title: Converted old Rep2

city: Saarbrücken

first: Tobias

last: Fehlmann

address: Campus E2 1

country: Germany

institute: Saarland University

description:
single-nuclei RNA
Converted_counttable.txt.gz

data_processing:
Raw sequence reads for each sample and dataset were downsampled to 2x200 million PE reads in a PE read-sensitive manner using the seqtk toolkit (v1.3, parameters: sample -s100 <fwd_reads.fq.gz> 200000000) before mapping reads using the mapping software zUMIs (Parekh et al., 2018) (v2.5, relevant parameters: sequence_files: file1: base_definition: -BC(1-16) -UMI(17-27) file2: base_definition: -cDNA(1-91); filter_cutoffs: numb_bases:1 phred: 20 UMI_filter: num_baes: 1 phred: 20; barcodes: barcode_num: 40000 barcode_file: null automatic: yes BarcodeBinning: 1 nReadsperCell: 100 counting_opts: introns:yes downsampling: 0 strand: 0 Ham_Dist: 0 write_ham: no primaryHit: yes twoPass: yes read_layout: SE). We supplied the current version of the mm10 reference genome and gencode annotation (vM22) for the mapping. We targeted for each sample and dataset the top 40,000 cell barcodes, and counted reads in introns and exons, given the considerable amount of intronic reads in nuclear transcripts (following the official 10X guidelines). Cells/nuclei were called using the defaultDrops function (parameters: expected=7000, upper.quant:0.98, lower.prop=0.1).
Genome_build: GRCm38
Supplementary_files_format_and_content: The processed files contain for each library type the concatenated UMI count matrices obtained from the intron+exon matrices of the zUMIs pipeline.

source_name: Hippocampus

growth_protocol: Animals  Aged C57BL/6J male wild-type mice (18 months old) were obtained from the National Institute on Aging, and young C57BL/6J males (2 months of age) were purchased from The Jackson Laboratory. Mice were housed under a 12-h light–dark cycle in pathogen-free conditions in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health.    All animal procedures were approved by the VA Palo Alto Committee on Animal Research and the institutional administrative panel of laboratory animal care at Stanford University. Male mice were used for all experiments.

extract_protocol:
Nuclei were isolated as described previously in (Habib et al., 2017). Per replicate, we isolated nuclei from the flash-frozen, whole hippocampus of the right hemisphere. All reagents were placed on ice. A lab dish was placed on ice and frozen tissue was placed on the dish before being covered with 1 ml lysis buffer from the Nuclei EZ Prep Kit (Sigma-Aldrich, St. Louis, USA). Tissue was cut into 2 mm-small pieces and the tissue-buffer mix was transferred on a 2 ml tissue grinder placed on ice. 1 ml of fresh lysis buffer was used to rinse the lab dish and then loaded on the tissue grinder as well (2 ml total volume). Tissues were homogenized with 25 strokes with pastel A followed by 15 strokes with pastel B. Tissue homogenate was transferred to a fresh tube on ice, and the tissue grinder was rinsed with 2 ml of fresh lysis buffer, which were subsequently added to said tube (4 ml total volume), followed by 5 min incubation on ice. Nuclei were pelleted with 5 min centrifugation at 500g, before resuspending the pellet with 4 ml fresh lysis buffer. Following further 5 min centrifugation at 500g, lysis buffer was removed and the pellet washed with 4 ml PBS. Nuclei were pelleted and resuspended in 500 μl PBS containing 2 U/ml Protector RNase Inhibitor (Sigma-Aldrich, St. Louis, USA). Nuclei solution was parsed through a 40 μm mesh. Nuclei were counted and concentrations adjusted to ~1 mio nuclei/ml.
Reagents of the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10X Genomics, Pleasanton, USA) were thawed and prepared according to the manufacturer's protocol. Nuclei/master mix solution was adjusted to target 7,500 nuclei per sample and loaded on a standard Chromium Controller (10X Genomics, Pleasanton, USA), according to the manufacturer’s protocol. All reaction and quality control steps, including library construction (using Chromium Single Cell 3′ Library Construction Kit v3) were conducted according to the manufacturer’s protocol and with recommended reagents, consumables and instruments. Quality control of cDNA and libraries was conducted using a Bioanalyzer (Agilent, Santa Clara USA) at the Stanford Protein and Nucleic Acid Facility (http://pan.stanford.edu/index.html).    Native libraries: Illumina sequencing of 10X snRNA-seq libraries was performed by Novogene Co. Inc. (Sacramento, US; https://en.novogene.com/). Multiplexed libraries were sequenced with 2x150-bp paired-end (PE) reads in a single S4 lane on a Illumina Novaseq S4 (Illumina, San Diego, USA), targeting 215 million reads per library. Base calling, demultiplexing and generation of FastQ files was conducted by Novogene.    Converted libraries: Libraries generated using the Chromium 10X Single Cell 3’ (version 3) kit require a conversion step using the MGIEasy Universal Library Conversion kit (Part Number: 1000004155, MGI Tech Co., Ltd, Shenzhen, China) before DNA nanoballs (DNBs) can be generated. For each library, 50 ng of final 10X library product was further amplified using 5 cycles of PCR to incorporate a 5’ phosphorylation on the forward strand only. Purified PCR product was then denatured and mixed with a “splint” oligonucleotide that is homologous to both P5 and P7 adapter regions of the library to generate a circle. A ligase reaction was then performed to create a complete ssDNA circle of the forward strand. Then an exonuclease digest was performed to remove single stranded non-circularized DNA molecules and splint oligonucleotide. DNBs were then prepared using the MGI CoolMPS PE100 beta sets now available as commercial products (Part Number 1000016935, MGI Tech Co., Ltd, Shenzhen, China). Rolling circle replication time for DNB preparation was set to 25 min. DNBs were loaded onto DNBSEQ-G400 4-lane flowcell provided with the PE100 sequencing set by manual pipetting of DNBs mixed with loading buffer into flowcell lanes using the MGIDL-200H DNB loader as described in the CoolMPS High-throughput Sequencing Set User Manual provided with the kit. Sequencing primers in the MGI CoolMPS PE100 kit were replaced with the primers provided in the High-Throughput Pair-End Sequencing Primer Kit (App-A) (Part Number 1000004156, MGI Tech Co., Ltd, Shenzhen, China) according to the instructions described in the Primer Kit manual. Sequencing was performed with a customized program with 28 sequencing cycles (cell barcode) for the 1st strand and 98 cycles of cDNA followed by 8 cycles of the sample barcode reading for the 2nd strand.  Base calling and generation of FastQ files on the DNBSEQ-G400 was performed using the software release for CoolMPS (BasecallLite version_1.0.7.84).

tag: group

value: CoolMPS-compatible

value: 18 months (old)


title: TPC1-NC rep3 RNA-seq

status: Public on Nov 05 2020

type: SRA

city: Shenyang

first: Jiahui

last: Guo

address: No. 155 Nanjing Bei Street

country: China

institute: The First Hospital of China Medical University

library_source: transcriptomic

data_processing:
Illumina Casava1.8 software used for basecalling.
Hisat2 v2.0.5 was used for reads mapping to the reference genome
featureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene. FPKM was futher calculated to estimated the expression levels of genes.
Genome_build: hg_38
Supplementary_files_format_and_content: gene_fpkm.txt

molecule: total RNA

organism: Homo sapiens

source_name: thyroid cancer cells

growth_protocol: TPC1 cells were maintained in DMEM with 10% FPS

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

treatment_protocol: Cells were transfected with si-circ_0000839 or NC siRNAs for 60 hours.

tag: cell line

tag: cell type

value: papillary thyroid cancer

tag: treatment

value: siRNAs transfection with NC siRNAs

tag: genotype

value: control


title: Converted old Rep1

organism: Mus musculus

tag: age


title: scRNA_MEF_batch1

city: Beijing

first: Fan

state: Beijing

address: 1 Beichen West Road, Chaoyang District

department: The State Key Laboratory of Stem Cell and Reproductive Biology

institute: Institute of Zoology, Chinese Academy of Sciences

description:
barcodes_and_annotations.xlsx.gz
MEF_to_XEN_countMatrix.csv.gz

data_processing:
Single-cell data were cleaned up to remove cells in which a few kinds of spike-in RNA were detected and then normalized with the R package RUVSeq to remove batch effects. The mean value and dispersion were considered when variable genes were picked up for principal component analysis and t-distributed stochastic neighbor embedding. The transcriptional factor gene correlation network was constructed by calculating the correlations between gene pairs, and only edges with correlations less than –0.4 (negatively correlated) or greater than 0.4 (positively regulated) were retained.
Genome_build: mm9
Supplementary_files_format_and_content: MEF_to_XEN_countMatrix.csv.gz: comma-delimited text file of count matrix ; barcodes_and_annotations.xlsx.gz: Excel data file including sequences of barcodes used in scRNA-seq and annotations of cells.

molecule: polyA RNA

source_name: Chemically-induced mouse embryonic fibroblasts

growth_protocol: MEFs were cultured with high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% GlutaMAX, 1% nonessential amino-acids (NEAAs), 0.055 mM 2-mercaptoethanol, and 1% penicillin-streptomycin.

extract_protocol: Individual cell was manually picked after digestion, lysed and subjected to cDNA synthesis. Single-cell cDNA was then amplified and fragmented as published steps. The sequencing library was constructed and sequenced with paired-end 150-bp reads on an Illumina HiSeq X-Ten platform. Raw reads were firstly separated by cell barcodes, then trimmed, and aligned to the mm9 mouse transcriptome and de-duplicated by UMIs information as described previously.

treatment_protocol:
For XEN induction, the medium was changed to
XEN reprogramming medium the next day, and it was changed every 4 days for 12 days.

tag: strain


title: TA_D51_6 RNA-seq

city: New York

first: Zhaoning

last: Wang

state: New York

address: 701 West 168th Street Hammer 14 - 1410

department: Genetics and Development

country: USA

institute: Columbia University

description: biological replicate 3

data_processing:
Quality control of RNA-Seq data was performed using FastQC Tool (Version 0.11.4).
Sequencing reads were aligned to mouse GRCm38 (mm10) reference genome using HiSTAT2 (Version 2.0.4) with default settings and --rna-strandness F.
Aligned reads were counted using featurecount (Version 1.6.0) per gene ID.
Differential gene expression analysis was performed with the R package edgeR (Version 3.30.1) using the GLM approach. For each comparison, genes with more than 1 CPM (Count Per Million) in at least three samples were considered as expressed and were used for calculating normalization factor. Cutoff values of absolute fold change greater than 2.0 and false discovery rate less than 0.01 were used to select differentially expressed genes between sample group comparisons. Normalized gene CPM values were used to calculate RPKM (Reads Per Kilobase per Million mapped reads) values, which were then used for heatmap plotting.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files including raw count values and RPKM values for each sample, and differential expression analysis results from edgeR.

source_name: Tibialis Anterior (TA) muscle

growth_protocol: Animals were housed in a 12 h light/dark cycle in a temperature-controlled room in the Animal Research Center of UT Southwestern, with ad libitum access to water and food.

extract_protocol:
Mice were euthanized and TA muscles from WT and D51 mice were dissected. RNA was extracted using the RNeasy mini kit (QIAGEN, 74104) according to the manufacturer's protocol.
Stranded mRNA-Seq libraries were generated using the KAPA mRNA HyperPrep Kit (Roche, KK8580) following manufacturer’s protocol.

value: Delta 51

value: 1 month

tag: tissue

value: Tibialis Anterior (TA) muscle


title: Converted young Rep1

value: 2 months (young)


title: TA_D51 snRNA-seq

data_processing:
Library strategy: Single Cell RNA-Seq (Single Nucleus RNA-Seq)
The Cell Ranger Single-Cell Software Suit (10xGenomics) was used to perform sample demultiplexing, barcode processing and single-cell 3′ gene counting.
The cDNA reads were aligned to the mm10/GRCm38 premRNA reference genome to generated gene-barcode matrix.
R package Seurat was used for downstream quality filtering and data analysis. Potential cell doublets and low quality cells were removed from the data.
R package SoupX was used to identify and remove ambient RNA from the dataset. Original and SoupX strained feature-barcode matrices were provided as processed data.
Data were normalized and scaled, taken number of genes (nGene) and unique molecular/ identifiers (nUMI) into consideration.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text (txt) files of normalized gene expression value for nuclei analyzed; Tab separated value (tsv) files and matrix (mtx) files for filtered gene barcode matrices generated from CellRanger primary analysis from raw sequencing data.

