title: Nile rat Female 16 weeks

status: Public on May 01 2022

city: Zhunan, Miaoli County

first: Ben-Yang

last: Liao

address: 35 Keyan Rd.

department: Institute of Population Health Sciences

country: Taiwan

institute: National Health Research Institutes

data_processing:
Illumina Real Time Analysis 3 (RTA3) software was used for basecalling
The bases with low quality and sequences from adapters in raw data were removed using program fastp (v.0.22.0)
Cleaned paired-end reads of FASTQ files derived from various tissue samples were analyzed using kallisto (v.0.46.1)
The kallisto index was built with default parameters from the reference transcriptome file for quantification in each organism.
kallisto computed the transcript abundances as TPMs (Transcripts Per Kilobase Million). The TPM value of the longest transcript was assigned to each gene.
genome build: GRCm39
genome build: mArvNil1.pat.X
processed data files format and content: tab-delimited text files include Ensembl Gene ID and normalized TPM values for each sample.

organism: Arvicanthis niloticus

source_name: Olfactory epithelium

extract_protocol:
The olfactory epithelium was dissected out from the nasal cavity for each sample. The total RNA was isolated by TRIzol Reagent and subsequently purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany). The quantity and purity of the isolated RNA were measured by NanoDrop ND-1000 Spectrophotometer before cDNA library construction.
The cDNA libearies were constructed by TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). Briefly, mRNA was purified from total RNA (1 μg) by oligo(dT)-coupled magnetic beads and fragmented into small pieces under elevated temperature. The first-strand cDNA was synthesized using reverse transcriptase and random primers. After the generation of double-strand cDNA and adenylation on 3’ ends of DNA fragments, the adaptors were ligated, and the samples were purified with AMPure XP system (Beckman Coulter, Beverly, USA). The quality of the libraries was assessed on the Agilent Bioanalyzer 2100 system and a Real-Time PCR system.

value: olfactory epithelium

value: 16 weeks


title: Nile rat Male 16 weeks

type: SRA

library_source: transcriptomic

molecule: total RNA

tag: tissue

tag: Sex

value: male

tag: age


title: Nile rat Female 12 weeks

value: female

value: 12 weeks


title: Mouse Female 8 weeks

organism: Mus musculus

tag: strain

value: 8 weeks


title: WT AF tissue 4

status: Public on Jul 06 2022

type: RNA

city: Philadelphia

first: Makarand

middle: V

last: Risbud

state: PA

address: 1025 Walnut Street

department: Orthopaedic Surgery

country: USA

institute: Thomas Jefferson University

description: Total RNA from WT AF tissue; sst-rma normalization; DABG p<0.05; p<0.05; FC 2.0

hyb_protocol: Affymetrix Gene chips, Mouse Clariom S, were hybridized with 2.5 μg fragmented and biotin-labeled cDNA in 100 μl of hybridization cocktail.

scan_protocol: Array scanning was performed on an Affymetrix GeneChip Scanner 3000 7G using Command Console Software (Affymetrix)

data_processing: Raw data were processed and .CHP files were generated by sst-rma normalization from Affymetrix .CEL files, and quality control of the expeirment was performed in Transcriptome Analysis Console (TAC) v4.0.2 (Affymetrix). The experimental group was compared to the control group in the TAC, including all probe sets where at least 50% of the samples had a DABG (detected above background) p < 0.05. Inclusion cutoffs were defined at a 2.00-fold change and p-value < 0.05.

label: biotin

source_name: WT AF

label_protocol: Single-stranded cDNA was generated, then fragmented and biotin-labeled using the GeneChip WT Plus kit according to ABI protocol (Thermo Fisher Scientific).

growth_protocol: Mice wildtype (WT) or heterozygous (N153S) for Sting1em1Jmin were obtained at 12 weeks-of-age from the Jackson Laboratory and aged to 6 months-of-age at Thomas Jefferson University.

extract_protocol: NP and AF tissues were micro-dissected from caudal and lumbar intervertebral discs of 4 WT control and 4 N153S mice and immediately placed in RNAlater® Reagent (INVITROGEN). Tissues were homogenized with a Pellet Pestle Motor (Sigma Aldrich, Z359971) and RNA was extracted from tissue lysates using RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions.

value: Annulus Fibrosus

value: 6 months

tag: genotype

value: WT


title: N153S AF tissue 4

description: Total RNA from N153S AF tissue; sst-rma normalization; DABG p<0.05; p<0.05; FC 2.0

source_name: N153S AF


title: Nile rat Female 8 weeks


title: P106

status: Public on Apr 29 2022

city: Stockholm

first: Kristina

last: Broliden

state: Solna

address: Visionsgatan 4, J7:20

department: Dept. of Medicine Solna

country: Sweden

institute: Karolinska Institutet

data_processing:
Base calling and demultiplexing of the Bcl2 files was performed using the bcl2fastq program.
Indexing of the human reference genome (hg38/GRCh38) was done using STAR (Spliced Transcripts Alignment to a Reference).
The resulting fastq files were aligned and uniquely mapped reads were counted and mapped to annotated exons using data from UCSC (University of Santa Cruz) genome browser (http://genome.ucsc.edu/) as reference.
Read counts were normalized to counts per million (CPM) and log2 scale with the edgeR package in Bioconductor.
Genome_build: hg38/GRCh38
Supplementary_files_format_and_content: Comma seperated files with raw gene counts and TMM-CPM-log2 normalized values respectively for each gene in each sample
Supplementary_files_format_and_content: Comma seperated file with metadata,

molecule: polyA RNA

organism: Homo sapiens

source_name: Ectocervix

extract_protocol:
The ectocervical biopsies that were stored in RNA-later were thawed and RNA was isolated and purified using AllPrep DNA/RNA Mini Kit (QIAGEN, Hilden, Germany) and the QIAcube Connect (QIAGEN). Poly-A containing mRNA transcripts were purified, fragmented and converted to cDNA. The cDNA fragments were amplified, barcoded and sequenced using NextSeq 550 (Illumina, San Diego, CA, USA). All ectocervical biopsies displayed good quality of isolated RNA and generated >10 million reads each.
We used the low sample (LS) library prep workflow from the TruSeq Stranded mRNA Reference Guide (1000000040498 v00). The samples were split into six seperate runs and sequenced with single end 75 bp reads

tag: design

value: Control

tag: subjectid


title: P105


title: P086


title: N153S AF tissue 3


title: N153S NP tissue 4

description: Total RNA from N153S NP tissue; sst-rma normalization; DABG p<0.05; p<0.05; FC 2.0

source_name: N153S NP

value: Nucleus Pulposus


title: WT AF tissue 2


title: WT AF tissue 3


title: WT NP tissue 4

description: Total RNA from WT NP tissue; sst-rma normalization; DABG p<0.05; p<0.05; FC 2.0

source_name: WT NP


title: P117


title: P080


title: P087


title: P083


title: Nile rat Male 12 weeks


title: WT AF tissue 1


title: WT NP tissue 3


title: P082


title: N153S NP tissue 3


title: P079


title: P078


title: P081


title: P077


title: P075


title: P074


title: WT NP tissue 2


title: N153S AF tissue 1


title: N153S NP tissue 2


title: P072


title: N153S AF tissue 2


title: P067


title: P073


title: WT NP tissue 1


title: P070


title: P069


title: P066


title: P064


title: P071


title: P062


title: P059


title: P061


title: N153S NP tissue 1


title: P053


title: P057


title: P050


title: P045


title: P043


title: P044


title: P046


title: P042


title: P068


title: P040


title: P036


title: P034


title: P065


title: P039


title: P031


title: P029


title: P025


title: P027


title: P026


title: P022


title: P021


title: P018


title: P017


title: P019


title: P014


title: P020


title: P012


title: P052


title: P008


title: P048


title: P041


title: P002


title: P004


title: P033


title: P003


title: P001


title: SC_High Fat Diet 14 Weeks

status: Public on Mar 10 2022

city: Ann Arbor

first: Lindsey

last: Muir

state: MI

address: 1600 Huron Parkway

institute: University of Michigan

data_processing:
Raw spatial transcriptomics sequencing data were processed using the 10X Genomics SpaceRanger (version 1.0.0) pipeline
Processed spatial transcriptomics data is filtered by removing barcodes not associated with (not overlapping) the tissue
Raw single-cell RNA-sequencing data were processed using the 10X Genomics CellRanger (version 4.0.0) pipeline
Processed single-cell RNA-seq data is filtered by removing barcodes that correspond to background noise (free-floating mRNA from lysed or dead cells), as determined by a cell-calling heuristic in the 10X genomics software pipeline.
Genome_build: mm10-3.0.0
Supplementary_files_format_and_content: Processed data files are in the Hierarchical Data Format (HDF5 or H5) which is a compressed binary format. The file contains a matrix stored in Compressed Sparse Column (CSC) format containing absolute transcript counts reported as unique molecular identifier (UMI) counts, as well as the features and spot-barcodes/cell-barcodes associated with the matrix rows and columns, respectively, which are stored as HDF5 groups
Supplementary_files_format_and_content: Tissue positions lists are also provided as CSV files supplementing the spatial transcriptomics data. These files contain a table with rows corresponding to tissue spots and columns specifying the tissue spot coordinates among other details. More information can be found here: https://support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/output/images

source_name: epidydymal white adipose tissue

extract_protocol:
For spatial transcriptomics, extraction and library construction was performed according to manufacturer protocols with permeablization optimized for adipose tissue (10X genomics, fresh frozen tissue). Stromal vascular cells that were magnetically enriched for CD45 were used for single cell RNA-seq samples after isolation from enzymatically digested adipose tissue. Single cell RNA-seq samples underwent additional surface protein staining followed by extraction and processing through a standard single cell Chromium workflow.
spatial transcriptomics and scRNA-seq

treatment_protocol: Obese mice were fed 60% high-fat diet for the stated durations.

