions or over the whole genome in 100-nt-bins
Genome_build: hg19, mm10
Supplementary_files_format_and_content: begraph files were generated with bedtools genomecov

growth_protocol: S phase synchronized IMR-5 cells by double Thymidine block

extract_protocol:
For spike-in experiments (ChIP-Rx) 10% of fixed NIH-3T3 mouse cell lines were added before lysis. Cells were treated with 1% formaldehyde for 5 min at RT following 5 min of incubation with glycine. After cell lysis (5 mM PIPES pH 8.8, 5 mM KCl, 0.5% NP40), nuclei were resuspended in RIPA buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1% Triton-X-100, 0.1% deoxycholic acid (DOC), 0.1% SDS, 1 mM EDTA containing protease and phosphatase inhibitor cocktail) and DNA was fragmented to a size <500 bp using a Covaris M220 sonifier. Antibodies were bound to Protein A/G-Dynabeads® (Invitrogen) and immunoprecipitated. After extensive washing, chromatin was eluted with 1% SDS and crosslinking was reverted overnight. Chloroform/phenol extraction was used for DNA purification. Purified DNA was end-repaired, A-tailed, ligated to Illumina adaptors, size-selected (200 bp) and purified with a gel extraction kit. DNA fragments were amplified by 16 cycles of PCR and library size and amount of library was specified with the Fragment Analyzer (Agilent). The library was subjected to Illumina NextSeq 500 sequencing according to the manufacturer’s instructions. After base calling with Illumina’s FASTQ Generation software v1.0.0 (NextSeq 500 sequencing), high quality PF-clusters were selected for further analyses.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 16 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: 4 h treatment with 1 µM MLN8237 or 1 µM MK5108 or DMSO

value: neuroblastoma cells

tag: treatment

value: DMSO, MLN

tag: chip antibody


title: al458-c

description: RNA-seq


title: H3S10P_MLN_ChIP-Seq_rep3

description: spike normalized ChIP-Sequencing (ChIP-rx)

value: MLN

value: pH3S10 (rabbit, Abcam, ab177218)


title: MX20


title: H3S10P_DMSO_ChIP-Seq_rep3

value: DMSO


title: al-456-c

description: RNA-seq


title: H3.3_DMSO_ChIP-Seq_rep3

value: rabbit monoclonal H3.3 (EPR17899) Lot N° GR3263393-3)


title: MX17


title: MX15

description: multiple.dmr.results.csv.gz


title: MX14

description: multiple.dmr.results.csv.gz


title: MX18


title: H3.3_MLN_ChIP-Seq_rep3


title: 3A2A m6A-IP rep 3

city: Chicago

first: Allen

last: Zhu

state: Illinois

address: 929 E 57th St, Room E331

institute: University of Chicago

description: m6A IP poly A RNA

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were first mapped to the mycoplasma genome to check for contamination and remove any reads (parameter -k 7), then mapped to the mm10 whole genome using Hisat2 v2.1.0 with parameters -k 1
For m6A-seq, our lab's software packages RADAR (https://scottzijiezhang.github.io/RADARmanual/Mannual.html) and MeRIPtools (https://scottzijiezhang.github.io/MeRIPtoolsManual/index.html) were used to call peaks. QNB was used to perform differential methylation analysis.
For RNA-seq, we used the input samples only. DESeq2 was used to count reads for each gene and to perform differential expression analysis.
Genome_build: mm10
Supplementary_files_format_and_content: DEG-all.csv: DESeq2
Supplementary_files_format_and_content: allPeakResults.csv: MeRIPtools
Supplementary_files_format_and_content: m6A-diff-Me-Peaks.txt: QNB

source_name: Mouse Embryonic Stem Cells

growth_protocol: The R1 ESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts in ES medium containing DMEM supplemented with 15% FBS, 1 mM L-glutamine, 0.1 mM mercaptoethanol, 1% non-essential amino acid , 1% Pen/Strep, nucleosides 1,000 U /ml leukemia inhibitory factor, 3mM CHIR99021 and 1mM PD0325901.