extract_protocol:
Mice were euthanized and TA muscles were dissected. Nuclei were extracted from muscle, stained with Hoechst and purified using FACS.
Purified nuclei were used for single nucleus RNA-seq using 10xGenomics Chromium platform. snRNA-seq libraries were generated using Chromium Next GEM Single Cell 3’ Gene Expression v3.1 kit (10xGenomics) according to the manufacturer’s protocol.


title: WT mESCs - RCOR2 ChIP-seq rep2

city: Clayton

first: Monika

last: Mohenska

state: VIC

address: 15 Innovation Walk

country: Australia

institute: Monash University

library_source: genomic

data_processing:
TINC-seq data processing:    Raw FASTQ files were quality trimmed with Trimmomatic v0.36, parameters SE -phred 33 SLIDINGWINDOW:3:15 MINLEN:36. FASTQ files were then mapped to a GENCODE’s GRCm38 with default parameters. Following this, sambamba was used to convert the mapped .sam files into .bam files. The .bam files which were then sorted and indexed with sambamba using default parameters. Unlike conventional DNA-pull down techniques such as ChIP or genome wide sequencing experiments, TINC-seq targets a very small fraction of the genome (in theory only one region). Due to the low input fraction and the fact that mostly similar sequences should be pulled down, the method is prone to more noise than experiments which include a vast proportion of the genome. For this reason reads were then quality filtered for blacklisted regions as well as repeat masker regions. Peak calling was then performed with macs2 v2.1.1.20160309 on pooled (by macs2) TALE1 or TALE2 replicates over the input, with the --no-model parameter, reads of MAPQ>10 were considered, and a FDR cut-off of 0.01 was applied. HOMER’s (v3.2) mergePeaks function was applied to calculated overlapping peaks with the parameter -d given. Motif enrichment analysis was performed on peaks called for each TALE sample with meme v5.0.1. The bedtools2 v2.25.0, function getfasta was used to convert the enriched peak regions of each TALE into fasta files. These were then utilised for the motif enrichment analysis with the parameters -dna -revcomp -minw 8 -maxw18.
ChIP-seq data processing:    Data was first quality trimmed with Trimmomatic v0.36 (Bolger, Lohse, and Usadel 2014) with the parameters SE (single-end) or PE (paired-end) -phred 33 SLIDINGWINDOW:3:15 MINLEN:36. FASTtQ files were then mapped to a customized GENCODE’s GRCm38, which included the RCOR2-HA, and mCherry ORF sequences (Rcor2 (C-3xHA-tag; VectorBuilder), mCherry (ORF:IRES:mCherry:T2A:puromycin)) with Bowtie2 v2.3.4.1, with default mapping parameters. Sambamba was used to convert mapped .sam files into .bam files, then sort and index them with default parameters. Reads were then quality filtered for blacklisted regions, and reads with MAPQ>10 were retained using samtools v1.8 and GNU parallel. This was followed by peak calls macs2 v2.1.1.20160309, using the parameters --keep-dup 1 -g 1.87e9. Biological replicates of samples and negative controls were pooled with  macs2, and then using HOMER’s mergePeak function, peaks within 700bp of their peak centre were merged. HOMER’s mergePeak function was then used to determine all variations of co-occupancy between RCOR2 and published (OCT4, SOX2, NANOG, LSD1, NuRD complex, REST/CoREST complex and ESC enhancers) data sets using the parameter -d dist, followed by genomic annotation of all regions using HOMER’s annotatePeaks.pl function (default parameters). This provided the final RCOR2 ChIP-seq peak set.
ATAC-seq data processing:    Data was first quality trimmed with Trimmomatic v0.36, with the parameters PE -phred 33 SLIDINGWINDOW:3:15 MINLEN:36. FASTQ files were then mapped to GENCODE’s GRCm38 with Bowtie2 v2.3.4.1, with default mapping parameters. Sambamba was used to convert mapped .sam files into .bam files, and then sort and index them with default parameters. Reads were then quality filtered for blacklisted regions, and reads with MAPQ>10 were retained using samtools v1.8 and GNU parallel. No peak calling was required for the study.
RNA-seq data processing:    Raw RNA-seq reads in the form of FASTQ format were demultiplexed with sabre using parameters pe -c -u -m 1 -l 8 -n -a 1, for the multiplexing indexes. Trimmomatic v0.36 was ised for quality trimming, with the parameters SE -phred 33 SLIDINGWINDOW:3:15 MINLEN:36. FASTQ files were then mapped to a customized GENCODE’s GRCm38, which included the RCOR2-HA, and mCherry ORF sequences (Rcor2 (C-3xHA-tag; VectorBuilder), mCherry (ORF:IRES:mCherry:T2A:puromycin)), with STAR v2.5.2b, using the parameters --outSAMattributes All --alignIntronMax 1000000 --alignEndsType EndToEnd, samples were sorted with Sambamba, and default parameters. Read counts were then generated with Subread’s featureCount function, using default parameters.
bigWig files were generated using deeptools2. Library size normalised (CPM) BigWig files were generated for all samples with parameters -bs 1 --minMappingQuality 10 --exactsScaling --ignoreDuplicates. For TINC-seq and ChIP-seq samples, the step was followed by generation of mean BigWig samples between the replicates and removal of the input sample (TINC-seq) or negative control (ChIP-seq). The parameters used were --scaleFactors 1:1 -bs 1.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig; raw counts

molecule: genomic DNA

source_name: WT mESCs - RCOR2 ChIP-seq

growth_protocol: ESCs were cultured in gelatin coated dishes at 37°C, 5%  CO2 in ESC media (KnockOutTM DMEM (Life Technologies) containing 15% Fetal Bovine Serum (Thermo Scientific), GlutaMAX Supplement, MEM Non-Essential Amino Acids Solution , Penicillin-Streptomycin, 0.1% (v/v) 2-Mercaptoethanol, 1000 U/mL Leukemia Inhibitory Factor, and 2i (1mM PD0325901 (StemMACS) and 3mM CHIR99021 (StemMACS)).

extract_protocol:
Approximately 30 x 10^6 cells were used per ChIP. The samples were pre-cleared with 100 μL of Pierce Control Agarose Resin (Thermo Fisher Scientific) for 1 h  at 4 °C and immunoprecipitations were performed with 30 μL of Pierce Anti-HA Agarose (Thermo Fisher Scientific) per sample rotating overnight at 4 °C.
Libraries were prepared using the ThruPLEX DNA-seq 12S kit (Rubicon Genomics, Cat#R400429). After limited PCR amplification, libraries were purified using Agencourt AMPure XP beads (Beckman Coulter, Cat#A63880), according to the manufacturer’s recommendations. 75 bp paired end sequencing was performed on an Illumina HiSeq 3000.

tag: strain background

value: RCOR2-HA expressing

value: embryonic stem cells (ESCs)

tag: antibody


title: NET-seq: spt6-YW, 30C 2

city: Boston

first: Fred

last: Winston

state: MA

address: 77 Avenue Louis Pasteur, Room 239

department: Genetics

institute: Harvard Medical School

description:
full genotype name:
MATα RPB3-(FLAG)x3::NatMx spt6-YW ura3-52 his4-912δ lys2-128δ
NET-seq library from an spt6-YW strain shifted to 30°C for 80 minutes.

data_processing:
Library strategy: NET-seq
Removal of adapter sequences from the 3′ end of the read and 3′ quality trimming were performed using cutadapt (Martin, 2011). Reads were aligned to the S. cerevisiae genome using Tophat2 without a reference transcriptome (kim2013), and uniquely mapping alignments were selected using SAMtools (Li et al., 2009). Coverage of the 5′-most base of the read, corresponding to the 3′-most base of the nascent RNA and the active site of elongating RNA polymerase, was extracted using bedtools genomecov (Quinlan and Hall, 2010), and normalized to the total number of uniquely mapping alignments. The quality of raw, cleaned, non-aligning, and uniquely aligning reads was assessed using FastQC (Andrews, 2014).
Genome_build: S. cerevisiae R64-2-1, S. pombe ASM294v2
Supplementary_files_format_and_content: BigWigs of library-size normalized coverage for each strand.

organism: Saccharomyces cerevisiae

growth_protocol: Yeast cultures were grown in YPD at 30ºC or were shifted to growth at 37ºC for 80 minutes before further processing or collection of the cells, as described previously (Cheung et al., 2008)

extract_protocol:
DOI: 10.1038/nature09652
RNA was extracted as previously described (Churchman, 2011).

tag: rna population

value: nascent RNA


title: TA_WT snRNA-seq

value: WT


title: NET-seq: spt6-YW, 37C 1

description:
full genotype name:
MATα RPB3-(FLAG)x3::NatMx spt6-YW ura3-52 his4-912δ lys2-128δ
NET-seq library from an spt6-YW strain shifted to 37°C for 80 minutes.


title: Converted young Rep2


title: NET-seq: spt6-YW, 30C 1

source_name: NET-seq library from an spt6-YW strain shifted to 30°C for 80 minutes.


title: TA_D51_5 RNA-seq

description: biological replicate 2


title: TA_D51_4 RNA-seq

description: biological replicate 1


title: F1B: fatostatin_RNAseq

city: Los Angeles

first: Jessica

middle: Erin

last: Rexach

state: CA

address: 2506 Gonda Bldg, 695 Charles E. Young Dr. South

department: Neurology

institute: UCLA

data_processing:
STAR 2.7.2b alignment + mapping to transcriptome
rsubread 1.34.6 featureCounts gene quantification
filter genes without an assigned ensembl gene identifier
filter genes where 80% of the samples are not > 5
cqn 1.32.0 normalization using transcript lengths from biomaRt, with lengthMethod = "smooth"
Genome_build: GRCm38, GencodeM11
Supplementary_files_format_and_content: matrix of gene name x sample counts data in csv format

growth_protocol: Mouse primary embryonic microglial cultures were prepared from P1 C57BL/6J pups as previously described (Saura et al., 2003) and cultured in DMEM-F12 supplemented with 2% heat inactivated FBS, penicillin/streptomycin (100 U/mL) and beta-mercaptoethanol (50 microM) for 24 hours prior to drug treatment.

extract_protocol:
RNA was isolated from primary mouse microglia using NucleoSpin RNA Plus (Takara)
Lexogen QuantSeq 3' Forward kit

treatment_protocol: Fatostatin was purchased from Selleckchem.  Primary microglia were treated with fatotastatin (5μM, 72h)  or corresponding DMSO vehicle control as indicated.

value: primary microglia culture

tag: treatment time (hours)


title: TPC1-NC rep2 RNA-seq


title: NET-seq: wild type, 37C 1

description:
full genotype name:
MATα RPB3-(FLAG)x3::NatMx ura3-52 his4-912δ lys2-128δ
NET-seq library from a wild type strain shifted to 37°C for 80 minutes.

value: wild type


title: NET-seq: wild type, 37C 2

source_name: NET-seq library from a wild type strain shifted to 37°C for 80 minutes.


title: TPC1-NC rep1 RNA-seq


title: TA_WT_3 RNA-seq

description: biological replicate 3


title: scRNA_MEF_batch5


title: NET-seq: wild type, 30C 2

description:
full genotype name:
MATα RPB3-(FLAG)x3::NatMx ura3-52 his4-912δ lys2-128δ
NET-seq library from a wild type strain shifted to 30°C for 80 minutes.


title: TPC1-si-circ_0000839 rep2 RNA-seq

value: siRNAs transfection with si-circ_0000839

value: hsa_circ_0000839 silencing


title: TA_WT_2 RNA-seq

description: biological replicate 2


title: TA_WT_1 RNA-seq

description: biological replicate 1


title: NET-seq: wild type, 30C 1

source_name: NET-seq library from a wild type strain shifted to 30°C for 80 minutes.