value: white adipose

tag: status

value: obese

tag: diet

value: High Fat

tag: time point

value: 14 Weeks


title: SC_High Fat Diet 8 Weeks

value: 8 Weeks


title: P006


title: P024


title: SC_Normal Diet

value: lean

value: Normal Diet


title: Pat23-B

status: Public on Jan 30 2022

city: GOYANG

first: JIHYUN

last: KIM

address: 323 ILSAN-RO

country: South Korea

institute: National cancer center

data_processing:
Single-cell RNA sequencing data were quantified using the 10X software package cellranger to map sequencing data to version 1.2.0 of the build of the hg19 reference genome supplied by 10X Genomics
Gene count matrix was generated from unique molecular identifiers (UMIs) that produced from cellranger count.
We filtered out cells that if any cells meet one of the following thresholds: 1) cells that lower variation than average variation with all genes; 2) cells that have zero UMI count 90% of all genes; 3) cells that greater than 10% expression originating from mitochondrial or hemoglobin genes; 4) cells that have UMIs lower than 100 or larger than 20000.
Genome_build: Hg19
Supplementary_files_format_and_content: tab-delimited text files include UMI count values for each Sample

source_name: Cancer cells

extract_protocol:
Human stomach tissues were cut into approximately 1 mm3 pieces and add the 5 ml of dispase in Hanks’ Balanced Salt Solution (STEMCELL Cat# 07913) and 5 mg collagenase type IV (Sigma Cat# C5138) and incubate at 37 °C for 2 h with gently shaking. After the sample was passed through a 100 m cell strainer (Falcon), the suspended cells in PBS (with1 mM EDTA) were centrifuged at 720 g for 5 min. To remove RBC in the sample, cell pellets were re-suspended PBS (with1 mM EDTA) and centrifuged through a discontinuous 50% Percoll® (Sigma Cat# P4937) gradient, at 470 g for 20 min, with minimum acceleration and no deceleration. The sample between layers PBS and 50% Percoll® was collected re-suspended in PBS for scRNA-seq.
10x signle cell 3' v2 kit
scRNA-seq

value: cancer stomach

tag: diagnosis

value: AGC

tag: lauren classification

value: Diffuse


title: Pat24-B


title: Pat24-A

source_name: Adjacent normal cells

value: normal stomach


title: Pat21-A


title: Pat20-B


title: Pat22-B

value: EGC

value: Intestinal


title: Pat22-A


title: Pat20-A


title: Pat19-A


title: Pat19-B


title: Pat18-B


title: Pat18-A


title: Pat17-B


title: P005

value: female


title: Pat17-A


title: Pat16-A


title: Pat16-B


title: Pat15-A


title: Pat15-B


title: Pat14-A


title: Pat14-B


title: Pat13-A


title: Pat13-B

value: ECG


title: Pat23-A


title: Pat11-B


title: Pat12-B


title: Pat11-A


title: Pat10-A


title: Pat10-B


title: Pat09-B


title: Pat08-B


title: Pat09-A


title: Pat04-A


title: Pat02-B


title: Pat08-A


title: Pat01-B


title: Pat02-A


title: Pat07-B


title: 170kb Enhancers + SynHoxA assemblon in WT mESCs, 24h of RA+SAG differentiation, replicate 2, RNA-Seq

status: Public on Dec 16 2021

city: Seattle

first: Sudarshan

last: Pinglay

state: WA

address: 3720 15th Ave NE

institute: University of Washington

description:
A8_WT_compE_RnHoxA_48hRA/SAG_rep2
featurecounts_WTlines_synHoxAWTaligned.txt
HCWTLBGXF_n01_mb362

data_processing:
Basecall conversion to fastq files was performed using Picard
Alignment: Fastq files were aligned to the genome (mm10_synHoxA or mm10_synHoxA_delHoxA custom genomes) using HISAT2, using options: -p 20 -q --rna-strandness F.
Computing Abundance: Mapped reads were assigned to annotated genes using the featureCount function in Rsubread, using options -s 2.
Differential gene expression analysis: Read counts were normalized using the ‘rlog’ or regularized log transformation in DESeq2 and used as inputs for the Principal Component Analysis (PCA). The log2 fold change (FC) and adjusted p-value in gene expression levels between 24h, 48h, and 96h vs. 0h was estimated using DESeq2 and plotted using ggplot. Normalized counts from DESeq2 were used to generate transgene/endogenous ratio plots, with the counts from endogenous locus being reduced by half to normalize the copy number between HoxA clusters. RNAseq track visualization was performed using combine tracks tool in IGV on bigwigs generated with deeptools bamCoverage and subsequent editing in Adobe Illustrator.
Genome_build: mm10_synHoxA or mm10_synHoxA_delHoxA custom genomes
Supplementary_files_format_and_content: Tab Separated Text File: Unnormalized Read Counts at annotated genes

source_name: ES-derived embryoid bodies with variant synthetic Hox clusters

growth_protocol: mESCs were cultured on plastic tissue culture plates coated with 0.1% gelatin (EMD Millipore ES-006-B) at 37°C and 5% CO2 in ‘80/20’ medium comprising 80% 2i medium and 20% mESC medium. mESCs were grown at 37°C and 8% CO2 before differentiation experiments.  2i medium was made from a 1:1 mix of Advanced DMEM/F12 (Gibco 12634010) and Neurobasal-A (Gibco 10888022), containing 1X N-2 supplement (Gibco 17502048), 1X B-27 supplement (Gibco 17504044), 2mM Glutamax (Gibco 35050061), 0.1mM Beta-Mercaptoethanol (Gibco 31350010), 103 units/mL LIF (Millipore, ESG1107), 1 μM MEK1/2 inhibitor (Stemgent, PD0325901), and 3 μM GSK3 inhibitor (R&D Systems, CHIR99021). mESC medium was made from Knockout DMEM (Gibco 10829018), containing 15% Fetal Bovine Serum (Gemini), 0.1 mM Beta-Mercaptoethanol (Gibco 31350010), 1X MEM Non Essential Amino Acids (Gibco 11140050), Glutamax (Gibco 35050061), 1X Nucleosides (Millipore ES-008-D) and LIF (Millipore ESG1107).

extract_protocol:
Cells were collected at 0h or 24h, 48h, or 96h after RA/SAG treatment. RNA was extracted with the TRIzol LS Reagent (Life Technologies) followed by purification with the RNAeasy mini kit (Qiagen). RNA integrity was checked with the Agilent High Sensitivity RNA Screentape (Agilent, 5067-5579).
500 ng of RNA was used to prepare libraries and spiked-in with ERCC Exfold Spike-in mixes (Thermo Fisher 4456739). The TruSeq Stranded mRNA Library Preparation kit (Illumina 20020594) was used to prepare RNA-seq libraries. Library size was checked on the High Sensitivity DNA ScreenTape (Agilent 5067-5584). The KAPA library amplification kit was used to quantify libraries on the Bio-Rad CFX96 or the Roche Lightcycler 480 before pooling libraries. Libraries were sequenced on the Illumina NextSeq 500 using V2.5 chemistry (75 cycles, single-end 75 bp) or on the Illumina NovaSeq 6000 using the SP Reagent Kit (100 cycles, single-end 100bp) at the Genomics Core Facility at NYU.

treatment_protocol: Trypsinized (Gibco) mESCs were plated in AK medium (Advanced DMEM/F12:Neurobasal (1:1) medium (Gibco), 7% KnockOut SR (vol/vol) (Gibco), 2 mM L-glutamine, 0.1 mM ß-mercaptoethanol and penicillin–streptomycin (Gibco)) to induce formation of embryoid bodies (EBs), at 37°C, 5% CO2. 3.5x105 cells were plated in 100 mm suspension plates (Corning) for RNA-seq experiments, while 3.5x106 cells were plated in 245 mm x 245 mm square plates (Corning) for ChIP-seq experiments. After 2 days (0h timepoint), the EBs were split 1:2 and plated in fresh AK medium supplemented with 1 μM all-trans retinoic acid (RA) and 0.5 μM smoothened agonist (SAG) (Millipore 566660). For CHIR differentiation, in place of RA/SAG, CHIR99021 was added at 3 µM concentration

tag: treatment

value: 24h of RA+SAG differentiation

tag: cell type

value: Embryoid Bodies


title: Pat01-A


title: Pat07-A


title: Pat06-B


title: Tnmd WT ScxGFP+ cell #10

status: Public on Nov 08 2021

city: Regensburg

first: Xin

last: Lu

address: Universitätsstraße 31

country: Germany

institute: University of Regensburg

description:
Raw_FeatureCounts.counts
WTSC10

data_processing:
after demultiplexing, with raw FASTQ files, which were submitted to an in-house single cell mRNA analysis pipeline.
The raw sequence data from six knock-out (KO) and six wild type (WT) single cells were trimmed with BBDuk, removing remaining adapter sequences and poor-quality bases at the end of each read.
Read decontamination was performed using BioBloom Tools 2.0.13 with filters for the genomes of Mus musculus (mm38), Homo sapiens (hg38), Escherichia coli (BL21), Mycoplasma pneumoniae (M129), Sphingobium sp. (SYK-6), Bradyrhizobium japonicum (USDA 110), Pichia pastoris (GS115), Malessia globosa (CBS 7966), Aspergillus fumigatus (Af293), and a set of viral genomes (RefSeq, 5k+ genomes).
All reads that did not map exclusively to mm38 (Ensembl version 96, GRCm38 DNA primary assembly) were defined as likely contaminations and discarded from downstream processing. Sequence quality per sample were evaluated before as well as after trimming and decontamination using FastQC 0.11.9 and, in addition, all samples were analyzed as a collective with MultiQC 1.9.
Next, the cleaned sample reads were aligned to the reference genome mm38 with STAR 2.5.1b. Reads mapping uniquely in exonic regions were counted per gene and per sample using featureCounts from Subread 2.0.0. Further quality characteristics such as library complexity, using Preseq 2.0.3, and the genomic origin of the reads and the 5’-3’-bias, both with QualiMap 2.2.2d, were assessed. The final counts table of the 12 samples were used further for differential expression analysis.
Genome_build: GRCm38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: uninjured Achilles tendon

growth_protocol: No growth protocol.

extract_protocol:
Uninjured Achilles tendons were enzymatically digested for 8 hours with collagenase, filtered (Alberton et al. 2015) resuspended in PBS, picked based on GFP expression using a micromanipulator and stored in Smart-Seq2 lysis buffer at -80°C).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Enzymatic digestion for 8 hours with collagenase, filtration and resuspension in PBS.

value: C57BL/6 background

value: Tenogenic-primed Scx-expressing cell

value: Tnmd WT ScxGFP+

value: untreated


title: Tnmd WT ScxGFP+ cell #5

description:
Raw_FeatureCounts.counts
WTSC05


title: Tnmd WT ScxGFP+ cell #2

description:
Raw_FeatureCounts.counts
WTSC02


title: Pat06-A


title: Pat05-B


title: Pat05-A


title: Tnmd Knockout ScxGFP+ cell #10

description:
Raw_FeatureCounts.counts
KOSC10

value: Tnmd-/- ScxGFP+


title: Tnmd Knockout ScxGFP+ cell #9

description:
Raw_FeatureCounts.counts
KOSC09


title: Pat03-B


title: Pat04-B


title: Pat03-A


title: Tnmd Knockout ScxGFP+ cell #7

description:
Raw_FeatureCounts.counts
KOSC07


title: Pat12-A


title: Tnmd Knockout ScxGFP+ cell #1

description:
Raw_FeatureCounts.counts
KOSC01


title: 170kb Enhancers + SynHoxA assemblon in WT mESCs, 48h of RA+SAG differentiation, replicate 2, RNA-Seq

description:
A8_WT_compE_RnHoxA_24hRA/SAG_rep2
featurecounts_WTlines_synHoxAWTaligned.txt
H5L3MBGXC_n01_mb253

value: 48h of RA+SAG differentiation


title: 170kb Enhancers + SynHoxA assemblon in WT mESCs, 24h of RA+SAG differentiation, replicate 1, RNA-Seq

description:
A8_WT_compE_RnHoxA_48hRA/SAG_rep1
featurecounts_WTlines_synHoxAWTaligned.txt
HCWTLBGXF_n01_mb361


title: Tnmd WT ScxGFP+ cell #4

description:
Raw_FeatureCounts.counts
WTSC04


title: 170kb Enhancers + SynHoxA assemblon in WT mESCs, 48h of RA+SAG differentiation, replicate 1, RNA-Seq

description:
A8_WT_compE_RnHoxA_24hRA/SAG_rep1
featurecounts_WTlines_synHoxAWTaligned.txt
H5L3MBGXC_n01_mb247


title: sample38: Peritonium tissue (Tumor)

status: Public on Oct 05 2021

city: Singapore

first: Kie Kyon

last: Huang

state: Select State/Province

address: 8 College Road

country: Singapore

institute: Duke-NUS Graduate Medical School

description: *** Raw files were not submitted due to patient privacy concerns ***

data_processing:
Cellranger v3.0 was used to align FASTQ sequencing reads to the hg38 reference transcriptome, generating single cell feature counts for each sample
Using Seruat version 3.0, each sample was considered for genes/features shared by 3 or more cells, and cells showing 500 or more features and less than 6000 features. Cells with mitochondrial RNA percentages of >20 were filtered out
Genome_build: hg38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every cell for each sample