extract_protocol:
Cells and RNA was harvested using Trizol reagent. Total RNA was extracted and underwent one round of poly-A Dynabeads purification (ThermoFisher). RNA was then fragmented, and Input saved. Fragmented RNA then underwent m6A-immunoprecipitation. Illumina TruSeq v2 RNA Sample Prep Kit (Cat#20020595) was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols
m6A MeRIP-seq

value: R1 cell line

value: embryonic stem cell

value: inner cell mass

value: Mettl3/Wtap KD; R-3A2A METTL3/WTAP


title: MX12

value: MeDIP

value: methoxychlor


title: MX23


title: 3A2A m6A-IP rep 2

description: m6A IP poly A RNA


title: H3_DMSO_ChIP-Seq_rep3

value: H3 (rabbit, Abcam, ab1791)


title: MX10

description: obesity.dmr.results.csv.gz

value: obesity


title: 3A2A m6A-IP rep 1

description: m6A IP poly A RNA


title: S2P_MK_ChIP-Seq_rep2

value: MK


title: MX7

description: obesity.dmr.results.csv.gz


title: MX13

value: multiple disease


title: MX11

description: kidney.dmr.results.csv.gz

value: kidney


title: H3_MLN_ChIP-Seq_rep3

value: neuroblastoma cells


title: MX8

description: obesity.dmr.results.csv.gz


title: MX5

description: multiple.dmr.results.csv.gz


title: A10_MK_ChIP-Seq_rep2

value: A10 Antibody, Santa Cruz Lot No:J2219


title: AL453CL-resub

description: RNA-seq

value: Hydrodynamic tail-vein injection method

value: Cell line

value: 8-10 passages


title: MX19

description: kidney.dmr.results.csv.gz


title: MX9

description: multiple.dmr.results.csv.gz


title: MX3

description: multiple.dmr.results.csv.gz


title: ZR751shLucif_rna_rep1

city: Nashville

first: Thomas

middle: Paul

last: Stricker

state: TN

address: 1161 21st Ave South, CC3309A

department: Pathology, Microbiology and Immunology

institute: Vanderbilt University

description:
RNAseq
3040-TPS-206
RNAseq_fpkm_table

data_processing:
FastQC v.0.11.4
trimming fastq-mcf, ea-utils, v1.1.2
alignmment Tophat (v2.0.13)
quantified using cufflinks (v2.2.1)
normalized using cuffnorm (v2.2.1)
Genome_build: hg19
Supplementary_files_format_and_content: RNAseq_fpkm_table

source_name: ZR75-1 cell line

growth_protocol: ZR751 cells (RRID CVCL_0588) were obtained from the Lannigan laboratory41 and grown in RPMI (Sigma Aldrich #R8758500ml) supplemented with 10% heat-inactivated FBS (Corning™ #35016CV), 0.002% insulin (Sigma Aldrich #11376497001) and 50 IU penicillin, 50 mg/mL streptomycin (Corning™ #MT30001CI). The cell culture incubator parameters were as follows: 37 ˚C, 95% relative humidity, and 5% CO2 concentration.