title: Myo_GFP_4

status: Public on Nov 04 2020

city: Brisbane

first: James

last: Hudson

state: QLD

address: 300 Herston Rd

institute: QIMR Berghofer Medical Research Institute

description:
Ribo-depleted RNA
20200506_MIP56.BCATvsGFP_CPM
20200506_MIP56.BCATvsGFP
20200506_MIP56.BCATvsGFP_Countmatrix

data_processing:
FASTQ files were quality trimmed with Trimmomatic
Reads were mapped to hg38 or mm10 using RNA STAR
Count matrix was calculated using Htseq tools
EdgeR was used for all comparisons
Genome_build: GRCh38/hg38 or GRCm38/mm10

source_name: Enzyme-dissociated cells

extract_protocol:
Adult CD-1 (ICR) mice were anaesthetized with an i.p injection of ketamine (100 mg/kg) and Xylazil (12.5 mg/kg) 3 days post-surgery. Hearts were quickly dissected and washed in perfusion buffer (120.4 mmol/l NaCl, 14.7 mmol/l KCl, 0.6 mmol/l KH2PO4, 0.6 mmol/l Na2HPO4, 1.2 mmol/l MgSO4•7H2O, 4.6 mmol/l NaHCO3, 10 mmol/l Na-HEPES, 30 mmol/l taurine, 5.5 mmol/l glucose and 10 mM 2,3-Butanedione 2-monoxime). The aorta was then cannulated with a 21-gauge cannula, secured with 3-0 silk suture and perfused with 37◦C, oxygenated perfusion buffer using a Langendorff apparatus (4ml/min). Once all blood was washed from the heart, digestion buffer (200 µg/ml Liberase DH (Roche) in perfusion buffer) was passed through the heart for ~8 minutes (until the hearts appeared waxy in color and flaccid to the touch). During perfusion, atria and excess tissue was removed. After enzymatic digestion, hearts were minced with fine scissors into small pieces and triturated to release cells. Cell isolates were passed through a 100 µm cell strainer and centrifuged at 30xg for 3 minutes at room temperature. Supernatant (containing the non-myocyte cells) was collected and the myocyte pellet was washed with perfusion buffer and re-centrifuged. The supernatant was again collected and the purified myocyte pellet was lysed with 1 ml of Trizol (ThermoFisher). The non-myocyte supernatant was centrifuged at 500xg for 5 minutes and FACS was performed.
For enzymatically isolated cells, ribosomal RNA was depleted with Ribo Zero Gold (Illumina), RNA quality ascertained using a MultiNA bioanalyser (Shimadzu) and cDNA generated with SuperScript II Reverse Transcriptase (ThermoFisher). Libraries were created with TruSeq Stranded Total RNA kits (Illumina) and read with HiSeq SR Cluster v4 kit (Illumina) on a HiSeq 2500 sequencer. Each sample contained ~45 million 50-bp single-end reads.

treatment_protocol: 8-week-old adult CD-1 male mice were housed in standard conditions with ad libitum access to normal chow and water. The mice were anaesthetised in a stinger box with 2% isoflurane (Bayer), then intubated and ventilated with 0.25 L/min oxygen with a tidal volume of 250 μL and a respiration rate = 133 strokes/min (Minivent, Harvard Apparatus). Mice were positioned on a heated surgical mat to maintain body temperature. Once fully anaesthetised, the ventrolateral left chest was shaved, and a lateral thoracotomy was performed at the 4th intercostal space. The pericardium was separated using fine nosed tweezers. A 7-0 prolene suture was used to permanently ligate the left anterior descending coronary artery. MI was verified by observing blanching of the myocardium. A Hamilton syringe with a 30-gauge needle was used to inject 1x1011 AAV particles (i.e. caYAP, caBCAT or GFP control). Viruses were diluted in sterile PBS to a final injection volume of 20 μL for all injection groups; 4 separate injections of 5 μL were injected around the infarct penumbra. Following injection, the chest wall was closed using 4-0 silk suture and the overlying skin sutured with 6-0 prolene suture. The mice were supplied with a s.c. injection of buprenorphine (0.1 mg/kg) and carprofen (10 mg/kg) and allowed to recover. Starting from the day of the surgery, mice received BrdU injections (100 mg/kg, i.p.) every second day for 6 doses.

value: Cardiomyocyte

tag: Sex

value: male


title: Myo_GFP_3


title: Myo_BCAT_5

growth_protocol: Briefly, HES3 and H9 hESCs (WiCell) or hiPSC (Sendai-virus reprogrammed CD34+ cells, ATCC) were maintained as TypLE (ThermoFisher Scientific) passaged cultures using mTeSR-1 (Stem Cell Technologies)/Matrigel (Millipore). hPSCs were seeded at 2´104 cells/cm2 in Matrigel-coated flasks and cultured for 4 days using mTeSR-1. They were then differentiated into cardiac mesoderm using RPMI B27- medium (RPMI1640 GlutaMAX+ 2% B27 supplement without insulin, 200 μM L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penicillin/Streptomycin (all ThermoFisher Scientific) containing 5 ng/ml BMP-4 (RnD Systems), 9 ng/ml Activin A (RnD Systems), 5 ng/ml FGF-2 (RnD Systems) and 1 μM CHIR99021 (Stem Cell Technologies) with daily medium exchange for 3 days. Subsequently, they were specified into a cardiomyocyte/stromal cell mixture using RPMI B27- containing 5 μM IWP-4 (Stem Cell Technologies) followed by another 7 days of RPMI B27+ (RPMI1640 GlutaMAX + 2% B27 supplement with insulin, 200 μM L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate and 1% Penicillin/Streptomycin) with medium exchange every 2-3 days. The differentiated cells were then cultured in RPMI B27+ until digestion at 15 days using 0.2% collagenase type I (Sigma) in 20% fetal bovine serum (FBS) in PBS (with Ca2+ and Mg2+) for 60 min at 37°C, followed by 0.25% trypsin-EDTA for 10 min. The cells were filtered using a 100 mm mesh cell strainer (BD Biosciences), centrifuged at 300 x g for 3 min, and resuspended at the required density in CTRL medium: α-MEM GlutaMAX, 10% FBS, 200 μM L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate and 1% Penicillin/Streptomycin.

extract_protocol:
Adult CD-1 (ICR) mice were anaesthetized with an i.p injection of ketamine (100 mg/kg) and Xylazil (12.5 mg/kg) 3 days post-surgery. Hearts were quickly dissected and washed in perfusion buffer (120.4 mmol/l NaCl, 14.7 mmol/l KCl, 0.6 mmol/l KH2PO4, 0.6 mmol/l Na2HPO4, 1.2 mmol/l MgSO4•7H2O, 4.6 mmol/l NaHCO3, 10 mmol/l Na-HEPES, 30 mmol/l taurine, 5.5 mmol/l glucose and 10 mM 2,3-Butanedione 2-monoxime). The aorta was then cannulated with a 21-gauge cannula, secured with 3-0 silk suture and perfused with 37◦C, oxygenated perfusion buffer using a Langendorff apparatus (4ml/min). Once all blood was washed from the heart, digestion buffer (200 µg/ml Liberase DH (Roche) in perfusion buffer) was passed through the heart for ~8 minutes (until the hearts appeared waxy in color and flaccid to the touch). During perfusion, atria and excess tissue was removed. After enzymatic digestion, hearts were minced with fine scissors into small pieces and triturated to release cells. Cell isolates were passed through a 100 µm cell strainer and centrifuged at 30xg for 3 minutes at room temperature. Supernatant (containing the non-myocyte cells) was collected and the myocyte pellet was washed with perfusion buffer and re-centrifuged. The supernatant was again collected and the purified myocyte pellet was lysed with 1 ml of Trizol (ThermoFisher). The non-myocyte supernatant was centrifuged at 500xg for 5 minutes and FACS was performed.
For enzymatically isolated cells, ribosomal RNA was depleted with Ribo Zero Gold (Illumina), RNA quality ascertained using a MultiNA bioanalyser (Shimadzu) and cDNA generated with SuperScript II Reverse Transcriptase (ThermoFisher). Libraries were created with TruSeq Stranded Total RNA kits (Illumina) and read with HiSeq SR Cluster v4 kit (Illumina) on a HiSeq 4000 sequencer. Each sample contained ~45 million 50-bp single-end reads.


title: Myo_GFP_1


title: TPC1-si-circ_0000839 rep3 RNA-seq

value: papillary thyroid cancer


title: Myo_GFP_2


title: Myo_BCAT_3


title: NB4-ATRA-1

type: RNA

city: San Giovanni Rotondo (FG)

first: Fabrizio

last: Bianchi

state: Outside U.S. & Canada

address: Viale Padre Pio 7

country: Italy

institute: Fondazione IRCCS Casa Sollievo della Sofferenza

description: APL-derived NB4 cells line bears the t(15;17) translocation and expresses the fusion protein PML-RARα

hyb_protocol: Samples were hybridized over-night.

scan_protocol: Arrays were scanned using the Affymetrix GeneChip Hybridization Oven 640, Fluidic Station 450 and Scanner 3000 7G (Thermo Fisher Scientific) to generate the raw data files (.CEL files).

data_processing: Quality control and normalization of Affymetrix .CEL files were performed using TAC software (v4.0; Thermo Fisher Scientific) by performing “Gene level SST-RMA” summarization method with human genome version hg38 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.26/). Gene expression data were log2 transformed before analyses.

label: biotin

source_name: APL cell line, ATRA 1uM treated, 120h

label_protocol: RNA samples have been labeled for array hybridization according to manufacturer’s instruction (Affymetrix).

growth_protocol: NB4 cells were grown in RPMI-1640 (Corning, Corning, NY, USA) supplemented with 10% heat-inactivated FBS (Thermo Fisher Scientific, Santa Clara, CA, USA), 2 mM L-Glutamine (Corning), 100 μg/mL penicillin, and 100 μg/mL streptomycin (Corning). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2.

extract_protocol: Before RNA extraction, death cells were removed using Lympholyte® cell separation media (Cedarlane, Burlington, Canada). Briefly, NB4 were stratified on a density gradient centrifugation media (1:1 ratio) and centrifuged at 2000 rpm for 10 min without brake. Live cells were washed three times in complete growth media, and cell pellets were finally resuspended in Invitrogen™ TRIzol™ Reagent (0.75 mL of TRIzol™ Reagent per 0.25 mL of sample; ~ 7.5×106 cells; Thermo Fisher Scientific) and stored at -80 °C.

treatment_protocol: All the experiments were performed treating cells with 1 μM all-trans retinoic acid (ATRA; Merck KGaA, Darmstadt, Germany) for 120 h. As ATRA powder was dissolved in DMSO, this solvent was used as a control vehicle in untreated cells (final concentration <1%).

value: blood

tag: tumor type

value: Acute promyelocytic leukemia (APL)


title: MRI Control 1

city: Amherst

first: Gregory

middle: Alan

last: DeIulio

address: 240 Thatcher Road

department: Biochemistry and Molecular Biology

institute: Umass Amherst

data_processing:
Data was denovo assembeled using Trinity denovo assembler version 2.4.0, using default parameters
Data was mapped back to the assembly using bowtie version 1.0.0 and expression was called using RSEM version 1.2.29, using all default parameters
Supplementary_files_format_and_content: Data consists of gene ID and FPKM as called by RSEM including transcripts per million, expected count, length, effective length

organism: Ocimum basilicum

source_name: Uninfected Leaf

extract_protocol:
Four disks per true leaf were sampled from both genotypes at 12, 24 and 72 hpi and immediately flash frozen in liquid nitrogen. The water control leaves were harvested at 12 hpi only. Total RNA was extracted from freshly ground tissue using the Spectrum™ Plant Total RNA Kit (Sigma Aldrich).
RNA samples were used to generate sequence libraries using a library prep kit from the New England Biolabs (NEB #E7530).

treatment_protocol: A 1 mL subsample from the filtered inoculum was pipetted into an eppendorf tube and frozen at -80C to serve as pathogen control. The remaining inoculum was centrifuged at 1,000g for 1 min and diH20 decanted. The resulting sporangia pellet was resuspended in diH2O and inoculum adjusted to 1 x 105 sporangia/mL. Plants were inoculated at the 6-leaf (3 true leaf set) growth stage with approximately ~1 mL/leaf and plants were incubated at 100% relative humidity. MRI and SB22 were sprayed with sterile diH2O in triplicate to serve as the mock inoculated control.