source_name: peritoneum

extract_protocol:
Single cell were  dissociated from gastric tissue sample using tumor dissociation kit from Miltenyi Biotec
10x Genomics Single Cell 5′ Library kit

value: Peritonium tissue (Tumor)


title: Tnmd WT ScxGFP+ cell #14

description:
Raw_FeatureCounts.counts
WTSC14


title: sample35: Primary Gastric Tissue (Normal)

source_name: Stomach

value: Primary Gastric Tissue (Normal)


title: sample34: Primary Gastric Tissue (Tumor)

value: Primary Gastric Tissue (Tumor)


title: Tnmd WT ScxGFP+ cell #1

description:
Raw_FeatureCounts.counts
WTSC01


title: Tnmd Knockout ScxGFP+ cell #20

description:
Raw_FeatureCounts.counts
KOSC20


title: sample33: Primary Gastric Tissue (Tumor)


title: sample30: Primary Gastric Tissue (Tumor)


title: sample32: Primary Gastric Tissue (Tumor)


title: sample29: Primary Gastric Tissue (Tumor)


title: sample39: Primary Gastric Tissue (Tumor)


title: sample40: Primary Gastric Tissue (Tumor)


title: sample31: Primary Gastric Tissue (Normal)


title: sample27: Primary Gastric Tissue (Tumor)


title: Tnmd Knockout ScxGFP+ cell #3

description:
Raw_FeatureCounts.counts
KOSC03


title: sample26: Primary Gastric Tissue (Tumor)


title: sample36: Primary Gastric Tissue (Tumor)


title: sample24: Primary Gastric Tissue (Tumor)


title: sample23: Primary Gastric Tissue (Normal)


title: sample21: Primary Gastric Tissue (Normal)


title: sample28: Primary Gastric Tissue (Tumor)


title: sample19: Peritonium tissue (Tumor)


title: sample20: Peritonium tissue  (Tumor)

value: Peritonium tissue  (Tumor)


title: sample25: Primary Gastric Tissue (Normal)


title: sample22: Primary Gastric Tissue (Tumor)


title: sample17: Primary Gastric Tissue (Tumor)


title: sample14: Primary Gastric Tissue (Tumor)


title: sample13: Primary Gastric Tissue (Tumor)


title: sample18: Primary Gastric Tissue (Tumor)


title: sample7: Primary Gastric Tissue (Tumor)


title: sample16: Primary Gastric Tissue (Tumor)


title: sample9: Primary Gastric Tissue (Normal)


title: sample6: Primary Gastric Tissue (Normal)


title: sample3: Primary Gastric Tissue (Tumor)


title: sample12: Primary Gastric Tissue (Tumor)


title: sample15: Primary Gastric Tissue (Tumor)


title: patient_NSC018_tumor_nodule3

status: Public on Sep 16 2021

city: Boston

first: Mahmoud

middle: Adel

last: Bassal

state: Massachusetts

address: 3 Blackfan Circle

department: Hematology and Oncology

institute: Beth Israel Deaconess Medical Center

description: polyA RNA

data_processing:
Transcripts were mapped to the mouse transcriptome following an established bioinformatics pipeline (https://github.com/indrops/indrops) for inDrop experiments to create a raw gene-cell counts matrix (Klein et al, Cell, 2015) with the slight modification of adding an additional filtering step to remove lower quality base calls using Trimmomatic (v0.36, [45]) with the parameters “LEADING:28 SLIDINGWINDOW:4:26 MINLEN:15” after the “identifying abundant barcodes” step. Human and mouse samples were processed in a similar manner except mice samples were aligned to GRCm38.81 and human to GRCh38.91.
Genome_build: GRCm38.81(mouse), GRCh38.91(human)
Supplementary_files_format_and_content: Gene_vs_Cell raw counts matrix (single cell matrix)

source_name: non-small cell lung tumor

extract_protocol:
Human lung tumor tissue was dissociated for single cell RNA sequencing using the Tumor Dissociation kit by Miltenyi Biotec (cat. no. 130-095-929). Briefly, the tissue was placed in a petri dish on ice and cut into small pieces of 2-4 mm. The pieces were infused with the RPMI/enzyme mix (Miltenyi Biotec), transferred to a gentleMACS C tube containing RPMI/enzyme mix, attached to the sleeve of the gentleMACS Octo Dissociator and run using the “37_h-TDK2” program. After termination of the program, the cells were spun down at 300 x g for 10 min at 4°C, resuspended in RPMI-2% FBS, passed through a 70µm strainer and centrifugation was repeated. The cell pellet was treated with 1 ml ACK solution for 7 min at room temperature, the lysis stopped with 4 ml RPMI-2% FBS. After centrifugation, the cells were resuspended in 1 ml of RPMI-2 % FBS and passed through a cell strainer to obtain a single-cell suspension. Immediately before transcriptome barcoding using the inDrop platform, cells were pelleted at 300 x g for 5 min at 4°C in a swing-bucket rotor, gently resuspended in 1x PBS with 0.1% w/v BSA, manually counted on a hemocytometer and diluted to 100 k cells/ml. The final cell suspension included 15% v/v OptiPrep (Sigma-Aldrich, cat. no. D1556).
Single cell barcoding and library preparation were performed using the inDrops protocol as described in Zilionis et 2017, Nature Protocols, with modifications summarized in Table S6 of Zilionis et al., 2019 (related to this GEO record).

tag: disease state

value: Non-small-cell lung carcinoma (NSCLC)

value: non-small cell lung tumor

tag: gender

value: M

tag: stage aj cc 8th ed

tag: purification

value: RBC-depletion


title: sample8: Primary Gastric Tissue (Tumor)

tag: tissue


title: sample10: Primary Gastric Tissue (Tumor)


title: sample1: Primary Gastric Tissue (Normal)


title: patient_NSC018_tumor_nodule2

description: polyA RNA

tag: age


title: sample11: Primary Gastric Tissue (Normal)


title: Pt5_superficial_cancer

status: Public on Aug 09 2021

city: Seoul

first: Hye Young

last: Jeong

address: Banpodaero 222

institute: Catholic University

description: Single-cell RNA-seq

data_processing:
Sample demultiplexing, barcode processing and UMI counting were performed by using the official 10x Genomics pipeline Cell Ranger (v3.1.0) (https://support.10xgenomics.com). Briefly, raw base call files generated by Illumina sequencers were demultiplexed into reads in FASTQ format using the bcl2fastq developed by Illumina (https://github.com/brwnj/bcl2fastq). The raw reads were trimmed from the 3’ end to get the recommended number of cycles for read pairs (Read1: 26 bp; Read2: 98 bp). The reads of each library were then processed separately using the “cellranger count” pipeline to generate a gene-barcode matrix for each library. During this step, the reads were aligned to a human reference genome (version: hg19). Cell barcodes and UMIs associated with the aligned reads were subjected to correction and filtering.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *_CountMatrix.txt: Tab-delimited text files include UMI count values for each Sample; raw counts.

source_name: superficial layer of gastric cancer of patient5

extract_protocol:
The single cells were isolated from fresh tumor tissues and paired normal gastric tissues. The tissues were cut to small pieces and minced with scalpels in a culture dish. Small pieces were washed with PBS and transfer the tissue pieces into the gentleMACS C tubes (Macs, CA, USA) and tumor dissociated using a Human Tumor Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell suspensions were filtered using a 100μm filter, and dissociated cells were pelleted and resuspended in PBS. Then we performed dead cell remove in single cell using a Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) per manufacturer guidelines.
The 10x Genomics Chromium platform was used to capture and barcode the cells to generate single-cell Gel Beads-in-Emulsion (GEMs) by following the manufacturer’s protocol. Briefly, along with the reverse transcription master mix, cell suspensions were loaded onto 10x Genomics Single Cell 30 Chips. During this step, cells were partitioned into the GEMs along with gel beads coated with oligonucleotides. These oligonucleotides enable mRNA capture inside the droplets by 30 bp oligo-dT after cell lysis and provide barcodes to index cells (16 bp) as well as transcripts (10 bp UMI). Following reverse transcription, cDNAs with both barcodes were amplified, and a library was constructed using the Single Cell 30 Reagent Kit (v2 chemistry) for each sample. The resulting libraries were sequenced on an Illumina HiSeq4000 System in a 2 × 100 bp paired-end mode.

tag: patient

value: diffuse type gastric cancer

value: superficial layer of gastric cancer


title: Pt4_superficial_cancer

description: Single-cell RNA-seq

source_name: superficial layer of gastric cancer of patient4


title: sample2: Primary Gastric Tissue (Tumor)


title: Pt5_deep_cancer

description: Single-cell RNA-seq

source_name: deep layer of gastric cancer of patient5

value: deep layer of gastric cancer


title: Pt3_superficial_cancer

description: Single-cell RNA-seq

source_name: superficial layer of gastric cancer of patient3


title: Pt3_normal

description: Single-cell RNA-seq

source_name: normal gastric tissue of patient3

value: normal gastric tissue


title: Pt5_normal

description: Single-cell RNA-seq

source_name: normal gastric tissue of patient5


title: Pt4_deep_cancer

description: Single-cell RNA-seq

source_name: deep layer of gastric cancer of patient4


title: Pt2_deep_cancer

description: Single-cell RNA-seq

source_name: deep layer of gastric cancer of patient2


title: Pt4_normal

description: Single-cell RNA-seq

source_name: normal gastric tissue of patient4


title: Pt1_deep_cancer

description: Single-cell RNA-seq

source_name: deep layer of gastric cancer of patient1


title: Pt2_superficial_cancer

description: Single-cell RNA-seq

source_name: superficial layer of gastric cancer of patient2


title: Pt3_deep_cancer

description: Single-cell RNA-seq

source_name: deep layer of gastric cancer of patient3


title: sample5: Primary Gastric Tissue (Tumor)


title: patient_NSC016_tumor_nodule3

description: polyA RNA

value: F


title: sample4: Primary Gastric Tissue (Normal)


title: patient_NSC018_tumor_nodule1

description: polyA RNA


title: Micro-C_∆CTCF_IAA_rep1

status: Public on Jul 15 2021

city: Berkeley

first: Xavier

last: Darzacq

state: CA

address: 475D Li Ka Shing Center

department: Molecular and Cell Biology

institute: University of California, Berkeley

description:
Homozygous cell line with endogenous CTCF loci tagged with an auxin-inducible degron
CTCF-AID_pool.mcool

library_source: genomic

data_processing:
Library strategy: Micro-C
ChIP-Seq quality check: FastQC
ChIP-Seq read alignment: Bowtie (-n 2, -m 1)
ChIP-Seq peak calling: MACS2 (--nomodel --extsize 300); merged inputs used as controls
ChIP-Seq heatmaps: deepTools (version 2.4.1) bamCoverage (--binSize 50 --normalizeTo1x 2150570000 --extendReads 300 --ignoreDuplicates -of bigwig) to normalize read numbers to 1x sequencing depth, obtaining read coverage per 50-bp bins across the whole genome (bigWig files). We then used the bigWig files to compute read numbers across 6 kb centered on  ChIP-Seq MACS2 peak summits (computeMatrix reference-point --referencePoint=TSS --upstream 3000 --downstream 3000 --missingDataAsZero --sortRegions=no). We sorted the output matrices by decreasing UT enrichment, calculated as the total number of reads within a MACS2 called ChIP-seq peak. Finally, heatmaps were created with the plotHeatmap tool (--averageTypeSummaryPlot=mean --colorMap='Blues' --sortRegions=no).
Micro-C allValidPairs were generated by HiC-Pro package (>2.10 version)
Micro-C mcool files were converted from allValidPairs by Cooler package
RNA-seq read alignment: STAR aligner
RNA-seq browser track: deepTools
mNET-seq read alignment: STAR aligner
mNET-seq browser track: deepTools
Genome_build: mm10
Supplementary_files_format_and_content: ChIP-Seq bigWig files: read coverage per 50-bp bins across the whole genome (read numbers normalized to 1x sequencing depth; merged duplicates)
Supplementary_files_format_and_content: mcool file contains all valid Micro-C pairs stored in HDF5 format with mutiple resolutions
Supplementary_files_format_and_content: RNA-seq bigWig files: read coverage per 10-bp bins across the whole genome (read numbers normalized to TPM; merged duplicates; split strands)
Supplementary_files_format_and_content: mNET-seq bigWig files: read coverage per 10-bp bins across the whole genome (read numbers normalized to TPM; merged duplicates; split strands)

molecule: genomic DNA

source_name: Embryonic stem cells

growth_protocol: JM8.N4 mESCs were grown on gelatin-coated plates in knock-out DMEM supplemented with 15% FBS, non-essential amino acids, glutamax, Penicillin-streptomycin, 2-mercapoethanol and LIF. mES cells were fed daily and passaged every 2-3 days by trypsinization.