extract_protocol:
Cells were harvested at steady-state using the RNAEasy Kit (Qiagen 74104). ChIPs were performed for two biological replicates, for one experimental repetition. Cells were grown to 80% confluency, washed 3 times in ice-cold PBS (8g NaCl, 0.2g KCl, 1.44g Na2HP04, 0.24gKH2PO4, H2O up to 1L, adjusted to pH 7.4 with HCl) and then fixed for 10 minutes at room temperature using 7% formaldehyde (50ml cold PBS, 2.5ml formaldehyde Sigma Aldrich 252549), followed by quenching with 2.5 ml of 2.5M glycine (93.8g glycine Sigma Aldrich G7126 in 500ml H2O) for two minutes at room temperature. After aspirating and washing with 50 ml cold PBS, we lysed the cells using 20ml Farnham lysis buffer (5mM HEPES pH 8.0, 85mM KCl, 0.5% NP-40) and 400ul protease inhibitor cocktail (PIC, Roche 11873580001) in order to scrape the cells off (Corning™ 3008) into a 50ml conical tube (Corning 352098). These tubes were spun down at 2,000 rpm for five minutes at 4° Celsius.     Nuclei lysis buffer (50mM Tris-HCl ph8, 10mM EDTA pH8, 1% SDS), 1X PIC, and 10mM sodium butyrate (Sigma Aldrich B5887) were added to a concentration of ~2e7 cells per 400 ul and resuspended until homogenous. Chromatin was sonicated using a Covaris LE220 for 35 minutes, then centrifuged at max speed for 10 minutes at 4°C to obtain supernatant. Per 0.1 ml of supernatant, we diluted with 0.9ml ChIP dilution buffer (50mM Tris-HCl pH 8, 0.167M NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate), 0.5ml RIPA-150 (50mM Tris-HCl pH8, 0.15M NaCl, 1mM EDTA pH8, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), 28ul 50X PIC, and 14ul 1M sodium butyrate.      Anti-ERα (3ul/IP), anti-H3K4me1 (1ul/IP), and anti-SP1 (3ul/IP) were linked to 100ul/IP, 60ul/IP, and 100ul/IP magnetic anti-rabbit Dynabeads respectively with RIPA-150 up to 500ul for 6 hours at 4°C for 6 hours in low-bind tubes (Eppendorf Z666505), and then incubated with 150ug of chromatin overnight at 4°C. Immunoprecipitants were washed with RIPA-150, RIPA-500 (50mM Tris-HCl pH8, 0.5M NaCl, 1mM EDTA pH8, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate) twice, RIPA-LiCl (50mM Tris-HCl pH8, 1mM EDTA pH8, 1% Nonidet P-40, 0.7% sodium deoxycholate, 0.5M LiCl2) twice, and 1X TE Buffer pH8 (10mM Tris-HCl pH8, 1mM EDTA pH8) twice for 5 minutes each. Chromatin-IPs were eluted from the beads in 200 ul freshly made Direct Elution Buffer (10mM Tris-HCl pH8, 0.3M NaCl, 5mM EDTA pH8, 0.5% SDS), and then treated with 1ul 1mg/ml RNase A (Fisher Scientific FEREN0531) at 65°C with shaking for 4 hours to reverse crosslinks. This was followed by 3ul proteinase-K (Sigma-Aldrich 3115879001) overnight at 55°C. DNA was purified using phenol–chloroform extraction. Samples were transferred to a spun-down 2ml phase lock gel tube (Qiagen 129056) and an equal volume of phenol/chloroform/isoamyl alcohol (Sigma Aldrich P3803100ML) was added. This was spun at room temperature for 5 minutes at 14,000g, and the sample was moved to a new 1.5ml tube.  One tenth volume sodium acetate (Invitrogen AM9740), 1ul glycogen (Roche 10901393001), twice volume 100% ethanol (Sigma Aldrich E7023500ML) was added, and the samples were incubated at -80°C for 30 minutes. The sample was spun at max speed for 30 minutes at 4°C, and the supernatant was carefully aspirated. The pellet was washed with 1ml cold 70% ethanol, and spun at max speed for 30 minutes at 4°C. The supernatant was aspirated, and the spin was repeated a final time. The supernatant was removed, and pellet was allowed to dry. The pellet was then resuspended in 25ul elution buffer (Qiagen 19086) and subsequently quantified by Qubit 2.0 Fluorometer.
Standard Illumina ChIP-seq Library Kits (IP-202-1012, IP-202-1024) were used to build sequencing libraries for two biological replicates per condition for one experimental repetition, with inputs used as control. . RNA quality was assessed in VANTAGE via Invitrogen Qubit and Agilent BioAnalyzer and samples with RIN >7 were used. RNA libraries were generated with two biological replicates of 2 μg RNA using Illumina’s TruSeq Stranded Total RNA Sample Prep Kit (20020597).

treatment_protocol: Oligos to use for shRNA were designed and ordered from Sigma/Genosys at the Molecular Cell Biology Core at Vanderbilt. The oligos were annealed, phosphorylated, and ligated into pSuper42–44 for transformation into DH5α cells. QIAprep Spin Miniprep Kit (Qiagen 27104) was used to isolate the vector, which was transfected into ZR751 cells and assessed by qPCR for KD.  KDs that worked were then isolated with QIAprep Spin Miniprep Kit (Qiagen 27104), digested, and ligated into pLVTH45 (Addgene 12262) for transformation into STBL3 cells. A QIAGEN Plasmid Plus Maxi Kit (Qiagen 12963) isolated the pLVTH for transfection into 293T cells, from which lentivirus was collected. The oligo sequence used to silence MLL3 was 5° - CCGGCGCACCTTATAGTAAACAGTTCTCGAGAACTGTTTACTATAAGGTGCGTTTTT -3°, taken from The RNAi Consortium.46 Negative control Luciferase shRNA Control was donated by the Lannigan laboratory.47 Cells were stably transduced at 100,000 cells per well in a 6-well plate (Corning 3516) with 4 ul lentivirus, and subsequently flow sorted for GFP expression and propidium iodide (Sigma Aldrich P4864) staining after 3 days. qPCR was performed in technical triplicate to check shRNA KD 3 days after transduction. Experiments were performed in multiple, but early (<=10) passages of the stably transduced cell lines. The antibodies used for ChIP-seq were anti-ERα (Santa Cruz Biotechnology sc-543X), anti-H3K4me1 (Abcam ab8895), anti-SP1 (Abcam ab13370), and sheep anti-rabbit IgG Dynabeads M-280 (Invitrogen™ 11203D).