tag: infection time

value: Control

tag: basil cultivar

value: Mirihani

value: Leaf Disc

tag: developmental stage

value: 6 leaves


title: SB22 Control 1

value: SB22, Rutgers University


title: SB22 Control 3


title: MRI Control 3


title: TPC1-si-circ_0000839 rep1 RNA-seq


title: MRI Control 2


title: Myo_BCAT_1


title: NB4-ATRA-2


title: NB4-DMSO-1

source_name: APL cell line, dmso treated, 120h


title: All_CellLines

city: Pittsburgh

first: Fangyuan

last: Chen

state: PA

address: 204 Craft Avenue

institute: University of Pittsburgh

data_processing:
Illumina NovaSeq6000 System used for basecalling to generate bcl files
FASTQ files were generated with cellanger(v 3.0.2) mkfastq from bcl files
Output files (including .bam) were generated with cellanger(v 3.0.2) count
Spliced and unspliced transcripts were respectively quantified with velocyto CLI (v0.17.17) run10X, using cellanger(v 3.0.2) count outputs, which generated .loom file readable with scvelo.read(), containing matrix of both spliced and unspliced RNA expression.
To derive scRNA_RawCounts.csv: cells and genes are filtered: 1.doublets were detected using scrublet and filtered out; 2. only cells with no fewer than 2000 genes, no fewer than 8,000 counts, no more than 100,000 counts and no more than 15% mitochondria reads percent are kept; 3. only genes expressing in no fewer than 2 cells are kept.
To derive _3000genes_logNormCounts.csv: from scRNA_RawCounts.csv, the top 3000 highly variable genes are selected using scvelo.pp.filter_genes_dispersion (flavor='seurat'); this filtered matrix is then normalized for spliced/unspliced transcripts respectively by dividing the sum of spliced/unspliced reads of all genes per cell, and calculated as log1p (natural logarithm (base e) of (1 + a number)).
Genome_build: GRCh38
Supplementary_files_format_and_content: scRNA_RawCounts.csv: comma-separated values (CSV) file including raw counts per gene per cell of the single cell library, after empirical filtering lowly expressed genes, low quality cells and potential doublets; scRNA_3000genes_logNormCounts.csv: comma-separated values (CSV) file including log normalized counts of top 3000 most variable genes per cell; scRNA_metaInfo.csv: comma-separated values (CSV) file including scRNA-seq library quality metrics, and annotations of cell type and cell cycle phase for each cell from computation.

source_name: Cell lines from American Type Culture Collection (ATCC) or Brita Jacobson Laboratory (University of Colorado)

growth_protocol: Each cell line was grown in corresponding regular media (supplementary table 1), in 37°C, with 95% air and  5% carbon dioxide.

extract_protocol:
Nine groups of cells, each with viability > 90% based upon Trypan Blue staining and Invitrogen automated cell counting, were fixed separately in 90% methanol at 4°C for 15 minutes before being equally mixed (around 1000 cells per group) and temporarily stored at -80°C. The cell suspension was rehydrated before submitting for library preparation.
The rehydrated cell suspension was processed using 10X Genomics 3' Chromium v3.0 platform at University of Pittsburgh Genomics Core

tag: cell line source tissue

value: breast, embryonic kidney

value: mixture of 8 cell lines


title: NB4-DMSO-2

tag: cell line


title: Myo_BCAT_2

tag: cell type


title: T15 angus cattle pituitary 30 months

city: Changchun

first: YuWen

last: Huang

state: Jilin

address: College of Animal Science

institute: Jilin University

data_processing:
Using sequencing-by-synthesis technology, the Illumina high-throughput sequencing platform（Illumina HiSeq2500,150bp）encodes and sequences cDNA libraries, generate a large amount of high-quality raw data. Filter Raw Data Only retain the reads with a double-ended pairing greater than 60bp after filtering, use the Perl script to process the adaptor sequence and Reads containing plot N, and delete the low-quality data to obtain pure data. Clean data in each sample can reach 6.30 GB, and most base quality scores reach or exceed Q30. Quality control of the paired-end reads on clean reads in this study yielded 129.18 GB of clean data. All downstream analyses were based on these clean data. Convert the original sequence into a clean read sequence. These clean read sequences are then mapped to the reference genome sequence. Here, we use a specific genome (Bos taurus reference genome, version UMD 3.1.1) as a reference for sequence alignment and subsequent analysis, allowing up to two base mismatches, and by using HISAT2 and StringTie to effectively compare, Assemble and evaluate Clean Data.
Based on the Hisat2 alignment of each sample read and the reference genome sequence, GATK software was used to identify single-base mismatches between the sequenced sample and the reference genome to identify potential single nucleotide polymorphism (SNP) sites. The SNP loci and the transformation type proportions were counted. The SNPs and Indel with SNPEff were annotated to obtain the alternative splicing results and corresponding expression level of each sample using ASprofile software. When performing the gene expression analysis, the number of mapped reads and the transcript length in each sample were standardized so that the number of fragments truly reflected the expression level of the transcript. StringTie was used as a high-flow algorithm, and the number of transcribed fragments per thousand bases of transcripts per thousand fragments (FPKM) was used to measure the transcription and indicate the gene or gene expression levels. DEseq was used for differential expression analysis between sample groups to obtain groups of genes that were differentially expressed between biological conditions. FDR<0.05 and fold changes ≥2 were considered differentially regulated in the control group.
Genome_build: Bos taurus reference genome, version UMD 3.1.1
Supplementary_files_format_and_content: Here, we use a specific genome (Bos taurus reference genome, version UMD 3.1.1) as a reference for sequence alignment and subsequent analysis, allowing up to two base mismatches, and by using HISAT2 and StringTie to effectively compare, Assemble and evaluate Clean Data.

organism: Bos taurus

source_name: pituitary

growth_protocol: Nine Angus cattle at the ages of 6, 18 and 30 months from the Changsha Institute of Animal Science were selected and divided into 3 groups according to age, with 3 cows per group. Six-month-old cows are freshly weaned calves; 18-month-old cows are sexually mature cows that meet the requirements for priming, and 30-month-old cows have completed producing their first litters. The selected cattle were healthy and disease-free, and their body weights and physiological states were similar in each group.

extract_protocol:
The collected tissue samples were processed by grinding with liquid nitrogen, and the total RNA in the tissue samples was extracted using TRIzol reagent. The concentration and integrity of the total RNA were determined using 1.5% (w/v) agarose gel electrophoresis. To avoid amplifying the RNA samples, the total RNA of each sample needed to be >4.0 μg, with an RNA integrity number of 8.0. The PolyATract mRNA separation system (Promega) was used to further isolate the poly-a-containing mRNA from each sample of total RNA.
Reverse transcriptase and random primers were used to convert RNA fragments into first-strand cDNA, then DNA polymerase I and RNase H were used to synthesize the second-strand cDNA. After the end repair and ligand ligation, the product was purified, and PCR amplification yielded the final cDNA library.

treatment_protocol: External observation and rectal touches before slaughter confirmed that all cows were not in heat. Before slaughter, electric shock caused stun, making it unconscious, and then cut off its common carotid artery to bleed, and immediately died. All animal protocols were carried out according to the guidance of the Animal Care Committee at Jilin University. All ethical assessments are carried out in accordance with the requirements of China's Laboratory Animal Management Regulations. The upper hypothalamus and pituitary glands of the cows were collected, frozen in liquid nitrogen until RNA isolation, and numbered according to age and tissue type.

value: 30 months

value: pituitary


title: T16 angus cattle hypothalamus 30 months

source_name: hypothalamus

value: hypothalamus


title: T14 angus cattle hypothalamus 30 months


title: T11 angus cattle pituitary 18 months

value: 18 months


title: T17 angus cattle pituitary 30 months


title: T13 angus cattle pituitary 30 months


title: T12 angus cattle hypothalamus 18 months


title: SB22 Control 2


title: T10 angus cattle hypothalamus 18 months


title: T08 angus cattle hypothalamus 18 months


title: T09 angus cattle pituitary 18 months


title: T04 angus cattle hypothalamus 6 months

value: 6 months


title: T06 angus cattle hypothalamus 6 months


title: T07 angus cattle pituitary 18 months


title: T03 angus cattle pituitary 6 months


title: T02 angus cattle hypothalamus 6 months


title: T18 angus cattle hypothalamus 30 months


title: daz3tree1 4 hour C2H4 RNAseq, rep1

city: Austin

first: Hong

last: Qiao

state: TEXAS

address: 2506 speedway, NMS5.324

department: Department of Molecular Biosciences

institute: The University of Texas at Austin

description: ethylene treated for 4 hours

data_processing:
Illumina Casava1.7 software used for basecalling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to TAIR10 whole genome using TopHat version 2.0.9 with default parameters
Differential expressed genes were calculated by Cuffdiff version 2.0.1
Genome_build: tair10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample, generated by Cuffdiff version 2.0.1

organism: Arabidopsis thaliana

source_name: whole seedling

growth_protocol: Arabidopsis seeds were  dark-grown at 24 ºC for 3 days

extract_protocol:
RNA was harvested using Trizol reagent from seedlings.
Libraries were prepared according to NEB instructions of the RNA Library Kit (NEB #E7420S/L)

treatment_protocol: Treated with air (hydrocarbon-free air (Zero grade air, AirGas)) or ethylene (hydrocarbon-free air with 10 parts per million  (ppm) ethylene) for 4 hours

value: whole seedling

tag: treatment

value: after ethylene treatment

value: daz3tree1 in COL background


title: daz3tree1 4 hour C2H4 RNAseq, rep2

description: ethylene treated for 4 hours

tag: genotype


title: daz3tree1 4 hour air RNAseq, rep2

description: air treated for 4 hours

tag: age

value: after air treatment


title: daz3tree1 4 hour air RNAseq, rep1

description: air treated for 4 hours


title: TREE1p::TREE1-GFP C2H4 rep1

description: ethylene treated for 4 hours

data_processing:
Illumina Casava1.7 software used for basecalling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to TAIR10 whole genome using bwa with default parameters
For each TREE1 binding in each condition,mapped reads were pooled across ChIP-seq replicates as described (Gjoneska, Pfenning et al.Nature, 2015)
Peaks significantly enriched in ChIP-seq tags were identified by Model-based Analysis for ChIP-Seq (MACS, version 1.4.2 20120305).
Genome_build: tair10
Supplementary_files_format_and_content: the bed and txt files are generated using MACS (version 1.4.2 20120305) software.

source_name: Shoot tissue of pTREE1:TREE-GFP seedling after ethylene treatment

extract_protocol:
TREE1 proteins were extracted from seedlings and TREE1-DNA complexes were isolated with antibody.
Libraries were prepared according to NEB instructions of the ChIP-seq Library Kit (NEB #E6240S/L)

value: 3 days

value: ethylene

value: Shoot tissue of seedling

tag: chip antibody

value: anti-green fluorescent protein (life technologies:A11122)


title: T01 angus cattle pituitary 6 months

tag: tissue


title: TREE1p::TREE1-GFP air input

description: air treated for 4 hours

source_name: Shoot tissue of pTREE1:TREE-GFP seedling after air treatment

value: air


title: TREE1p::TREE1-GFP C2H4 rep2

description: ethylene treated for 4 hours


title: TREE1p::TREE1-GFP C2H4 input

description: ethylene  treated for 4 hours


title: TREE1p::TREE1-GFP air

description: air treated for 4 hours


title: TREE1ox 4 hour air RNAseq, rep2

description: air treated for 4 hours

data_processing:
Illumina Casava1.7 software used for basecalling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to TAIR10 whole genome usingTopHat version 2.0.9 with default parameters
Differentially expressed genes were calculated by Cuffdiff version 2.0.1
Genome_build: tair10
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample, generated by Cuffdiff version 2.0.1. "Shoot" samples are re-analysis of: GSM2209777 and GSM2209778.