extract_protocol:
Micro-C protocol was performed as the following steps: 1) digest crosslinked chromatin by MNase; 2) Repair fragment ends with biotin-dNTP; 3) Proximal ligation and purge unligated ends; 4) Purify ligated dinucleosomal DNA.
Purified DNA with biotin-dNTPs was captured by Dynabeads® MyOne™ Streptavidin C1. Standard library preparation protocol including end-repair, A-tailing, and adapter ligation was performed on beads with individual enzymes purchased from Lucigen or NEBnext UltraII kit. Sequencing library was amplified by Kapa HiFi PCR enzyme with lowest possible cycles to reduce PCR duplicates. Sequencing library was sequenced paired-end 50x50 in Illumina HiSeq 4000 sequencer.

treatment_protocol: Degron cell lines were treated for 3 hours with either ethanol only (control, untreated) or with 500 uM, ethanol-dissolved auxin.

tag: cell line

tag: genotype

value: FLAG-BFP-mAID-CTCF / RAD21-SNAPf-V5 / osTir

value: 500 mM auxin in ethanol, 3 hrs

tag: chip antibody


title: Micro-C_∆WAPL_IAA_rep1

description:
Homozygous cell line with endogenous WAPL loci tagged with an auxin-inducible degron
WAPL-AID_pool.mcool

value: FLAG-Halo-CTCF / RAD21-SNAPf-V5 / HA-BFP-mAID-WAPL / osTir


title: Micro-C_∆RAD21_IAA_rep1

description:
Homozygous cell line with endogenous RAD21 loci tagged with an auxin-inducible degron
RAD21-AID_pool.mcool

value: FLAG-Halo-CTCF / RAD21-BFP-mAID-V5 / osTir


title: RNA-seq_∆WAPL_IAA_rep2

description: Homozygous cell line with endogenous WAPL loci tagged with an auxin-inducible degron

data_processing:
ChIP-Seq quality check: FastQC
ChIP-Seq read alignment: Bowtie (-n 2, -m 1)
ChIP-Seq peak calling: MACS2 (--nomodel --extsize 300); merged inputs used as controls
ChIP-Seq heatmaps: deepTools (version 2.4.1) bamCoverage (--binSize 50 --normalizeTo1x 2150570000 --extendReads 300 --ignoreDuplicates -of bigwig) to normalize read numbers to 1x sequencing depth, obtaining read coverage per 50-bp bins across the whole genome (bigWig files). We then used the bigWig files to compute read numbers across 6 kb centered on  ChIP-Seq MACS2 peak summits (computeMatrix reference-point --referencePoint=TSS --upstream 3000 --downstream 3000 --missingDataAsZero --sortRegions=no). We sorted the output matrices by decreasing UT enrichment, calculated as the total number of reads within a MACS2 called ChIP-seq peak. Finally, heatmaps were created with the plotHeatmap tool (--averageTypeSummaryPlot=mean --colorMap='Blues' --sortRegions=no).
Micro-C allValidPairs were generated by HiC-Pro package (>2.10 version)
Micro-C mcool files were converted from allValidPairs by Cooler package
RNA-seq read alignment: STAR aligner
RNA-seq browser track: deepTools
mNET-seq read alignment: STAR aligner
mNET-seq browser track: deepTools
Genome_build: mm10
Supplementary_files_format_and_content: ChIP-Seq bigWig files: read coverage per 50-bp bins across the whole genome (read numbers normalized to 1x sequencing depth; merged duplicates)
Supplementary_files_format_and_content: mcool file contains all valid Micro-C pairs stored in HDF5 format with mutiple resolutions
Supplementary_files_format_and_content: RNA-seq bigWig files: read coverage per 10-bp bins across the whole genome (read numbers normalized to TPM; merged duplicates; split strands)
Supplementary_files_format_and_content: mNET-seq bigWig files: read coverage per 10-bp bins across the whole genome (read numbers normalized to TPM; merged duplicates; split strands)

extract_protocol:
RNAs were extracted using the standard TRIzol RNA extraction protocol and then depleted rRNA with NEBNext rRNA Depletion Kit (NEB, #E6310).
The rRNA-depleted RNAs were then subjected to the RNA-seq library construction using the protocol of NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (NEB, #E7765).


title: RNA-seq_∆WAPL_UT_rep2

value: Ethanol, 3 hrs


title: Pt1_superficial_cancer

description: Single-cell RNA-seq

source_name: superficial layer of gastric cancer of patient1


title: patient_NSC016_tumor_nodule1

description: polyA RNA


title: RNA-seq_∆WAPL_UT_rep1


title: patient_NSC016_tumor_nodule2

description: polyA RNA


title: RNA-seq_∆RAD21_UT_rep1

description: Homozygous cell line with endogenous RAD21 loci tagged with an auxin-inducible degron


title: RNA-seq_∆WAPL_IAA_rep1


title: Pt1_normal

description: Single-cell RNA-seq

source_name: normal gastric tissue of patient1


title: RNA-seq_∆RAD21_IAA_rep1


title: RNA-seq_∆CTCF_UT_rep1

description: Homozygous cell line with endogenous CTCF loci tagged with an auxin-inducible degron


title: Micro-C_∆WAPL_UT_rep1

description:
Homozygous cell line with endogenous WAPL loci tagged with an auxin-inducible degron
WAPL-UT_pool.mcool


title: Micro-C_∆WAPL_UT_rep2


title: RNA-seq_∆CTCF_IAA_rep2


title: Micro-C_∆WAPL_IAA_rep3


title: RNA-seq_∆CTCF_IAA_rep1


title: RNA-seq_∆RAD21_IAA_rep2


title: Micro-C_∆CTCF_UT_rep2

description:
Homozygous cell line with endogenous CTCF loci tagged with an auxin-inducible degron
CTCF-UT_pool.mcool


title: Micro-C_∆RAD21_UT_rep1

description:
Homozygous cell line with endogenous RAD21 loci tagged with an auxin-inducible degron
RAD21-UT_pool.mcool


title: Micro-C_∆RAD21_IAA_rep4


title: RNA-seq_∆RAD21_UT_rep2


title: Micro-C_∆WAPL_IAA_rep2


title: mNET-seq_∆WAPL_IAA_rep1

data_processing:
Library strategy: mNET-seq
ChIP-Seq quality check: FastQC
ChIP-Seq read alignment: Bowtie (-n 2, -m 1)
ChIP-Seq peak calling: MACS2 (--nomodel --extsize 300); merged inputs used as controls
ChIP-Seq heatmaps: deepTools (version 2.4.1) bamCoverage (--binSize 50 --normalizeTo1x 2150570000 --extendReads 300 --ignoreDuplicates -of bigwig) to normalize read numbers to 1x sequencing depth, obtaining read coverage per 50-bp bins across the whole genome (bigWig files). We then used the bigWig files to compute read numbers across 6 kb centered on  ChIP-Seq MACS2 peak summits (computeMatrix reference-point --referencePoint=TSS --upstream 3000 --downstream 3000 --missingDataAsZero --sortRegions=no). We sorted the output matrices by decreasing UT enrichment, calculated as the total number of reads within a MACS2 called ChIP-seq peak. Finally, heatmaps were created with the plotHeatmap tool (--averageTypeSummaryPlot=mean --colorMap='Blues' --sortRegions=no).
Micro-C allValidPairs were generated by HiC-Pro package (>2.10 version)
Micro-C mcool files were converted from allValidPairs by Cooler package
RNA-seq read alignment: STAR aligner
RNA-seq browser track: deepTools
mNET-seq read alignment: STAR aligner
mNET-seq browser track: deepTools
Genome_build: mm10
Supplementary_files_format_and_content: ChIP-Seq bigWig files: read coverage per 50-bp bins across the whole genome (read numbers normalized to 1x sequencing depth; merged duplicates)
Supplementary_files_format_and_content: mcool file contains all valid Micro-C pairs stored in HDF5 format with mutiple resolutions
Supplementary_files_format_and_content: RNA-seq bigWig files: read coverage per 10-bp bins across the whole genome (read numbers normalized to TPM; merged duplicates; split strands)
Supplementary_files_format_and_content: mNET-seq bigWig files: read coverage per 10-bp bins across the whole genome (read numbers normalized to TPM; merged duplicates; split strands)

extract_protocol:
Nascent RNAs were extracted as the following steps: 1) Chromatin isolation; 2) Chromatin and RNA digestion; 3) Immunoprecipitation of Pol II complex; 4) RNA phosphorylation with T4 PNK; 5) RNA purification; 6) Size-selection for small RNAs.
mNET-seq RNA libraries were prepared according to protocol of NEBNext small RNA library prep kit (NEB, #E7330).