value: breast cancer cell line ZR75-1

value: shLucif/control


title: ZR751shMLL3_rna_rep2

description:
RNAseq
3040-TPS-214
RNAseq_fpkm_table


title: WT m6A-IP rep 3

description: m6A IP poly A RNA

value: R1 cell line

value: embryonic stem cell

value: Mettl3/Wtap KD; R-WT METTL3/WTAP


title: ZR751shMLL3_rna_rep1

description:
RNAseq
3040-TPS-213
RNAseq_fpkm_table


title: B5, HEK293, in vivo (SSII)

city: Groningen

first: Danny

last: Incarnato

state: Netherlands

address: Nijenborgh 7

department: Molecular Genetics

country: Netherlands

institute: University of Groningen

data_processing:
Library strategy: SHAPE-MaP
All the relevant data analysis steps have been conducted using RNA Framework v2.6.9 (PMID: 29893890; http://www.rnaframework.com). Reads pre-processing and mapping was performed using the rf-map module (parameters: -ctn -cmn 0 -cqo -cq5 20 -b2 -mp "--very-sensitive-local"). Reads were trimmed of terminal Ns and low-quality bases (Phred < 20). Reads with internal Ns were discarded. Mapping was performed using the “very-sensitive-local” preset of Bowtie2. The mutational signal was derived using the rf-count module (parameters: -m -rd), enabling the right re-alignment of deletions. Grid search of optimal slope/intercept pairs was performed using the rf-jackknife module (parameters: -rp “-md 600 -nlp” -x) and ViennaRNA package v2.4.11, by disallowing isolated base-pairs, setting the maximum base-pairing distance to 600 nt and enabling the relaxed structure comparison mode (a basepair i/j is considered to be correctly predicted if any of the following pairs exist in the reference structure: i/j; i-1/j; i+1/j; i/j-1; i/j+1).
Supplementary_files_format_and_content: Additional data files (RC/XML files for analysis with RNA Framework [http://www.rnaframework.com]) are available at: http://www.incarnatolab.com/datasets/2A3_Marinus_2020.php

source_name: Total RNA from HEK293 cells, in vivo treated with B5

extract_protocol:
Cells pellets were lysed by direct addition of 1 mL ice-cold QIAzol, followed by a brief incubation at 56°C for 5 min to completely dissolve cell aggregates. 200 µl of chloroform were added, followed by vigorous vortexing for 15 sec. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12,500 x g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 mL tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase at 37°C for 20 min.
Total RNA was first fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer [65 mM Tris-HCl pH 8.0; 95 mM KCl; 4 mM MgCl2], then purified with NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 2 µl (for SSII and HIV RT) or 2.5 µl (for TGIRT-III) NF H2O. Eluted RNA was supplemented with 0.5 µl 20 µM random hexamers and either 0.25 µl (for SSII) or 0.5 µl (for HIV RT and TGIRT-III) dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 5 µl. For HIV RT, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 40 mM MgCl2], 0.5 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 5 U HIV RT (Worthington Biochemical Corporation, cat. HIVRT). For TGIRT-III, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2], 0.25 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 50 U TGIRT-III RT (InGex, cat. TGIRT-50). For SSII, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 0.5 µl DTT 0.1 M, 0.25 µl MnCl2 120 mM, 5 U SUPERase•In™ RNase Inhibitor and 50 U SuperScript II RT (ThermoFisher Scientific, cat. 18064014). Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 37°C (for HIV RT), 2 h at 57°C (for TGIRT-III), or 3 h at 42°C (for SSII). SSII and HIV RT were heat-inactivated by incubating at 75°C for 20 min. As TGIRT-III tightly binds the cDNA-RNA complex, 0.5 µg of Proteinase K were added and reaction was incubated at 37°C for 20 min, after which 0.5 µl of a 1:2 dilution of protease inhibitor cocktail (Sigma Aldrich, cat. P9599) in water was added to stop the reaction. For SSII, the buffer exchange step was omitted. Instead, 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, cat. E6111L). Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), following manufacturer instructions.

treatment_protocol: HEK293 cells were grown in DMEM (4.5 g/L D-Glucose), supplemented with 10% heat-inactivated FBS, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 U/mL penicillin and 25 μg/mL streptomycin, at 37°C (5% CO2), to ~75% confluence. Cells were then covered with a thin layer of Trypsin-EDTA (0.25%) solution, incubated at room temperature for 1 min, then dissociated by pipetting up and down with complete medium. Cells were centrifuged at 180 x g for 1 min and medium discarded. Cells were then resuspended in 450 µl 1X PBS (pH 7.4) and 50 µl of each SHAPE compound were added, followed by gentle mixing by tapping the tube. Cells were allowed to react at 37°C for 15 min, with moderate shaking, after which 500 µl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 10,000 x g for 1 min (4°C) and supernatant discarded.


title: MX6

description: kidney.dmr.results.csv.gz

value: MeDIP