source_name: seedlings

value: Seedlings

tag: genetic background

value: COL


title: TREE1ox 4 hour air RNAseq, rep1

description: air treated for 4 hours


title: T05 angus cattle pituitary 6 months


title: Senescent_ESCs_4d_diff_rep4

city: Saint-Petersburg

first: Aleksandra

last: Borodkina

address: Tikhoretsky ave. 4

country: Russia

institute: Institute of Cytology RAS

data_processing:
Raw reads underwent quality filtering via the FilterByTile script from the BBtools package using the default options (version 38.75). The remaining reads were additionally filtered and trimmed with the use of trimFilter script from the FastqPuri package (version 1.0.7). In particular, trimming operation was applied for both ends of reads if they contained N’s or their quality was below the quality threshold set to 27, all reads shorter than 25 bases were discarded.
For transcript abundances estimating the salmon lightweight-mapping was applied (version 1.1.0). The mapping was performed in the selective alignment mode. The list of decoys was generated based on the Gencode human reference genome GRCh38.p13 (release 33) and used further for building the index on concatenated transcriptome and genome Gencode reference files (release 33) using k-mer size of 21. Mapping operations were run with additional flags --numBootstraps 30 --seqBias --gcBias —validateMappings.
Estimated gene counts, metadata and transcript ranges were loaded into R using tximeta (version 1.4.5) and summarized to a gene level. Resulting count matrix was filtered to contain rows having at least 10 estimated counts across all samples. Gene differential expression analysis and log fold change (LFC) estimation were computed using DESeq2 (version 1.26.0) with a design formula controlling for cells differentiation status. To strengthen DE analysis we correct LFC using combination of adaptive shrinkage estimator from the apeglm package (version 1.8.0) and specifying additional LFC threshold equal to 0.667. This testing produced s-values telling whether the LFC is greater in absolute value than the threshold (FSOS). Genes that had s-value smaller than 0.005 were defined as differentially expressed.
Genome_build: GRCh38.p13 (release 33)
Supplementary_files_format_and_content: «quants.tar.gz» archive contains salmon mapping results
Supplementary_files_format_and_content: «dec_PRL_project_env.Rdata.tar.gz» archive contains DESeqDataSet object with coldata, metadata and counts, and DESeqResults of DE testing obtained in the study

source_name: Endometrial stromal cells

growth_protocol: ESCs were isolated from desquamated endometrium.  Cells were cultured in DMEM/F12 (Gibco BRL, USA) at 37 ˚C in humidiﬁed incubator, containing 5 % CO2. Cultural media was supplemented with 10 % FBS (HyClone, USA), 1 % penicillin-streptomycin (Gibco BRL, USA) and 1 % glutamax (Gibco BRL, USA).

extract_protocol:
ExtractRNA reagent (Evrogen, Russia) was applied according to the manufacturer’s protocol.
CDNA was synthesized from total RNA using Mint-2 kit (Evrogen, Russia) according to the manufacturer’s protocol. The RNA libraries were prepared with the Qiaseq FX DNA Library kit (Qiagen, Germany). The whole transcriptome RNA sequencing was performed with the HiSeq 2500 sequencing platform (Illumina, San Diego, CA, USA) in the single-end mode and with a read length of 50 bp in a rapid run mode.

treatment_protocol: For senescence induction ESCs were exposed to 200 µM of H2O2 for 1 hour in serum-free media and cultured for 14 days for senescence stabilization in standard media, media was exchanged every third day. Decidualization induction  was performed when the cells reached 80 % density, the media was exchanged for serum-free media for the 24 h. The next day media was replaced by the fresh media containing 2 % of serum and 0.3 mM N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (Sigma-Aldrich, USA) , 10 nM β-Estradiol (Sigma-Aldrich, USA) and 1 µM medroxyprogesterone 17-acetate (Sigma-Aldrich, USA), such media was exchanged every second day.

value: Endometrial stromal cells

tag: senescence state

value: SEN

tag: day of differentiation


title: Senescent_ESCs_8d_diff_rep4


title: Senescent_ESCs_8d_diff_rep3


title: Senescent_ESCs_8d_diff_rep1


title: Senescent_ESCs_4d_diff_rep3


title: Senescent_ESCs_4d_diff_rep1


title: Senescent_ESCs_8d_diff_rep2


title: Senescent_ESCs_4d_diff_rep2


title: Senescent_ESCs_Non_diff_rep3


title: Senescent_ESCs_Non_diff_rep1


title: Senescent_ESCs_Non_diff_rep4


title: Senescent_ESCs_Non_diff_rep2


title: Young_ESCs_8d_diff_rep4

value: CTR


title: Young_ESCs_8d_diff_rep1


title: Young_ESCs_8d_diff_rep2


title: Young_ESCs_8d_diff_rep3


title: Young_ESCs_4d_diff_rep4


title: Young_ESCs_4d_diff_rep3


title: Young_ESCs_Non_diff_rep3


title: Young_ESCs_Non_diff_rep4


title: Young_ESCs_4d_diff_rep2


title: Young_ESCs_4d_diff_rep1


title: Young_ESCs_Non_diff_rep2


title: Chromatin-associated RNA-Seq siINTS1 CPSF73-AID IAA rep 1

city: Exeter

first: Steven

last: West

state: Devon

address: Stocker Rd

department: Living Systems Institute

country: United Kingdom

institute: University of Exeter

description:
single nucleotide bigwig coverage files were generated for total and strand separated reads
INTS1_siRNA_CPSF73_IAA_antisense.bw
INTS1_siRNA_CPSF73_IAA_sense.bw
INTS1_siRNA_CPSF73_IAA.bw

data_processing:
Adapters were removed from nuclear RNA-seq reads using Trim Galore! v0.4.4 (wrapper tool for Cutadapt v1.15).
Alignment of nuclear RNA-Seq reads with Hisat2 v2.1.0 to ensembl GRCh38.
Properly mapped single-end reads were extracted and indexed using samtools v1.4.1.
Replicates were merged using bamtools v2.4.0.
For stranded coverage analysis, sense and antisense reads were seperated from merged BAMs using samtools v1.4.1
Bigwig files of chromatin RNA-seq reads were generated from BAM files using the bamCoverage of Deeptools v3.0.2
Genome_build: GRCh38
Supplementary_files_format_and_content: Bigwig files of chromatin RNA-seq reads represent normalised read coverage (RPKM) at a single nucleotide resolution across the genome.

molecule: nuclear RNA

source_name: Chromatin-associated RNA-seq CPSF73-AID Doxycycline (DOX) inducible (TIR1) minus DOX plus auxin

growth_protocol: HCT116 cell lines were maintained in DMEM with 10% fetal bovine serum, pen/strep at 37 °C and 5% CO2.

extract_protocol:
Isolated nuclei were lysed into an insoluble chromatin pellet and soluble nucleoplamic supernatant. Chromatin-associated RNA was then purified from the insoluble chromatin pellet using Trizol reagent (see Eaton et al. 2018 for detailed method).
Isolated nuclei were lysed into an insoluble chromatin pellet and soluble nucleoplamic supernatant. Chromatin-associated RNA was then purified from the insoluble chromatin pellet using Trizol reagent (see Eaton et al. 2018 for detailed method).
Chromatin-associated RNA was depleted of ribosomal RNA using Illumina Ribo-Zero (Illumina #20020597). Library was constructed using Illumina TruSeq® Stranded Total RNA (Illumina #20020597).

treatment_protocol: The sample was transfected with a silencer select INTS1 siRNA 72h prior to collection in media without any pen/strep using Lipofectamine RNAiMAX (Life Technologies). Auxin was added to cell media at a final concentration of 500 µM  3 h  prior to collection.

tag: genotype/variation

value: Auxin treated:siINTS1 incubated nuclear RNA

tag: molecule subtype

value: Chromatin-associated RNA


title: Chromatin-associated RNA-Seq siINTS1 CPSF73-AID IAA rep 2


title: Chromatin-associated RNA-Seq siCONT CPSF73-AID IAA rep 2

description:
single nucleotide bigwig coverage files were generated for total and strand separated reads
csiRNA_CPSF73_IAA_antisense.bw
csiRNA_CPSF73_IAA_sense.bw
csiRNA_CPSF73_IAA.bw

treatment_protocol: The sample was transfected with a silencer select negative control siRNA 72h prior to collection in media without any pen/strep using Lipofectamine RNAiMAX (Life Technologies).  Auxin was added to cell media at a final concentration of 500 µM  3 h  prior to collection.

value: Auxin treated:siCONT incubated nuclear RNA


title: Chromatin-associated RNA-Seq siCONT CPSF73-AID IAA rep 1


title: H3K27ac_2

data_processing:
FASTQ files were quality trimmed with Trimmomatic (v 0.36.6)
Reads were then mapped with Bowtie2 (v 2.3.4.1) to GRCh37/hg19
Peaks were called with the callpeak function of MACS2 (v 2.1.1.0) with default parameters (q-value < 0.01). For H3K4me3 and H3K27ac files, broadpeaks were called using broad subcommand with --broad.cutoff= 0.1.
Replicate peaks were intersected using the intersect function of GenomicRanges (v1.34.0)
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Processed files are text files indicating peaks in bed format.

source_name: human cardiac 2D cultures

extract_protocol:
Cells were washed 2x with ice cold PBS and then scraped in 1mL PBS into a 1.5mL Eppendorf tube. Fixed cells were spun (200xg, 10 minutes) and treated with lysis buffer according to the MAGnify Chromatin Immunoprecipitation kit protocol (150 μL/3 million cells assuming 15x106 cells/flask). Chromatin was sheared using a Bioruptor UCD2000 sonicator with the following settings: 12x cycles of 30 seconds sonication with 90 seconds cooling. Chromatin was sonicated in 1.5 mL eppendorf tubes, and the volume of cell lysate in each tube during sonication was kept at 150 μL. Sheared chromatin in lysis buffer was stored at -80°C prior to ChIP. ChIP reactions were carried out with MAGnify Chromatin Immunoprecipitation System (ThermoFisher) reagents as per manufacturer’s instructions. Antibodies used in ChIP reactions were: 1 μg anti-TCF4 (Santa Cruz: sc-8631 X), 3 μL anti-H3K4me3 (Active Motif; 39159), 5 μg anti-H3K27ac (Active Motif; 39133).
ChIP-seq Libraries were created with TruSeq ChIP Library Preparation Kit (Illumina) with DNA size selected and quality controlled by Pippin Prep size selection (Sage Science). Libraries were read with HiSeq SR Cluster v4 kit (Illumina) on a HiSeq 2500 sequencer with 50-bp single-end reads.

treatment_protocol: 2D cardiac cells were treated for 24 hours with 5 µm CHIR.

value: 2D pluripotent stem cell derived cardiac cultures

tag: antibody

value: anti-H3K27ac (Active Motif; 39133)


title: H3K27ac_1


title: TCF7L2_1

value: anti-TCF4 (Santa Cruz:sc-8631 X)


title: Input_1


title: TCF7L2_2


title: H3k4me3_2

value: anti-H3K4me3 (Active Motif; 39159)


title: H3k4me3_1


title: Input_2


title: Nucleoplasmic RNA-Seq DIS3-AID minus auxin

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
NP_noIAA

data_processing:
RNA-seq: Adapters were removed from nuclear RNA-seq reads using Trim Galore! v0.4.4 (wrapper tool for Cutadapt v1.15).
RNA-seq: Alignment of nuclear RNA-Seq reads with Hisat2 v2.1.0 to ensembl GRCh38.
RNA-seq: Properly mapped single-end reads were extracted and indexed using samtools v1.4.1.
RNA-seq: Replicates were merged using bamtools v2.4.0.
RNA-seq: For stranded coverage analysis, sense and antisense reads were seperated from merged BAMs using samtools v1.4.1
RNA-seq: Bigwig files of nuclear RNA-seq reads were generated from BAM files using the bamCoverage of Deeptools v3.0.2
Genome_build: GRCh38
Supplementary_files_format_and_content: RNA-seq: Bigwig files of nuclear RNA-seq reads represent normalised read coverage (RPKM) at a single nucleotide resolution across the genome.
EPAP RNA-seq: Adapters were removed from nuclear RNA-seq reads using Trim Galore! V.0.6.5 (wrapper tool for Cutadapt v2.9).
EPAP RNA-seq: Poly(A) and poly(T) reads were manually selected and removed from adapter trimmed RNA-Seq reads using Trim Galore! V.0.6.5 (wrapper tool for Cutadapt v.2.9).
EPAP RNA-seq: Alignment of nuclear RNA-Seq reads with Hisat2 v2.1.0 to ensembl GRCh38.
EPAP RNA-seq: Properly mapped single-end reads were extracted and indexed using samtools v1.10
EPAP RNA-seq: For stranded coverage analysis, sense and antisense reads were seperated from merged BAMs using samtools v1.10
EPAP RNA-seq: Bigwig files of nuclear RNA-seq reads were generated from BAM files using the bamCoverage of Deeptools v3.4.2
Supplementary_files_format_and_content: EPAP RNA-seq: Bigwig files of nuclear RNA-seq reads represent normalised read coverage (RPKM) at a single nucleotide nucleotide across the genome.
ONT: Basecalling of raw Fast5 reads using guppy v2.3.7
ONT: Isolation and orientation of full-length reads using pychopper v2.0.2
ONT: Adapters removed from long-reads using porechop v0.2.4
ONT: Alignment of long ONT reads using minimap2 v2.16-r934-dirty; -a -k 15 -w 5 --splice -g 2000 -G 200k -A 1 -B 2 -O 2,32 -E 1,0 -C 9 -z 200 -u f --junc-bonus=9 --splice-flank=yes --no-long-join --secondary=no
ONT: Properly mapped ONT reads were extracted and indexed using samtools v1.10
ONT: Replicates were merged using bamtools v2.4.0.
ONT: For stranded coverage analysis, sense and antisense reads were seperated from merged BAMs using samtools v1.10
ONT: BigWig files of mapped ONT reads were generated from BAM files using the bamCoverage of Deeptools v3.4.2 with the 'BPM' option (equivalent to TPM for RNA-seq data).
Supplementary_files_format_and_content: ONT: Bigwig files of nuclear long-read ONT sequence represent normalised read coverage (TPM) of separate strands at a single nucleotide resolution across the genome.
mNET-Seq: Adapters were removed from nuclear RNA-seq reads using Trim Galore! v0.4.4 (wrapper tool for Cutadapt v.1.15).
mNET-Seq: Alignment with Hisat2 v2.1.0 to ensembl GRCh38. mNET-seq files used the paired-end parmameter '--fr' to select concordatly mapped pairs.
mNET-Seq: mNET-seq profiles were generated with the 3' most nucleotide of fragments (reverse complement of the 5' most nucleotide of the second read displayed with the strandedness of the first read) for concordantly mapped reads with the samflag pairs 99/147 and 83/163.
Supplementary_files_format_and_content: mNET-Seq: Final BAM files represent the mapped 3’ most read fragments at a single nucleotide resolution
ChIP-Seq: Adapters were removed from nuclear RNA-seq reads using Trim Galore! V.0.6.5 (wrapper tool for Cutadapt v2.9).
ChIP-Seq: Alignment of ChIP-Seq reads with Hisat2 v2.1.0 to ensembl GRCh38.
ChIP-Seq: Properly paired mapped paired-end reads were extracted and indexed using samtools v1.10
ChIP-Seq: Replicates were merged using bamtools v2.4.0.
ChIP-Seq: Bigwig files of ChIP-Seq reads were generated from BAM files using the bamCoverage of Deeptools v3.4.2
Supplementary_files_format_and_content: ChIP-Seq: Bigwig files of ChIP-Seq reads represent normalised read coverage (RPKM) at a single nucleotide nucleotide across the genome.