value: Polymerase II (8WG16 antibody)


title: mNET-seq_∆WAPL_UT_rep1


title: mNET-seq_∆RAD21_IAA_rep2


title: Micro-C_∆RAD21_IAA_rep3


title: Micro-C_∆RAD21_IAA_rep2


title: Micro-C_∆RAD21_UT_rep2


title: Micro-C_∆CTCF_IAA_rep4


title: RNA-seq_∆CTCF_UT_rep2


title: mNET-seq_∆RAD21_UT_rep1


title: Micro-C_∆CTCF_IAA_rep3


title: mNET-seq_∆CTCF_UT_rep1


title: Micro-C_∆CTCF_IAA_rep2


title: mNET-seq_∆WAPL_UT_rep2


title: Micro-C_∆CTCF_UT_rep1


title: mNET-seq_∆WAPL_IAA_rep2


title: HiC_WH7_d4-7_dTAG13

status: Public on Mar 15 2021

first: Andrea

middle: June

last: Kriz

state: MA

address: 185 Cambridge St

department: Molecular Biology

institute: Massachusetts General Hospital, Harvard Medical School

description:
processed data file: H_WH2_WH7_13.hic
processed data file: H_WH2_WH7_13.mus.hic
processed data file: H_WH2_WH7_13.cas.hic

data_processing:
ChIP-seq and Calibrated ChIP-seq: For uncalibrated ChIP-seq, input and ChIP-seq reads were aligned to the 129S1/SvJm (mus) and CAST/Eih (cas) genome as previously described (Kung et al., 2015; Minajigi et al., 2015). PCR duplicates were removed and uniquely mapped reads used for further analysis. Fpm-normalized bigwig tracks were generated for all uniquely mapped (comp), mus-specific (mus), and cas-specific (cas) reads. Heatmaps and metaplots were generated using deepTools functions computeMatrix and plotHeatmap with the reference point as the summit of the called ChIP peaks.Calibrated ChIP-seq analysis was performed as previously described (Hu et al., 2015). Briefly, paired-end reads were aligned to a concatenated mouse and human genome (mm9 + hg19) using Bowtie2 with ?--no-mixed? and ?--no-discordant? options. PCR duplicates were filtered out with SAMBAMBA (Tarasov et al., 2015). Uniquely aligned mouse reads were normalized to human spike-in by subsampling mm9 reads so that total number of hg19 reads was matched between samples and bigWig tracks generated from normalized samples. For calibration of RAD21 cChIP between RAD21-FKBPdegron clones, only reads within hg19 peaks (hg19 tracks combined) were counted for normalization purposes. Subsampling factors were further adjusted based on ratio of hg19 to mm9 reads in matched input samples. After normalization factor was determined, paired end reads were aligned to mus and cas genomes as previously described, except using Bowtie2 with the same parameters as above (with ?--no-mixed? and ?--no-discordant?) instead of Novoalign. PCR duplicates were filtered out with SAMBAMBA and allelic reads normalized using the same normalization factor as above.Input subtracted H3K27me3 calibrated ChIP coverage profiles were generated with SPP (Kharchenko et al., 2008) with smoothing using 1kb windows recorded every 500 bp, as previously described (Simon et al., 2013). For input subtraction, inputs from DMSO and dTAG-13 treated samples were combined to ensure equal profiles were subtracted from each calibrated IP.Heatmaps and metaplots were generated using deepTools functions computeMatrix and plotHeatmap with the reference point as the summit of the called ChIP peaks. Plots of calibrated H3K27me3 signal over upregulated or downregulated genes were calculated using ?scale-regions? with the entire gene scaled to 3kb. To compare the allelic densities of H3K27me3 over different classes of X-linked genes, we computed average densities over gene bodies using multiBigwigSummary from deepTools with default parameters. Non-expressed genes were defined as X-linked genes have 0 RPKM in at least one RNA-seq replicate of DMSO control. Genes subject to XCI were identified as the set of genes have allelic information (see RNA-seq analysis above), with escapees, Xist and Tsix excluded. Escapees were as defined previously (Wang et al., 2018). To compare allelic densities of H3K27me3 over S1 and S2 compartments, we defined S1 compartments as regions having positive value in the first Principal Component calculated in the WAPL-FKBPdegron DMSO control days 4-7 sample and S2 compartments as regions have negative value. S1 and S2 compartments were then binned into 100kb windows using bedtools makewindows, excluding windows that overlapped with unmappable regions as defined previously (Wang et al., 2018). We then we computed average densities over these 100kb bins using multiBigwigSummary from deepTools with default parameters.
RNA-seq: RNA-seq reads were aligned allele-specifically to the 129S1/SvJm (mus) and CAST/Eih (cas) genomes using TopHat2 (Kim et al., 2013) as previously described (Kung et al., 2015; Minajigi et al., 2015). PCR duplicates were removed and unique reads mapping to exons of each gene quantified by Homer (Heinz et al., 2010). DESeq 2 was used to call differentially expressed genes with a cut off of adjusted p-value < 0.1. An expression threshold of FKPM > 0.5 was also applied. Expressed genes with a minimum of 13 allelic-specific reads in at least one sample were counted as genes with allelic information (Colognori et al., 2019). For a given gene, percent mus was calculate as the percentage of allelic reads that were mus-specific (mus / (mus+cas) reads).
in situ Hi-C: Reads were trimmed using cutadapt with the options --adapter=GATCGATC (MboI ligation junction) and --minimum-length=20. Reads of each pair were individually mapped to the mus (Xi) and cas (Xa) reference genomes using novoalign and merged into Hi-C summary files and filtered using HOMER as previously described (Minajigi et al., 2015). Hi-C contact maps were generated from reads in the filtered HOMER mus and cas tag directories using the ?pre? command of Juicer tools with the genomeID mm9 (Durand et al., 2016b). The resulting Hi-C contact maps in .hic format were visualized and normalized with the ?Coverage Square Root? option in Juicebox version 1.8.8 (Durand et al., 2016a). Filtered reads from separate RAD21-FKBPdegron clones were combined before generating Hi-C contact maps.
CHART-seq: Input and ChIP-seq reads were aligned to the 129S1/SvJm (mus) and CAST/Eih (cas) genome as previously described (Kung et al., 2015). PCR duplicates were removed and uniquely mapped reads used for further analysis. Fpm-normalized bigwig tracks were generated for all uniquely mapped (comp), mus-specific (mus), and cas-specific (cas) reads. CHART libraries were sampled to equal read depth and input subtracted CHART coverage profiles were generated with SPP (Kharchenko et al., 2008) with smoothing using 1kb windows recorded every 500 bp, as previously described (Simon et al., 2013). After scaling, we computed base 2 logarithm of the ratio of the coverage of DMSO control divided by that of dTAG-13 using bigWigCompare from deepTools with --binSize 1000 and default parameters otherwise.
PRO-seq: Paired-end reads were aligned to a concatenated mouse and fly genome (mm9 + dm6) using Bowtie2 with ?--no-mixed? and ?--no-discordant? options. DMSO and dTAG-13 samples for each clone were normalized to drosophila spike-in by subsampling mm9 reads so that total number of dm6 reads was matched between samples. For RAD21-FKBPdegron clone G9, downsampling factors corresponded to library size (i.e., were equivalent to downsampling based on total number of reads). For RAD21-FKBPdegron clone C4, downsampling factors were 1 for DMSO and 0.73 for dTAG-13 although library sizes were similar, indicating that there were overall lower levels of nascent transcription in the dTAG-13 sample. Duplicates were not removed to preserve reads corresponding to TSS and promoter proximal PolII positions. Reads mapping to genes (full gene bodies) were quantified by Homer (Heinz et al., 2010), removing miRNA, snoRNA and scaRNAs. DESeq 2 was used to call differentially expressed genes with a cut off of adjusted p-value < 0.1. For analyses on all genes, no FPKM threshold was applied, but genes were required to have at least 1 count in one sample. An expression threshold of FKPM > 0.5 was applied for analyses on expressed genes. Log2FoldChange and log2baseMean from DESeq2 were plotted for indicated classes of genes. H3K27me3 genes were defined as genes overlapping H3K27me3 cChIP-seq peaks in their respective samples (DMSO and dTAG-13 peaks combined). H3K27me3 loop genes were defined as H3K27me3 genes overlapping H3K27me3-bound loop anchors called in RAD21-FKBPdegron dTAG-13 Hi-C maps (i.e., cohesin-independent H3K27me3 anchored loops). For overlap with RAD21 loop anchors, loops were called in respective DMSO Hi-C maps.For allelic analysis, PRO-seq reads not mapping to the fly genome (dm6) were aligned to mus and cas genomes using Bowtie2 with the same parameters as above (?--no-mixed? and ?--no-discordant?) and downsampled using the same factors as calculated above. Expressed genes with a minimum of 13 allelic-specific reads in at least one sample were counted as genes with allelic information (Colognori et al., 2019). For CDPs, normalized mus and cas counts divided by gene length were plotted.
Genome_build: mm9
Supplementary_files_format_and_content: ChIP-seq: bigWig (bw) files for all reads (.bw), regardless of whether they are allele-specific or not, cas-specific (.cas.bw) and mus-specific (.mus.bw). All bigWig files are fpm normalized and H3K27me3 bigWig files are additionally input normalized.
Supplementary_files_format_and_content: Calibrated ChIP-seq: bigWig (bw) files for all reads (.bw), regardless of whether they are allele-specific or not, cas-specific (.cas.bw) and mus-specific (.mus.bw). All bigWig files are normalized to spike-in HEK293 control and H3K27me3 bigWig files are additionally input normalized.
Supplementary_files_format_and_content: RNA-seq: Strand specific (cwig, Crick strand, wwig, Watson strand) bigWig (bw) files for all reads (.bw), regardless of whether they are allele-specific or not, cas-specific (.cas.bw) and mus-specific (.mus.bw). All bw files are fpm normalized. Raw exonic reads in each RNA-seq library were quantified and summarized in .txt files.
Supplementary_files_format_and_content: PRO-seq: Strand specific (cwig, Crick strand, wwig, Watson strand) bigWig (bw) files for all reads (.bw), regardless of whether they are allele-specific or not, cas-specific (.cas.bw) and mus-specific (.mus.bw). All bw files are downsampled according to Drosophila spike-in. Gene body counts in each PRO-seq library were quantified and summarized in .txt files.
Supplementary_files_format_and_content: In situ Hi-C: Compressed Hi-C matrix (.hic) files for all contacts genome-wide (.hic), regardless of whether they are allele-specific or not, cas-specific chrX (chrX.cas.his) contacts and mus-specific chrX (chrX.mus.hic) contacts. File can be loaded onto Juicebox to view either raw or normalized Hi-C interaction matrices at 5kb to 2.5Mb resolutions.

source_name: Hi-C, clone H7

growth_protocol: Undifferentiated mESCs were grown on γ-irradiated MEF feeders in medium containing DMEM, high glucose, GlutaMAX supplement, pyruvate, 15% Hyclone FBS (Sigma), 25 mM HEPES pH 7.2-7.5, 1x MEM non-essential amino acids, 1x Pen/Strep, 0.1 mM βME, and 500 U/mL ESGRO recombinant mouse Leukemia Inhibitory Factor (LIF) protein (Sigma, ESG1107) at 37°C with 5% CO2. To induce differentiation in EBs, mESCs were trypsinized and resuspended in the same media without LIF. For the first 3 days of differentiation, EBs were grown in suspension. On day 4 and subsequent days, EBs were grown on gelatinized plates until harvested at day 7 or day 10.