source_name: Nuclear RNA-seq DIS3-AID Sleeping Beauty (TIR1) minus auxin

growth_protocol: HCT116 cell lines were maintianed in DMEM with 10% fetal bovine serum, pen/strep at 37 °C and 5% CO2.

extract_protocol:
Isolated nuclei were lysed into an insoluble chromatin pellet and soluble nucleoplamic supernatant. Nucleoplasmic RNA was then purified using RNA phenol chloroform and ethanol precipitated (see Eaton et al. 2020 for detailed method).
Nucleoplasmic poly(A)+ RNA was selected, rRNA depleted using RiboCop rRNA Depletion Kit (Lexogen #037) and used to generate libraries using the QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina (Lexogen #016.24).

treatment_protocol: Control: Treated for 3 h with ethanol (absolute 1ul/ml media).

value: Control:auxin untreated nuclear RNA

value: Nucleoplasmic RNA


title: Young_ESCs_Non_diff_rep1


title: Nucleoplasmic EPAP labelled RNA-Seq DIS3-AID minus auxin

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
NP_noIAA_EPAP

source_name: Nuclear RNA-seq DIS3-AID Sleeping Beauty (TIR1) minus auxin EPAP

treatment_protocol: Control: Treated for 3 h with ethanol (absolute 1ul/ml media). After extraction, nucleoplasmic RNA was treated with EPAP (Life Technologies) for 30 minutes at 30°C

value: Control:auxin untreated EPAP labelled nuclear RNA


title: Chromatin-associated EPAP labelled RNA-Seq DIS3-AID plus auxin

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
Chr_IAA_EPAP

source_name: Chromatin-associated RNA-seq DIS3-AID Sleeping Beauty (TIR1) plus auxin EPAP

extract_protocol:
Isolated nuclei were lysed into an insoluble chromatin pellet and soluble nucleoplamic supernatant. Chromatin-associated RNA was then purified from the insoluble chromatin pellet using Trizol reagent (see Eaton et al. 2020 for detailed method).
Chromatin-associated poly(A)+ RNA was selected, rRNA depleted using RiboCop rRNA Depletion Kit (Lexogen #037) and used to generate libraries using the QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina (Lexogen #016.24).

treatment_protocol: Auxin was added to cell media at a final concentration of 500 µM  3 h  prior to collection. After extraction, chromatin-associated RNA was treated with EPAP (Life Technologies) for 30 minutes at 30°C

value: Auxin treated EPAP labelled nuclear RNA


title: Nuclear RNA-Seq INTS11-SMASh minus asunaprevir

description:
single nucleotide bigwig coverage files were generated for total reads and separate stranded reads
ints11_minus_ASN

extract_protocol:
Nuclear RNA was extracted from isolated nuclei using Trizol reagent.
Nuclear RNA was depleted of ribosomal RNA using Illumina Ribo-Zero Gold (Illumina #MRZE706). Library was constructed using Illumina TruSeq® Stranded Total RNA (Illumina #20020596)

treatment_protocol: Control: Treated for 48 h with DMSO.

value: Control:asunaprevir untreated nuclear RNA

value: Nuclear RNA


title: Nucleoplasmic EPAP labelled RNA-Seq DIS3-AID plus auxin

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
NP_IAA_EPAP

source_name: Nuclear RNA-seq DIS3-AID Sleeping Beauty (TIR1) plus auxin EPAP

treatment_protocol: Auxin was added to cell media at a final concentration of 500 µM  3 h  prior to collection. After extraction, nucleoplasmic RNA was treated with EPAP (Life Technologies) for 30 minutes at 30°C


title: Chromatin-associated RNA-Seq siCONT rep 2

description:
single nucleotide bigwig coverage files were generated for total reads and separate stranded reads
csiRNA_2

source_name: Chromatin-associated RNA-seq CPSF73-AID Doxycycline (DOX) inducible (TIR1) minus DOX minus auxin

extract_protocol:
Isolated nuclei were lysed into an insoluble chromatin pellet and soluble nucleoplamic supernatant. Chromatin-associated RNA was then purified from the insoluble chromatin pellet using Trizol reagent (see Eaton et al. 2018 for detailed method).
Chromatin-associated RNA was depleted of ribosomal RNA using Illumina Ribo-Zero (Illumina #20020597). Library was constructed using Illumina TruSeq® Stranded Total RNA (Illumina #20020597).

treatment_protocol: The sample was transfected with a silencer select negative control siRNA 72h prior to collection in media without any pen/strep using Lipofectamine RNAiMAX (Life Technologies). These samples were treated with 1ul/ml EtOH for 18h prior to extraction but this is not expected to have any effect.

value: CPSF73-AID_P2A_[HygromycinR/NeomycinR]_SV40-pA; AAVS1(TIR1; Puromycin). Note that AID-tagging CPSF73 is not expected to alter the behaviour of the cell line unless auxin treated

value: Control:auxin untreated:siCONT incubated nuclear RNA


title: Chromatin-associated EPAP labelled RNA-Seq DIS3-AID minus auxin

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
Chr_noIAA_EPAP

source_name: Chromatin-associated RNA-seq DIS3-AID Sleeping Beauty (TIR1) minus auxin EPAP

treatment_protocol: Control: Treated for 3 h with ethanol (absolute 1ul/ml media). After extraction, chromatin-associated RNA was treated with EPAP (Life Technologies) for 30 minutes at 30°C


title: Chromatin-associated RNA-Seq DIS3-AID minus auxin

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
Chr_noIAA

source_name: Chromatin-associated RNA-seq DIS3-AID Sleeping Beauty (TIR1) minus auxin


title: Chromatin-associated RNA-Seq siCONT rep 1

description:
single nucleotide bigwig coverage files were generated for total reads and separate stranded reads
csiRNA_1

treatment_protocol: The sample was transfected with a silencer select negative control siRNA 72h prior to collection in media without any pen/strep using Lipofectamine RNAiMAX (Life Technologies).  These samples were treated with 1ul/ml EtOH for 18h prior to extraction but this is not expected to have any effect.

value: CPSF73-AID_P2A_[HygromycinR/NeomycinR]_SV40-pA; AAVS1(TIR1; Puromycin).  Note that AID-tagging CPSF73 is not expected to alter the behaviour of the cell line unless auxin treated


title: 28851516_RNA

city: Daejeon

first: Jong Hwan

last: Kim

address: 125 Gwahak-ro, Yuseong-gu

department: Genome structure research center

country: South Korea

institute: Korea Research Institute of Bioscience and Biotechnology

description: patient 98 tumor tissue RNA_seq

data_processing:
Illumina Casava1.7 software used for basecalling.
The reads were mapped to hg19 whole genome using STAR v2.5.1.
Read counts were calculated using a STAR standard protocol.
Heat map was constructed using Mev (v. 4.9.0). Statistical analyses were performed using R (v. 3.3.0) and PYTHON (v. 2.7.6).
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include read count values for each sample

source_name: colorectal tumor tissue

extract_protocol:
Total RNA was isolated from the colorectal tumor tissues using the RNeasy RNA extraction Mini-Kit (Qiagen) and the quality of the RNA was confirmed by agarose gel electrophoresis.
TruSeq Stranded mRNA Sample Preparation Guide, Part #15031047 Rev. The sequencing library was prepared using the IlluminaTruSeq Stranded mRNA LT Sample Prep Kit (Illumina).
The mRNA derived from total RNA using poly-T oligo-attached magnetic beads was fragmented and converted into cDNA. Adapters were ligated to the cDNA and the fragments were amplified by PCR.

tag: patient number

value: patient 98

value: colorectal tumor


title: Chromatin-associated RNA-Seq DIS3-AID plus auxin

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
Chr_IAA

source_name: Chromatin-associated RNA-seq DIS3-AID Sleeping Beauty (TIR1) plus auxin

treatment_protocol: Auxin was added to cell media at a final concentration of 500 µM  3 h  prior to collection.

value: Auxin treated nuclear RNA

value: Chromatin-associated RNA


title: Nucleoplasmic RNA-Seq DIS3-AID plus auxin

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
NP_IAA

source_name: Nuclear RNA-seq DIS3-AID Sleeping Beauty (TIR1) plus auxin


title: Nuclear RNA ONT DIS3-AID plus auxin rep 2

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
dis3_2_auxin_GI; ONT MinION Enhanced; MinION Flowcell: R9.4.1; FLO-MIN106D

source_name: Nuclear RNA ONT cDNA-PCR Seq DIS3-AID Sleeping Beauty (TIR1) plus auxin

extract_protocol:
Nuclear RNA was extracted from isolated nuclei using Trizol reagent. PolyA+ RNA was then GI tailed using a Poly(A) Tail-Length Assay Kit (Thermo #764551KT)
GI-tailed nuclear RNA was selected by reverse transcription into cDNA using a custom designed VNP pimer. Libraries were then generated using the cDNA-PCR Sequencing kit (Oxford Nanopore Technologies #SQK-PCS109)

treatment_protocol: Auxin was added to cell media at a final concentration of 500 µM  3 h  prior to collection. Samples were GI-tailed prior to library construction.

tag: vnp rt-pcr primer


title: Chromatin-associated RNA-Seq siINTS1 rep 2

description:
single nucleotide bigwig coverage files were generated for total reads and separate stranded reads
INTS1_siRNA_2

treatment_protocol: The sample was transfected with a silencer select INTS1 siRNA 72h prior to collection in media without any pen/strep using Lipofectamine RNAiMAX (Life Technologies). These samples were treated with 1ul/ml EtOH for 18h prior to extraction but this is not expected to have any effect.

value: Control:auxin untreated:siINTS1 incubated nuclear RNA


title: Total mNET-Seq rep 2

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
mNET_noIAA_rep2

source_name: Total mNET-Seq FUS-AID Sleeping Beauty Doxycycline (DOX) inducible (TIR1)  minus auxin

extract_protocol:
Pol II associated RNAs were selected by IP of all forms of RNA Pol II CTD in solubilised chromatin (CMA601, MBL Technologies). A detailed description of the mNET-seq protocol can be found by Nojima, T., Gomes, T., Carmo-Fonseca, M., and Proudfoot, N.J. (2016). Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide. Nature Protocols 11, 413-428.
Libraries were produced from Pol II associated RNAs using NEBNEXT Small RNA Library Prep Set for Illumina (NEB #E7330S)

treatment_protocol: Untreated

value: FUS-AID_P2A_[HygromycinR/NeomycinR]_SV40-pA; AAVS1(TIR1; Puromycin).  Note that AID-tagging FUS is not expected to cause transcriptional differences in untreated samples like these.

value: Control:auxin untreated total CTD

value: Polymerase bound RNA


title: 36575736_RNA

description: patient 96 tumor tissue RNA_seq

value: patient 96


title: 13882828_RNA

description: patient 97 tumor tissue RNA_seq

value: patient 97


title: Nuclear RNA ONT DIS3-AID plus auxin rep 1

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
dis3_1_auxin_GI; ONT MinION Enhanced; MinION Flowcell: R9.4.1; FLO-MIN106D


title: Nuclear RNA ONT DIS3-AID minus auxin rep 2

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
dis3_2_minus_GI; ONT MinION Enhanced; MinION Flowcell: R9.4.1; FLO-MIN106D

source_name: Nuclear RNA ONT cDNA-PCR Seq DIS3-AID Sleeping Beauty (TIR1) minus auxin

treatment_protocol: Control: Treated for 3 h with ethanol (absolute 1ul per ml media). Samples were GI-tailed prior to library construction.

value: Control:auxin untreated Nuclear RNA


title: Total mNET-Seq rep 1

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
mNET_noIAA_rep1


title: 15370941_RNA

description: patient 95 tumor tissue RNA_seq

value: patient 95


title: Nuclear RNA ONT DIS3-AID minus auxin rep 1

description:
single nucleotide bigwig coverage files were generated for separate stranded reads
dis3_1_minus_GI; ONT MinION Enhanced; MinION Flowcell: R9.4.1; FLO-MIN106D

treatment_protocol: Control: Treated for 3 h with ethanol (absolute 1ul per ml media).  Samples were GI-tailed prior to library construction.


title: Chromatin-associated RNA-Seq siINTS1 rep 1

description:
single nucleotide bigwig coverage files were generated for total reads and separate stranded reads
INTS1_siRNA_1


title: Nuclear RNA-Seq INTS11-SMASh plus asunaprevir

description:
single nucleotide bigwig coverage files were generated for total reads and separate stranded reads
ints11_plus_ASN

treatment_protocol: Asunaprevir was added to cell media at a final concentration of 1 µM 48 h  prior to collection.

value: Asunaprevir treated nuclear RNA


title: 36536919_RNA

description: patient 91 tumor tissue RNA_seq

value: patient 91


title: VMZ.11_rep2

city: Seattle

first: Anneke

middle: Dixie

last: Kakebeen

state: WA

address: 5611 9th Ave NW

department: Biochemistry

institute: University of Washington

data_processing:
Trimming adaptors: Trim Galore!
psuedoalignment: kallisto quant
log2 normalized counts: DeSeq2::rlog
Genome_build: X.laevis transcriptome v9.2:
Supplementary_files_format_and_content: counts.csv: CSV file containing raw count output from kallisto for each sample
Supplementary_files_format_and_content: SupplementTable(090920).csv: CSV file containing DESeq2 rlog normalized counts for each sample including analysis metadata

organism: Xenopus laevis

source_name: VMZ

growth_protocol: Fertilized eggs were incubated in 1/3 MMR at 16°C until stage 10.5

extract_protocol:
When sibling embryos became stage 11, 11.5, 12, 12.5, 13, 14, 15, 16, 17, and 18, five or six DMZs were collected and dissolved in TRIzol Reagent for each time point. The time interval between the stages was approximately 30 minutes to an hour at room temperature. 20 animal caps and five VMZs were excised from stage 8 and 10.5, respectively. Animal caps and VMZs were dissolved in TRIzol when sibling embryos were at stage 11.
Standard illumina RNA-Seq library preparation at a core facility

treatment_protocol: 50 DMZ explants were isolated from stage 10.5 embryos using forceps, hair loop, and hair knife, then cultured in 1x Steinberg’s solution

tag: replicate

tag: Stage

value: Ventral Marginal Zone


title: DMZ.17_rep2

source_name: DMZ

value: Dorsal Marginal Zone


title: DMZ.18_rep2


title: Cap.11_rep2

source_name: Cap

value: Animal Cap


title: 36561326_RNA

description: patient 90 tumor tissue RNA_seq

value: patient 90


title: DMZ.16_rep2


title: DMZ.15_rep2


title: DMZ.13_rep2


title: 32466861_RNA

description: patient 87 tumor tissue RNA_seq

value: patient 87

value: colorectal tumor


title: 22329662_RNA

description: patient 86 tumor tissue RNA_seq

value: patient 86


title: DMZ.12_rep2


title: 36591682_RNA

description: patient 88 tumor tissue RNA_seq

value: patient 88


title: DMZ.12.5_rep2


title: 36528653_RNA

description: patient 84 tumor tissue RNA_seq

value: patient 84


title: 36418352_RNA

description: patient 85 tumor tissue RNA_seq

value: patient 85


title: DMZ.11.5_rep2


title: DMZ.11_rep2


title: VMZ.11_rep1


title: 36226407_RNA

description: patient 81 tumor tissue RNA_seq

value: patient 81


title: 36688548_RNA

description: patient 94 tumor tissue RNA_seq

value: patient 94


title: 19718956_RNA

description: patient 93 tumor tissue RNA_seq

value: patient 93


title: 33331470_RNA

description: patient 82 tumor tissue RNA_seq

value: patient 82


title: 36557389_RNA

description: patient 92 tumor tissue RNA_seq

value: patient 92


title: Cap.11_rep1


title: 36165160_RNA

description: patient 80 tumor tissue RNA_seq

value: patient 80


title: 36584172_RNA

description: patient 89 tumor tissue RNA_seq

value: patient 89


title: DMZ.14_rep2


title: 35962445_RNA

description: patient 78 tumor tissue RNA_seq

value: patient 78


title: 36365643_RNA

description: patient 83 tumor tissue RNA_seq

value: patient 83


title: DMZ.16_rep1

value: Dorsal Marginal Zone


title: 15694762_RNA

description: patient 79 tumor tissue RNA_seq

value: patient 79


title: 36115646_RNA

description: patient 77 tumor tissue RNA_seq

value: patient 77


title: DMZ.18_rep1


title: DMZ.17_rep1


title: DMZ.15_rep1


title: 36113789_RNA

description: patient 76 tumor tissue RNA_seq

value: patient 76

value: colorectal tumor


title: DMZ.14_rep1


title: DMZ.12_rep1


title: DMZ.13_rep1


title: DMZ.12.5_rep1


title: DMZ.11.5_rep1


title: U2OS_KRAB_HiC

first: Wei

last: Xie

address: Zhongguancun north street

institute: Tsinghua University

data_processing:
Basecalls performed using CASAVA version 1.8
For Hi-C sapples: sisHi-C sequencing reads were mapped, processed and iteratively corrected using HiC-Pro, a pipeline developed by Servant et al. Briefly, the read pairs were mapped to the hg19 reference genome in a two-step approach with bowtie2. Then the invalid read pairs including dangling end, self-circle ligation and duplicates were discarded. The genome was divided into bins of specific length to generate the contact maps. For global detection of contacts, a 100Kb bin size was used and a 40Kb bin size was used for examination of local domain level contacts. The raw contact counts are normalized with iterative correction.
For RNA-seq samples: RNA-seq reads were aligned to the hg19 genome assembly using tophat2 version 2.0.11and transcript abundance (FPKM) were calculated based on Refseq annotation using cufflinks version 2.0.2.
For ChIP-seq samples: The paired-end ChIP reads were aligned using Bowtie2. All unmapped reads, non-uniquely mapped reads, reads with low mapping quality (MAPQ < 20) and PCR duplicates were removed. For downstream analysis, we normalized the read counts by computing the numbers of reads per kilobase of bin per million of reads sequenced (RPKM) for 100-bp bins of the genome. To minimize the batch and cell type variation, RPKM values across whole genome were further Z-score normalized. To visualize the ChIP signals in the UCSC genome browser, we generated the RPKM values on a 100bp-window base.
Genome_build: hg19
Supplementary_files_format_and_content: The bedgraph files contains the values  counted by the number of reads falling into 200bp bin in the genome. The gene rpkm txt file contains FPKM value for all samples. with number for each bin. While the validpair txt file indicates the paired interaction reads for each sample.

source_name: U2OS cell line

growth_protocol: The U2OS (human bone osteosarcoma epithelial, female) cell were cultured in DMEM (Life Technologies) with high glucose in 10% FBS (fetal bovine serum, Life Technologies). All cells were cultured at 37°C and 5% CO2 in a humidified incubator.

extract_protocol: The sisHi-C library generation was performed as described previously (Du et al., 2017). Briefly, cells were fixed with 1% formaldehyde at room temperature (RT) for 10 min. Formaldehyde was quenched with glycine for 10 min at RT. Cells were washed with 1XPBS for two times and then lysed in 50ul lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 0.1 mM EDTA, 0.5% NP-40 and proteinase inhibitor) on ice for 50 min. After spinning at 3000 rpm/min in 4°C for 5 min, the supernatant was discarded with a pipette carefully. Chromatin was solubilized in 0.5% SDS and incubated at 62°C for 10 min. SDS was quenched by 10% Triton X-100 at 37°C for 30 min. Then the nuclei were digested with 50 U MboI at 37°C overnight with rotation. MboI was then inactivated at 62°C for 20 minutes. To fill in the biotin to the DNA, dATP, dGTP, dTTP, biotin-14-dCTP and Klenow were added to the solution and the reaction was carried out at 37°C for 1.5 hours with rotation. The fragments were ligated at RT for 6 hours with rotation. This was followed by reversal of crosslink and DNA purification. DNA was sheared to 300-500 bp with Covaris M220. The biotin-labeled DNA was then pulled down with 10ul Dynabeads MyOne Streptavidin C1 (Life Technology). Sequencing library preparation was performed on beads, including end-repair, dATP tailing and adaptor-ligation. DNA was eluted twice by adding 20ul water to the tube and incubation at 66°C for 20 minutes. 9-15 cycles of PCR amplification were performed with Extaq (Takara). Finally, size selection was done with AMPure XP beads and fragments ranging from 200bp to 1000bp were selected.

value: human bone osteosarcoma epithelial cell line (female)

value: transduced with PCP-GFP, sgRNA targeting to C19Q, dCas9-KRAB


title: U2OS_dCas9_HiC

value: transduced with PCP-GFP, sgRNA targeting to C19Q, dCas9


title: U2OS_KRAB_RNA

extract_protocol: 5 mg RNA was extracted using quick-RNA MiniPrep kit (Zymo) and then treated with DNase I (Fermentas) at 37°C for 1 hour. RNA was then purified using AMPure beads. Poly-A tailed mRNA was collected using DynabeadsTM mRNA purification kit (Invitrogen). Purified RNA was fragmented with RNA Fragmentation Buffer (NEB) at 95°C for 5 min. Reaction was stopped and RNA was purified by AMPure beads. First strand cDNA was synthesized with a commercial kit using both oligo dT and random primers (Invitrogen). Second strand cDNA was synthesized with second strand synthesis buffer (Invitrogen), MgCl2, DTT, dNTP, dUTP, RNase H (Fermentas), E. coli DNA ligase (NEB) and DNA polymerase I (NEB). DNA was purified after 2 hours incubation on thermomixer at 16°C. Purified DNA or cDNA was subjected to NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). The DNA was resuspended in 25 μL ddH2O, followed by end-repair/A-tailing with 3.5 μL End Prep buffer and 1.5 μL enzyme mix according to manufactory instruction. The ligation reaction was then performed by adding diluted 1.25 μL adaptors (NEB), 15 μL Ligation master mix, 0.5 μL Ligation enhancer, at 4°C for overnight. The ligation reaction was treated with 1.5μL USERTM enzyme according to the instruction and was purified by AMPure beads. The 1st round PCR was performed by adding 25 μL 2 × KAPA HiFi HotStart Ready Mix (KAPA biosystems) with primers of NEB Oligos kit, with the program of 98°C for 45 s, (98°C for 15 s and 60°C for 10 s) with 8-9 cycles and 72°C for 1 min. DNA was purified using 1xAMPure beads and a 2nd round PCR was performed with 25 μL 2xKAPA HiFi HotStart Ready Mix with Illumina universal primers, and same PCR program for 8 cycles. The final libraries were purified by 1xAMPure beads and subjected to next-generation sequencing. All libraries were sequenced on Illumina HiSeq 2500 or HiSeq XTen according to the manufacturer’s instruction.