extract_protocol:
ChIP-seq: ChIP-seq was performed as described previously with minor modifications (Del Rosario et al., 2019). Briefly, cells were trypsinized, resuspended in 1 million cells/mL of media and crosslinked with 1% formaldehyde for 10 min at room temperature with rotation. Cells were then quenched with 0.2 M glycine for 5 min. Crosslinked cells were washed once with cold PBS, pelleted, and snap frozen in an EtOH/dry ice bath. Cell pellets were stored at -80C until further processing. 5-10 million cross-linked cells per IP were thawed on ice and resuspended in 300ul Lysis Buffer 1 (25mM HEPES pH 7.2-7.5, 25mM EDTA, 0.5% SDS). Nuclei were sonicated (Qsonica Q800 Sonicator) in polystyrene tubes at 40% power setting, 30s ON/30s OFF for a total sonication (ON) time of 6 min. After sonication, 1.2 mL of Lysis Buffer 2 (25mM HEPES pH 7.2-7.5, 187.5mM NaCl, 1.25% Triton X-100, 0.625% Na-deoxycholate, 6.25% glycerol, 1x cOmplete EDTA-free protease inhibitor cocktail) was added. 30ul of Dynabeads Protein G (Thermo Fisher Scientific) per IP were washed 3 times with ChIP Wash Buffer 1 (25mM HEPES pH 7.2-7.5, 150mM NaCl, 5mM EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, 5% glycerol, 1x cOmplete EDTA-free protease inhibitor cocktail) and resuspended in 60ul ChIP Wash Buffer 1. Each IP was blocked with 20ul washed beads for 2 hours at 4C with rotation. The other 40ul of washed beads were incubated with antibody (5ul of RAD21 antibody or 2ul of H3K27me3 antibody/IP) for 2 hours at 4C. 5% input was taken from blocked lysates. Beads conjugated to antibody were added to blocked lysates and IP allowed to proceed overnight at 4C with rotation. IPs were then washed 3 times with cold ChIP Wash Buffer 1, 3 times with cold ChIP Wash Buffer 2 (25mM HEPES pH 7.2-7.5, 300mM NaCl, 5mM EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, 5% glycerol, 1x cOmplete EDTA-free protease inhibitor cocktail). And 1 time with cold TENG (50mM Tris-HCl pH 8, 1mM EDTA, 50mM NaCl, 5% glycerol, 1x cOmplete EDTA-free protease inhibitor cocktail). All washes were performed by inverting tubes and setting aside for ~2 minutes per wash. IPs were then resuspended in 500ul TENG with 100ug RNAse A (Thermo Fisher Scientific) and incubated for 30 min at 37C. IPs were then washed 2 times with cold ChIP Wash Buffer 2 and 2 times with cold TENG without protease inhibitors. IPs were resuspended in 200ul elution buffer (50mM Tris-HCl pH 7.5, 10mM EDTA, 1% SDS) and incubated 15 min at 65C. Eluate was then transferred to a new tube and 198ul TE buffer (Ambion) and 40ug Proteinase K (Sigma) added. Inputs were thawed, TE buffer added to equalize volume to IPs and 200ul elution buffer and 40ug Proteinase K added. Inputs and IPs were then incubated for 2h at 45C and crosslinks reversed overnight at 65C. DNA was purified with phenol-chloroform extraction and resuspended in 50ul TE. Efficiency of ChIP was assayed via qPCR, with samples displaying over 10-fold enrichment of positive regions over negative control prepared for sequencing. Libraries were prepared from ~500ng of input or IP DNA using the NEBNext® Ultra? II DNA Library Prep Kit for Illumina® (NEB) according to kit instructions. Experiments were performed in two biological replicates. Input and ChIP-seq libraries were sequenced on Illumina HiSeq, generating 20-50 million paired end 50 or 150 nucleotide reads per sample.
Calibrated ChIP-seq: Calibrated ChIP-seq was performed using the same protocol as ChIP-seq except that 50,000 HEK293 cells per 5 million mouse cells were mixed in at the initial lysis step. Cells were counted and equal numbers of cells used per IP.
in situ Hi-C: As described in Rao et al. (2014), 3-5 million cells were crosslinked with 1% formaldehyde for 10min. Crosslinked cell pellets were lysed with 300uL ice-cold HiC lysis buffer (10mM Tris HCl pH 8, 10mM NaCl, 0.2% Igepal CA630) with protease inhibitors (Roche). Cell suspension was incubated for 30min at 4C with rotation, then pelleted at 2500xg for 5min. Pelleted nuclei were then washed with 500ul ice-cold HiC lysis buffer and resuspended in 50uL 0.5% SDS solution and incubated at 62C for 10min. 145ul H2O and 25ul 10% Triton X-100 were added to quench SDS and nuclei incubated at 37C for 15min. 26.1ul of 10X NEB Buffer 2 and 100U of MboI restriction enzyme were added and nuclei digested overnight at 37C with rotation. MboI was then heat activated at 62C for 20min, then nuclei cooled to room temperature. To fill in MboI fragment overhangs and mark DNA ends with biotin, 50ul of fill-in master mix was added (37.5ul 0.4mM biotin-14-dATP, 1.5ul 10mM dCTP, 1.5ul 10mM dGTP, 1.5ul 10mM dTTP, 8ul 5U/ul DNA Polymerase I, Large (Klenow) Fragment) and mixed by pipetting. Nuclei were then incubated at 37C for 1h with rotation. To ligate biotinylated DNA ends together, 900ul ligation master mix was added (649ul H2O, 120ul 10X NEB T4 ligase buffer, 100ul 10% Triton X-100, 6ul of 20mg/ml BSA, 25ul of 400U/ul T4 DNA ligase). Reaction was mixed by inverting and incubated at room temperature for 4h with rotation. Reaction was then centrifuged at 1700xg for 5min at room temperature. Pelleted nuclei were resuspended in 750ul TES buffer (1mM EDTA, 10mM Tris-HCl pH 8, 0.5% SDS), 50ul Proteinase K (20mg/ml) added and incubated at 55C for 1h with rotation. To reverse crosslinks, 88.9ul 3M NaCl was added and crosslinks reversed by incubated overnight at 65C with shaking (Eppendorf thermomixer, 950rpm). Samples were cooled to room temperature and DNA extracted with phase-lock gel tubes (5Prime). RNA was digested from purifying DNA by adding 2ul RNAse A (20mg/ml) and incubating at 37C for 1h. DNA was then extracted with phase-lock gel tubes (5Prime) and resuspended in 130ul 1X Tris buffer (10mM Tris-HCl, pH 8). To ensure complete resuspension, DNA was then incubated at 37C for 15min. DNA was then sonicated to a size of 300-500bp using a Covaris with the following parameters: 140W, 10% duty cycle, 200 bursts/cycle, 100s. Samples were then size selected to 300-500bp using AMPure XP beads and eluted in 300ul 1X Tris buffer. To pulldown biotinylated ligation products, 30ul (per sample) of MyOne Streptavidin C1 beads were washed with 400ul of 1X Tween washing buffer (5mM Tris-HCl pH 7.5, 0.5mM EDTA, 1M NaCl, 0.05% Tween 20) and resuspended in 300ul 2X binding buffer (10mM Tris-HCl pH 7.5, 1mM EDTA, 2M NaCl). Beads were then added to purified DNA and sample incubated at room temperature for 15min with rotation. Beads were washed twice with 600ul 1X Tween Washing buffer. Subsequent library preparation steps were then done directly on beads.
CHART-seq: Xist CHART-seq was performed as previously described (Wang et al., 2018). Briefly, cells were trypsinized, resuspended in 1 million cells/mL of media and crosslinked with 1% formaldehyde for 10 min at room temperature with rotation. Cells were then quenched with 0.2 M glycine for 5 min. Crosslinked cells were washed once with cold PBS, pelleted, and snap frozen in an EtOH/dry ice bath. Cell pellets were stored at -80C until further processing. 25 million cross-linked cells per CHART were thawed on ice and resuspended in 1 mL cold sucrose buffer. (10 mM HEPES pH 7.5, 0.3 M sucrose, 1% Triton X-100, 100 mM potassium acetate, 0.1 mM EGTA) supplemented with 0.5 mM spermidine, 0.15 mM spermine, 1 mM DTT, 1x protease inhibitor cocktail (Sigma, P8340), 10 U/mL RNase inhibitor (Roche). The cell suspension was rotated for 10 min at 4°C, after which it was diluted with additional 2 mL cold sucrose buffer. This was added to a pre-chilled 15-mL glass Wheaton dounce tissue grinder (Thermo Fisher Scientific) that had been pre-rinsed once with RNaseZAP RNase decontamination solution (Thermo Fisher Scientific), three times with DEPC-treated water, and once with 2 mL sucrose buffer. Release of nuclei was accomplished by douncing cells 20 times with a tight pestle, and verified by staining with Trypan Blue Solution (Thermo Fisher Scientific). The nuclear suspension was then layered atop a cushion of 7.5 mL cold glycerol buffer (10 mM HEPES pH 7.5, 25% glycerol, 1 mM EDTA, 0.1 mM EGTA, 100 mM potassium acetate) supplemented with 0.5 mM spermidine, 0.15 mM spermine, 1 mM DTT, 1x cOmplete EDTA-free protease inhibitor cocktail (Roche), and 5 U/mL RNase inhibitor, and centrifuged at 1500 g for 10 min at 4°C. The purified nuclear pellet was resuspended in 6 mL PBS and further cross-linked with 3% formaldehyde for 30 min at room temp with rotation. Afterward, nuclei were pelleted by centrifugation at 1,000 g for 5 min at 4°C and washed three times with cold PBS. Nuclei were resuspended in in 1 mL cold nuclear extraction buffer (50 mM HEPES pH 7.5, 250 mM NaCl, 0.1 mM EGTA, 0.5% sarkosyl, 0.1% sodium deoxycholate, 5 mM DTT, 10 U/mL RNase inhibitor) and incubated for 10 min at 4°C with rotation. Nuclei were then centrifuged at 400 g for 5 min at 4°C and resuspended in 230 uL cold sonication buffer (50 mM HEPES pH 7.5, 75 mM NaCl, 0.1 mM EGTA, 0.5% sarkosyl, 0.1% sodium deoxycholate, 0.1% SDS, 5 mM DTT, 10 U/mL RNase inhibitor) to a final volume of ?270 uL. Nuclei were sonicated for 5 min at 4°C using a Covaris E220e (140W peak incident power, 10% duty factor, 200 cycles/burst). Sonicated chromatin was centrifuged at 16,000 g for 20 min at 4°C to remove debris. The supernatant (?220 uL) was mixed with 110 uL sonication buffer to a final volume of ?330 uL, which was split into two CHART reactions (Xist capture using antisense probes and control capture using sense probes). For two CHART reactions, 600 uL MyOne Streptavidin C1 beads (Thermo Fisher Scientific) were used, which were pre-washed with 1 mL DEPC-treated water, blocked with 600 uL blocking buffer (33% sonication buffer, 67% 2x hybridization buffer, 500 ng/uL yeast RNA [Thermo Fisher Scientific], 1% BSA) at room temp for 1 hr, washed with 1 mL DEPC-treated water again, and resuspended in 610 uL 1x hybridization buffer (33% sonication buffer, 67% 2x hybridization buffer [see below for composition]). For each CHART, 160 uL chromatin was mixed with 320 iL 2x hybridization buffer (50 mM Tris pH 7.0, 750 mM NaCl, 1% SDS, 1 mM EDTA, 15% formamide, 1 mM DTT, 1 mM PMSF, 1x cOmplete EDTA-free protease inhibitor cocktail, 100 U/mL RNase inhibitor) and pre-cleared with 60 uL pre-blocked MyOne Streptavidin C1 beads at room temp for 1 hr with rotation. Pre-cleared chromatin was separated from beads, with 1% saved as ?input? sample. The remaining pre-cleared chromatin (?500 uL) was mixed with 36 pmol of either antisense (Xist-targeting) or sense (control) biotinylated capture probes. We used a pool of 9 capture probes, which were selected from the 11 probes described previously (Simon et al., 2013), excluding probes X.3 and X.8. Hybridization was carried out at 37°C for 4 hr followed by incubation with 240 uL blocked MyOne Streptavidin C1 beads at 37°C for 1 hr. The beads were washed once with 1x hybridization buffer (33% sonication buffer, 67% 2x hybridization buffer) at 37°C for 10 min, five times with wash buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 2% SDS, 2 mM EDTA, 2 mM EGTA, 1 mM DTT) at 37°C for 5 min, and twice with elution buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.5% NP-40, 3 mM MgCl2, 10 mM DTT) at 37°C for 5 min. 10% of the final wash was saved as an ?RNA capture? sample to estimate the efficiency of Xist RNA capture afterward by qRT-PCR. CHART-enriched DNA was eluted twice in 200 uL elution buffer supplemented with 5 U/uL RNase H (New England BioLabs) at 37°C for 30 min. An ?input? sample and CHART DNA were treated with 0.5 mg/mL RNase A (Thermo Fisher Scientific) at 37°C for 1 hr and then with 1% SDS, 10 mM EDTA, and 0.5 mg/mL proteinase K (Sigma) at 55°C for 1 hr. Reversal of crosslinks was performed by addition of 150 mM NaCl (final 300 mM) and incubation at 65°C overnight. DNA was purified by phenol-chloroform extraction and further sheared to < 500-bp fragments using Covaris E220e (140W peak incident power, 10% duty factor, 200 cycles/burst). Sonicated DNA was purified using 1.8x Agencourt AMPure XP beads (Beckman Coulter). Libraries were sequenced on Illumina, generating ?50 million 150-nt paired-end reads per sample
RNA-seq: Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific) and mRNA isolated using the NEBNext Poly(A) Magnetic Isolation Module (NEB) according to the kit instructions. Libraries were sequenced on the Illumina HiSeq2500 (rapid run), generated 30-50 million 50 nt paired end reads per sample.
PRO-seq: PRO-Seq library construction was performed by the Nascent Transcriptomics Core at Harvard Medical School, Boston, MA. Aliquots of frozen (-80°C) permeabilized cells were thawed on ice and pipetted gently to fully resuspend. An aliquot was removed and diluted 1:10 in 1xPBS for cell counting using an automatic counter (Biorad TC-20) with counts spot checked by manual recount using a hemocytometer as needed. An aliquot of each dilution was also stained with Trypan Blue and counting repeated to determine permeabilization efficiency. For each sample, 1 million permeabilized cells were used for nuclear run-on. For normalization, 50,000 permeabilized Drosophila S2 cells were added to each sample of 1 million cells. Nuclear run on assays and library preparation were then performed essentially as described [Elrod, N.D., et al. (2019) Molecular Cell 76: 738-752.e7] with modifications noted below. The nuclear run-on buffer was prepared as 4X stock (20mM Tris (pH 8), 20mM MgCl2, 2mM DTT, 600mM KCl, 40uM/ea biotin-11-NTPs (Perkin Elmer), 20U SuperaseIN (Thermo)). The 4X stock was mixed 1:1 with 2% sarkosyl (Sigma) to yield 2X complete run-on mix. The 3' adapter (RNA oligo: 5'P-GAUCGUCGGACUGUAGAACUCUGAAC-3'InvdT) was pre-adenylated prior to use (5' DNA adenylation kit, NEB, according to the manufacturer's instructions). Adenylated oligo was purified by ethanol precipitation, resuspended in water and quality was modification confirmed by electrophoresis on a 15% TBE-Urea gel (Novex) next to appropriate controls. The adapter concentration was adjusted to 10uM and 1uL was used for each ligation which used T4 RNA ligase 2, truncated KQ (NEB) with 15% PEG-8000 in the reaction mix. Reactions were incubated at 16°C overnight. Each ligation reaction was then diluted with 180uL of betaine binding buffer (1.42g of betaine brought to 10mL with binding buffer and sterile filtered) containing 1uL of 100uM blocking oligo (TCCGACGATCCCACGTTCCCGTGG/3InvdT/). The blocking oligo is complementary to and overhangs the 3' end of the 3' adapter and reduces 5'/3' adapter dimer formation during the 5' adapter ligation step. The blocking oligo has modifications at its 3? end, to prevent extension by reverse transcriptase. Blocking oligo was also included in final wash solution prior to the 5' adapter ligation and in the ligation reaction itself, both at 3-fold molar excess over the initial amount of 3' adapter (i.e. 1uL of 30uM per reaction). The 5' adapter ligation was performed as in Elrod except PEG-8000 was increased to 15%. Libraries were prepared using the NEBNext® Ultra? II DNA Library Prep Kit for Illumina® (NEB) according to kit instructions. PRO-seq: After reverse transcription, cDNA was immediately amplified for 5-cycles (preCR step). A PCR cocktail consisting of NEBNext Ultra II Q5 master mix (NEB) and Illumina TruSeq PCR primers (RP-1, common; and RPI-X, indexing) was added to each cDNA and amplification was done according to manufacturer's suggested 2-step cycling conditions for NGS applications. To determine optimal library amplification, the preCRs were thawed on ice and an aliquot serially diluted. Each dilution was mixed with a PCR cocktail (Q5 DNA polymerase with optional high GC enhancer) containing 10uM/ea of Illumina universal P5 and P7 primers and amplified 15-cycles. The reactions were electrophoresed on a 2.2% agarose gel (SeaKem) cast with SYBR gold (Thermo) and analyzed to determine the optimal number of cycles for final amplification. preCRs were transferred from ice to a pre-heated thermal cycler block and amplified for the appropriate number of additional cycles to reach the total determined in the test amplification.

treatment_protocol: Degradation of RAD21 or WAPL in FKBPdegron lines during differentiation was induced by adding a 1:20,000 dilution of 10mM dTAG-13 in DMSO to cell culture media (500nM final concentration) for the indicated time frames. The small molecule dTAG-13 was synthesized as previously described (Erb et al., 2017; Nabet et al., 2018). For DMSO vehicle controls, the same volume of DMSO alone was added to cell culture media.

tag: strain

value: CAST/Ei x 129/Sv/Jae

tag: genotype/variation

value: WAPL-FKBP(degron)

value: ES cell (Tsix-stop)

value: 3 days dTAG-13 (500nM)

tag: differentiation day

value: day 7


title: RNA_WH7_d4-7_dTAG13.rep1

description: processed data file: WH7d4-7-comp-gene-D-13-rep1-rep2-WH2d4-7-D-13-table.nospace.noFPKM.txt

source_name: RNAseq, clone H7, rep1


title: RNA_WH7_d4-7_dTAG13.rep2

source_name: RNAseq, clone H7, rep2


title: 100_Wapl-C20_48h_RNA_rep2

status: Public on Oct 15 2020

city: Rotterdam

first: Ning Qing

last: Liu

address: Wytemaweg 80

department: Hematology

country: Netherlands

institute: Erasmus University Medical Center Rotterdam

description: Sample 100

data_processing:
ChIP-seq: Calibrated ChIP-seq data were analyzed based on a modified method described in previously (Orlando et al., 2014). Raw sequencing data was mapped to a concatenated reference genome (mm10 and hg19) using Bowtie 2 mapper (version 2.3.4.1) (Langmead et al., 2009). The mapped reads with mapping quality score <15 and the optical PCR duplicates were discarded using SAMtools (version 1.9) (Li et al., 2009). The coverage files (bigWig files) were generated by applying the above computed scaling factor using deepTools (version 2.5.4) (Ramírez et al., 2014), and further calibrated based on average enrichment of the ChIP-seq experiments under direct comparison using their spike-in reference.; Standard ChIP-seq data were mapped to a mm10 reference. The mapped reads with mapping quality score <15 and the optical PCR duplicates were discarded using SAMtools. The coverage files of uncalibrated ChIP-seq data and the data of pluripotency factors (due to absence of these factors in HEK293T cells) were generated using “normalize to 1X genome coverage” methods in deepTools. Peak calling was performed using MACS2 (version 2.1.1.20160309) (Liu, 2014) at a q-value cutoff of 0.01.
RNA-seq: Raw RNA-seq data were mapped against mm10 reference genome using a TopHat2 pipeline (version 2.1.1) (Kim et al., 2013). The mapped reads with mapping quality score <10 were discarded using SAMtools. The read coverage for each gene in “Mus Musculus GRCm38.92” annotation file was determined using a HTSeq tool (version 0.9.1). The coverage files were generated using “normalize to 1X genome coverage” methods in deepTools. The genes with at least 20 reads in both replicates were kept for further analysis. The filtered expression data were normalized based on the size factors of the individual samples using a DESeq2 package.
Hi-C: Raw Hi-C data were mapped with HiC-Pro (Servant et al., 2015), which performs mapping, identification of valid Hi-C pairs, generation of contact matrices and ICE normalization (Imakaev et al., 2012).
4C-seq: The raw sequence data was mapped using our 4C mapping pipeline (http://github.com/deWitLab/4C_mapping ).
TT-seq: Raw TT-seq data were mapped against mm10 reference genome using a TopHat2 pipeline (version 2.1.1) with the following settings adjusted for nascent RNA sequencing: (1) microexon-search, and (2) fr-firststrand. The mapped reads with mapping quality score <10 were discarded using SAMtools. The read coverage for each gene (the entire gene) in “Mus Musculus GRCm38.92” annotation file was determined using a HTSeq tool (version 0.9.1). The downstream analysis of TT-seq data was performed using the same settings as RNA-seq for proper data comparison using A DESeq2 package.
ATAC-seq: ATAC-seq data were were mapped against mm10 reference genome using BWA-MEM. The mapped reads with mapping quality score <15, as well as optical PCR duplicates, were discarded using SAMtools. The coverage files were generated using “normalize to 1X genome coverage” methods in deepTools. Alignment of ATAC-seq was performed the same as ChIP-seq data.
Genome_build: mm10
Supplementary_files_format_and_content: 1. ChIP-seq: bigWig files; 2. RNA-seq: bigWig files, HTSeq raw read count tables, DESeq2 normalized ratio tables; 3. Hi-C: Hi-C iced matrix, genome abs.bed file; 4. 4C-seq: interaction coverage file, viewpoint information file.

growth_protocol: E14Tg2a (129/Ola isogenic background) and the derived cell lines were cultured on 0.1% gelatin-coated plates in serum-free DMEM/F12 (Gibco) and Neurobasal (Gibco) medium (1:1) supplemented with N-2 (Gibco), B-27 (Gibco), BSA (0.05%, Gibco), 104 U of Leukemia Inhibitory Factor/LIF (Millipore), MEK inhibitor PD0325901 (1 µM, Selleckchem), GSK3-β inhibitor CHIR99021 (3 µM, Cayman Chemical) and 1-Thioglycerol (1.5x10-4 M, Sigma-Aldrich). The cell lines were passaged every 2 days in daily culture. During the protein depletion experiments, the cells were seeded overnight before the start of the time course in the following densities: For a 96 h time course, 2.5 k, 35 k, 150 k, and 400 k cells were seeded in 24-well, 6-well, 10-cm and 15-cm plates, respectively. For 24 h time course, 5 k, 0.5 M, and 4 M cells were seeded in chamber slide (ThermoFisher Scientific), 6-well and 15-cm plates, respectively. The media were refreshed every 2 days during a time course.