title: U2OS_KRAB_H3K4me3

extract_protocol: Cells were collected and crosslinked by 1% formaldehyde. Cells were suspended in cell lysis buffer B (50 mM Tris-HCl PH 8.0, 20 mM EDTA, 0.3% SDS, freshly added protease inhibitor) for 10 min on ice before sonication. 6mg beads (Invitrogen) were washed wiPBS+5 mg/mL BSA for three times and incubated with 5µg antibody at 4°Cfor 6-8 hours before adding chromatin. Sonicated chromatin was diluted in dilution buffer (16.7mM Tris-HCl PH 8.0, 1.1% Triton X-100, 1.2 mM EDTA, 167mM NaCl, freshly added protease inhibitor), and was incubate with pre-treated beads and antibodies by rotation at 4°C overnight. Then beads were washed with washing buffer (50mM Hepes PH8.0, 1% NP-40, 0.7% DOC, 0.5 M LiCl, freshly added protease inhibitor) for 5 times followed by wash with TE once. 100µL elution buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS) was added and beads were incubated in Thermo mixer at 65°C for 30 min with max speed. Supernatant was collected and treated with protease K at 55°C for 2 hours, then purified with DNA purification kit (Tiangen). RNA was removed by treatment of RNase at 37°C for 1 hour. DNA was then purified by AMPure Beads (Beckman) and subjected to DNA library preparation. Purified DNA or cDNA was subjected to NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). The DNA was resuspended in 25 μL ddH2O, followed by end-repair/A-tailing with 3.5 μL End Prep buffer and 1.5 μL enzyme mix according to manufactory instruction. The ligation reaction was then performed by adding diluted 1.25 μL adaptors (NEB), 15 μL Ligation master mix, 0.5 μL Ligation enhancer, at 4°C for overnight. The ligation reaction was treated with 1.5μL USERTM enzyme according to the instruction and was purified by AMPure beads. The 1st round PCR was performed by adding 25 μL 2 × KAPA HiFi HotStart Ready Mix (KAPA biosystems) with primers of NEB Oligos kit, with the program of 98°C for 45 s, (98°C for 15 s and 60°C for 10 s) with 8-9 cycles and 72°C for 1 min. DNA was purified using 1xAMPure beads and a 2nd round PCR was performed with 25 μL 2xKAPA HiFi HotStart Ready Mix with Illumina universal primers, and same PCR program for 8 cycles. The final libraries were purified by 1xAMPure beads and subjected to next-generation sequencing.ng.


title: U2OS_dCas9_RNA


title: U2OS_dCas9_H3K27ac

extract_protocol: Cells were collected and crosslinked by 1% formaldehyde. Cells were suspended in cell lysis buffer B (50 mM Tris-HCl PH 8.0, 20 mM EDTA, 0.3% SDS, freshly added protease inhibitor) for 10 min on ice before sonication. 7mg beads (Invitrogen) were washed wiPBS+5 mg/mL BSA for three times and incubated with 5µg antibody at 4°Cfor 6-8 hours before adding chromatin. Sonicated chromatin was diluted in dilution buffer (16.7mM Tris-HCl PH 8.0, 1.1% Triton X-100, 1.2 mM EDTA, 167mM NaCl, freshly added protease inhibitor), and was incubate with pre-treated beads and antibodies by rotation at 4°C overnight. Then beads were washed with washing buffer (50mM Hepes PH8.0, 1% NP-40, 0.7% DOC, 0.5 M LiCl, freshly added protease inhibitor) for 5 times followed by wash with TE once. 100µL elution buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS) was added and beads were incubated in Thermo mixer at 65°C for 30 min with max speed. Supernatant was collected and treated with protease K at 55°C for 2 hours, then purified with DNA purification kit (Tiangen). RNA was removed by treatment of RNase at 37°C for 1 hour. DNA was then purified by AMPure Beads (Beckman) and subjected to DNA library preparation. Purified DNA or cDNA was subjected to NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). The DNA was resuspended in 25 μL ddH2O, followed by end-repair/A-tailing with 3.5 μL End Prep buffer and 1.5 μL enzyme mix according to manufactory instruction. The ligation reaction was then performed by adding diluted 1.25 μL adaptors (NEB), 15 μL Ligation master mix, 0.5 μL Ligation enhancer, at 4°C for overnight. The ligation reaction was treated with 1.5μL USERTM enzyme according to the instruction and was purified by AMPure beads. The 1st round PCR was performed by adding 25 μL 2 × KAPA HiFi HotStart Ready Mix (KAPA biosystems) with primers of NEB Oligos kit, with the program of 98°C for 45 s, (98°C for 15 s and 60°C for 10 s) with 8-9 cycles and 72°C for 1 min. DNA was purified using 1xAMPure beads and a 2nd round PCR was performed with 25 μL 2xKAPA HiFi HotStart Ready Mix with Illumina universal primers, and same PCR program for 8 cycles. The final libraries were purified by 1xAMPure beads and subjected to next-generation sequencing.ng.


title: U2OS_KRAB_H3K27ac

extract_protocol: Cells were collected and crosslinked by 1% formaldehyde. Cells were suspended in cell lysis buffer B (50 mM Tris-HCl PH 8.0, 20 mM EDTA, 0.3% SDS, freshly added protease inhibitor) for 10 min on ice before sonication. 8mg beads (Invitrogen) were washed wiPBS+5 mg/mL BSA for three times and incubated with 5µg antibody at 4°Cfor 6-8 hours before adding chromatin. Sonicated chromatin was diluted in dilution buffer (16.7mM Tris-HCl PH 8.0, 1.1% Triton X-100, 1.2 mM EDTA, 167mM NaCl, freshly added protease inhibitor), and was incubate with pre-treated beads and antibodies by rotation at 4°C overnight. Then beads were washed with washing buffer (50mM Hepes PH8.0, 1% NP-40, 0.7% DOC, 0.5 M LiCl, freshly added protease inhibitor) for 5 times followed by wash with TE once. 100µL elution buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS) was added and beads were incubated in Thermo mixer at 65°C for 30 min with max speed. Supernatant was collected and treated with protease K at 55°C for 2 hours, then purified with DNA purification kit (Tiangen). RNA was removed by treatment of RNase at 37°C for 1 hour. DNA was then purified by AMPure Beads (Beckman) and subjected to DNA library preparation. Purified DNA or cDNA was subjected to NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). The DNA was resuspended in 25 μL ddH2O, followed by end-repair/A-tailing with 3.5 μL End Prep buffer and 1.5 μL enzyme mix according to manufactory instruction. The ligation reaction was then performed by adding diluted 1.25 μL adaptors (NEB), 15 μL Ligation master mix, 0.5 μL Ligation enhancer, at 4°C for overnight. The ligation reaction was treated with 1.5μL USERTM enzyme according to the instruction and was purified by AMPure beads. The 1st round PCR was performed by adding 25 μL 2 × KAPA HiFi HotStart Ready Mix (KAPA biosystems) with primers of NEB Oligos kit, with the program of 98°C for 45 s, (98°C for 15 s and 60°C for 10 s) with 8-9 cycles and 72°C for 1 min. DNA was purified using 1xAMPure beads and a 2nd round PCR was performed with 25 μL 2xKAPA HiFi HotStart Ready Mix with Illumina universal primers, and same PCR program for 8 cycles. The final libraries were purified by 1xAMPure beads and subjected to next-generation sequencing.ng.


title: DMZ.11_rep1


title: U2OS_WT_H3K9me3

extract_protocol: Cells were collected and crosslinked by 1% formaldehyde. Cells were suspended in cell lysis buffer B (50 mM Tris-HCl PH 8.0, 20 mM EDTA, 0.3% SDS, freshly added protease inhibitor) for 10 min on ice before sonication. 1mg beads (Invitrogen) were washed with PBS+5 mg/mL BSA for three times and incubated with 5µg antibody at 4°Cfor 6-8 hours before adding chromatin. Sonicated chromatin was diluted in dilution buffer (16.7mM Tris-HCl PH 8.0, 1.1% Triton X-100, 1.2 mM EDTA, 167mM NaCl, freshly added protease inhibitor), and was incubate with pre-treated beads and antibodies by rotation at 4°C overnight. Then beads were washed with washing buffer (50mM Hepes PH8.0, 1% NP-40, 0.7% DOC, 0.5 M LiCl, freshly added protease inhibitor) for 5 times followed by wash with TE once. 100µL elution buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS) was added and beads were incubated in Thermo mixer at 65°C for 30 min with max speed. Supernatant was collected and treated with protease K at 55°C for 2 hours, then purified with DNA purification kit (Tiangen). RNA was removed by treatment of RNase at 37°C for 1 hour. DNA was then purified by AMPure Beads (Beckman) and subjected to DNA library preparation. Purified DNA or cDNA was subjected to NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). The DNA was resuspended in 25 μL ddH2O, followed by end-repair/A-tailing with 3.5 μL End Prep buffer and 1.5 μL enzyme mix according to manufactory instruction. The ligation reaction was then performed by adding diluted 1.25 μL adaptors (NEB), 15 μL Ligation master mix, 0.5 μL Ligation enhancer, at 4°C for overnight. The ligation reaction was treated with 1.5μL USERTM enzyme according to the instruction and was purified by AMPure beads. The 1st round PCR was performed by adding 25 μL 2 × KAPA HiFi HotStart Ready Mix (KAPA biosystems) with primers of NEB Oligos kit, with the program of 98°C for 45 s, (98°C for 15 s and 60°C for 10 s) with 8-9 cycles and 72°C for 1 min. DNA was purified using 1xAMPure beads and a 2nd round PCR was performed with 25 μL 2xKAPA HiFi HotStart Ready Mix with Illumina universal primers, and same PCR program for 8 cycles. The final libraries were purified by 1xAMPure beads and subjected to next-generation sequencing.

value: wide-type


title: U2OS_dCas9_H3K4me3

extract_protocol: Cells were collected and crosslinked by 1% formaldehyde. Cells were suspended in cell lysis buffer B (50 mM Tris-HCl PH 8.0, 20 mM EDTA, 0.3% SDS, freshly added protease inhibitor) for 10 min on ice before sonication. 5mg beads (Invitrogen) were washed wiPBS+5 mg/mL BSA for three times and incubated with 5µg antibody at 4°Cfor 6-8 hours before adding chromatin. Sonicated chromatin was diluted in dilution buffer (16.7mM Tris-HCl PH 8.0, 1.1% Triton X-100, 1.2 mM EDTA, 167mM NaCl, freshly added protease inhibitor), and was incubate with pre-treated beads and antibodies by rotation at 4°C overnight. Then beads were washed with washing buffer (50mM Hepes PH8.0, 1% NP-40, 0.7% DOC, 0.5 M LiCl, freshly added protease inhibitor) for 5 times followed by wash with TE once. 100µL elution buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS) was added and beads were incubated in Thermo mixer at 65°C for 30 min with max speed. Supernatant was collected and treated with protease K at 55°C for 2 hours, then purified with DNA purification kit (Tiangen). RNA was removed by treatment of RNase at 37°C for 1 hour. DNA was then purified by AMPure Beads (Beckman) and subjected to DNA library preparation. Purified DNA or cDNA was subjected to NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). The DNA was resuspended in 25 μL ddH2O, followed by end-repair/A-tailing with 3.5 μL End Prep buffer and 1.5 μL enzyme mix according to manufactory instruction. The ligation reaction was then performed by adding diluted 1.25 μL adaptors (NEB), 15 μL Ligation master mix, 0.5 μL Ligation enhancer, at 4°C for overnight. The ligation reaction was treated with 1.5μL USERTM enzyme according to the instruction and was purified by AMPure beads. The 1st round PCR was performed by adding 25 μL 2 × KAPA HiFi HotStart Ready Mix (KAPA biosystems) with primers of NEB Oligos kit, with the program of 98°C for 45 s, (98°C for 15 s and 60°C for 10 s) with 8-9 cycles and 72°C for 1 min. DNA was purified using 1xAMPure beads and a 2nd round PCR was performed with 25 μL 2xKAPA HiFi HotStart Ready Mix with Illumina universal primers, and same PCR program for 8 cycles. The final libraries were purified by 1xAMPure beads and subjected to next-generation sequencing.ng.