extract_protocol:
1. ChIP-seq: All the ChIP-seq experiments, except for H3K27me3, were performed in presence of 10% HEK293T cells as an internal reference using a published protocol with small modifications (Liu et al., 2017). For chromatin preparation, the mouse embryonic stem cells were mixed with 10% HEK293T cells and cross-linked by a final concentration of 1% formaldehyde for 10 min. The cross-linking reaction was quenched using 2.0 M glycine. The cross-linked cells were then lysed and sonicated to obtain ~300 bp chromatin using Bioruptor Plus sonication device (Diagenode). For ChIP assays, antibodies were first coupled with Protein G beads (ThermoFisher Scientific), and then the sonicated chromatin were incubated overnight at 4oC with the antibody coupled Protein G beads. After over incubation, captured chromatin was washed, eluted and de-crosslinked. The released DNA fragments were purified using MiniElute PCR Purification Kit (Qiagen). The ChIP experiments were performed using the following antibodies: (1) WAPL (16370-1-AP, Proteintech), (2) CTCF (07-729, Merck Millipore), (3) RAD21 (ab154769, Abcam), (4) SOX2 (AF2018, R&D Systems), (5) OCT4 (AF1759, R&D Systems), (6) NANOG (RCAB002P-F, Cosmo Bio Co.), (7) MED1 (A300-793A, Bethyl Laboratories), (8) H3K4me3 (pAb-003-050, Diagenode), (9) H3K27ac (ab4729, Abcam), and (10) H3K27me3 (pAb-195-050, Diagenode); 2. RNA-seq: RNA was isolated following a standard TRIzol RNA isolation protocol (Ambion). The cells were lysed using 1 ml of TRIzol reagent, and 200 µl chloroform was added to the lysates. The mixture was vortexed and centrifuged at 12,000 g at 4°C for 15 min. Upper phase was homogenized with 0.5 ml of 100% isopropanol, incubated at room temperature for 10 min, and centrifuged at 4°C for 10 min. The resulted RNA pellet was washed with 75% ice-cold ethanol, dried at room temperature for 10 min, and resuspended in RNase-free water. The isolated RNA was treated with DNase using RNeasy Mini Kit (Qiagen); 3. Hi-C: Hi-C data were generated using as a previous published protocol (Rao et al., 2014) with minor modifications (Haarhuis et al., 2017). For each template, 10 million cells were harvested and crosslinked using 2% formaldehyde. Crosslinked DNA was digested in nucleus using MboI, and biotinylated nucleotides were incorporated at the restriction overhangs and joined by blunt-end ligation. The ligated DNA was enriched in a streptavidin pull-down; 4. 4C-seq was performed as previously described (Geeven et al., 2018; van de Werken et al., 2012) using a two-step PCR method for indexing described first in (Haarhuis et al., 2017). We used MboI as the first restriction enzyme and Csp6I as the second restriction fragment; 5. TT-seq libraries were prepared following a published protocol (Gregersen et al., 2020). The untreated and 500 nM treated WAPL-AID cells (6 and 24 h) were labeled using 2 mM 4SU for 10 min. Total RNA from the labeled cells was isolated and fragmented. Then 4SU labeled RNA fragments were biotinylated and enriched using streptavidin MicroBeads; 6. ATAC-seq libraries were prepared following a published protocol (Liu et al. 2017). Nuclei were isolated, permeabilized, and tagmented using in-house produced Tn5 transposase.
1. ChIP-seq: ChIP-seq libraries were prepared according to the protocol of KAPA HTP Library Preparation Kit (Roche); 2. RNA-seq libraries were prepared using a TruSeq Stranded RNA LT Kit (Illumina); 3. Hi-C libraries were prepared using a standard end-repair and A-tailing method; 4. 4C-seq libraries were prepared using a two-step PCR method for indexing described first in (Haarhuis et al., 2017); 5. TT-seq libraries were prepared using KAPA RNA HyperPrep Kits (Roche) and KAPA Dual-Indexed Adapter Kits (Roche); 6. The resulted DNA fragments underwent two sequential 9-cycle PCR amplification and the fragments < 700 bp were purified using SPRI beads (Liu et al., 2017).
Illumina Hi-C 2500 (ChIP-seq, RNA-seq and 4C-seq: 65 bp single-end), Illumina Hi-C X (Hi-C: 150 bp paired-end), Illumina NextSeq 550 (TT-seq, ATAC-seq: 75 bp paired-end)

treatment_protocol: WAPL and RAD21 depletion were induced by treating the cells with a final concentration of 500 µM IAA (I5148-10G, Sigma Aldrich). OCT4- and NANOG-FKBP proteins were depleted by adding a final concentration of 500 nM dTAG-13 molecule (requested from Dr. Nathanael S. Gray from Dana-Faber Cancer Institute) (Nabet et al., 2018). All the time series experiments were performed by inducing protein degradation at different time points and harvest the samples in the end of the time course.

tag: genetic modification

value: OsTir1; WAPL-AID-eGFP

value: Embyonic stem cell


title: HiC_RC4_d78h_DMSO

description:
processed data file: RAD21_D_d78h_all-10032020.hic
processed data file: RAD21_D_d78h_all-cas-10032020.hic
processed data file: RAD21_D_d78h_all-mus-10032020.hic

source_name: mESC

value: 8h DMSO


title: 65_Wapl-C6_0h_HiC

description:
65-66_WaplC6_hiC_10000_abs.bed
65-66_WaplC6_hiC_20000_abs.bed
65-66_WaplC6_hiC_40000_abs.bed

data_processing:
Library strategy: Hi-C
ChIP-seq: Calibrated ChIP-seq data were analyzed based on a modified method described in previously (Orlando et al., 2014). Raw sequencing data was mapped to a concatenated reference genome (mm10 and hg19) using Bowtie 2 mapper (version 2.3.4.1) (Langmead et al., 2009). The mapped reads with mapping quality score <15 and the optical PCR duplicates were discarded using SAMtools (version 1.9) (Li et al., 2009). The coverage files (bigWig files) were generated by applying the above computed scaling factor using deepTools (version 2.5.4) (Ramírez et al., 2014), and further calibrated based on average enrichment of the ChIP-seq experiments under direct comparison using their spike-in reference.; Standard ChIP-seq data were mapped to a mm10 reference. The mapped reads with mapping quality score <15 and the optical PCR duplicates were discarded using SAMtools. The coverage files of uncalibrated ChIP-seq data and the data of pluripotency factors (due to absence of these factors in HEK293T cells) were generated using “normalize to 1X genome coverage” methods in deepTools. Peak calling was performed using MACS2 (version 2.1.1.20160309) (Liu, 2014) at a q-value cutoff of 0.01.
RNA-seq: Raw RNA-seq data were mapped against mm10 reference genome using a TopHat2 pipeline (version 2.1.1) (Kim et al., 2013). The mapped reads with mapping quality score <10 were discarded using SAMtools. The read coverage for each gene in “Mus Musculus GRCm38.92” annotation file was determined using a HTSeq tool (version 0.9.1). The coverage files were generated using “normalize to 1X genome coverage” methods in deepTools. The genes with at least 20 reads in both replicates were kept for further analysis. The filtered expression data were normalized based on the size factors of the individual samples using a DESeq2 package.
Hi-C: Raw Hi-C data were mapped with HiC-Pro (Servant et al., 2015), which performs mapping, identification of valid Hi-C pairs, generation of contact matrices and ICE normalization (Imakaev et al., 2012).
4C-seq: The raw sequence data was mapped using our 4C mapping pipeline (http://github.com/deWitLab/4C_mapping ).
Genome_build: mm10
Supplementary_files_format_and_content: 1. ChIP-seq: bigWig files; 2. RNA-seq: bigWig files, HTSeq raw read count tables, DESeq2 normalized ratio tables; 3. Hi-C: Hi-C iced matrix, genome abs.bed file; 4. 4C-seq: interaction coverage file, viewpoint information file.

extract_protocol:
1. ChIP-seq: All the ChIP-seq experiments, except for H3K27me3, were performed in presence of 10% HEK293T cells as an internal reference using a published protocol with small modifications (Liu et al., 2017). For chromatin preparation, the mouse embryonic stem cells were mixed with 10% HEK293T cells and cross-linked by a final concentration of 1% formaldehyde for 10 min. The cross-linking reaction was quenched using 2.0 M glycine. The cross-linked cells were then lysed and sonicated to obtain ~300 bp chromatin using Bioruptor Plus sonication device (Diagenode). For ChIP assays, antibodies were first coupled with Protein G beads (ThermoFisher Scientific), and then the sonicated chromatin were incubated overnight at 4oC with the antibody coupled Protein G beads. After over incubation, captured chromatin was washed, eluted and de-crosslinked. The released DNA fragments were purified using MiniElute PCR Purification Kit (Qiagen). The ChIP experiments were performed using the following antibodies: (1) WAPL (16370-1-AP, Proteintech), (2) CTCF (07-729, Merck Millipore), (3) RAD21 (ab154769, Abcam), (4) SOX2 (AF2018, R&D Systems), (5) OCT4 (AF1759, R&D Systems), (6) NANOG (RCAB002P-F, Cosmo Bio Co.), (7) MED1 (A300-793A, Bethyl Laboratories), (8) H3K4me3 (pAb-003-050, Diagenode), (9) H3K27ac (ab4729, Abcam), and (10) H3K27me3 (pAb-195-050, Diagenode); 2. RNA-seq: RNA was isolated following a standard TRIzol RNA isolation protocol (Ambion). The cells were lysed using 1 ml of TRIzol reagent, and 200 µl chloroform was added to the lysates. The mixture was vortexed and centrifuged at 12,000 g at 4°C for 15 min. Upper phase was homogenized with 0.5 ml of 100% isopropanol, incubated at room temperature for 10 min, and centrifuged at 4°C for 10 min. The resulted RNA pellet was washed with 75% ice-cold ethanol, dried at room temperature for 10 min, and resuspended in RNase-free water. The isolated RNA was treated with DNase using RNeasy Mini Kit (Qiagen); 3. Hi-C: Hi-C data were generated using as a previous published protocol (Rao et al., 2014) with minor modifications (Haarhuis et al., 2017). For each template, 10 million cells were harvested and crosslinked using 2% formaldehyde. Crosslinked DNA was digested in nucleus using MboI, and biotinylated nucleotides were incorporated at the restriction overhangs and joined by blunt-end ligation. The ligated DNA was enriched in a streptavidin pull-down; 4. 4C-seq was performed as previously described (Geeven et al., 2018; van de Werken et al., 2012) using a two-step PCR method for indexing described first in (Haarhuis et al., 2017). We used MboI as the first restriction enzyme and Csp6I as the second restriction fragment.
1. ChIP-seq: ChIP-seq libraries were prepared according to the protocol of KAPA HTP Library Preparation Kit (Roche); 2. RNA-seq libraries were prepared using a TruSeq Stranded RNA LT Kit (Illumina); 3. Hi-C libraries were prepared using a standard end-repair and A-tailing method; 4. 4C-seq libraries were prepared using a two-step PCR method for indexing described first in (Haarhuis et al., 2017)


title: 66_Wapl-C6_24h_HiC


title: mNET-seq_∆RAD21_IAA_rep1


title: RNA_WH7_d4-7_DMSO.rep1

value: 3 days DMSO


title: 93_Wapl-C20_0h_RNA_rep1

description: Sample 93


title: HiC_RC4_d78h_dTAG13

description:
processed data file: RAD21_13_d78h_all-10032020.hic
processed data file: RAD21_13_d78h_all-cas-10032020.hic
processed data file: RAD21_13_d78h_all-mus-10032020.hic


title: mNET-seq_∆CTCF_IAA_rep1


title: mNET-seq_∆CTCF_IAA_rep2


title: mNET-seq_∆CTCF_UT_rep2


title: HiC_WH7_d4-7_DMSO

description:
processed data file: H_WH2_WH7_D.hic
processed data file: H_WH2_WH7_D.mus.hic
processed data file: H_WH2_WH7_D.cas.hic


title: RNA_WH7_d4-7_DMSO.rep2


title: QQ rep 9

status: Public on Sep 10 2020

city: Knoxville

first: Jonathan

last: Beever

state: Illinois

address: 2506 River Drive

department: Animal Sciences

institute: University of Tennessee

data_processing:
Adapter sequences and low-quality bases and reads were trimmed using Trimmomatic version 0.39, with the following parameters: HEADCROP:1 ILLUMINACLIP:2:30:7 LEADING:24 TRAILING:24 SLIDINGWINDOW:10:28 MINLEN:50
Reads were assessed for quality with FastQC version 0.11.9
Reads were mapped to the ARS-UCD 2.0 assembly of the bovine genome using STAR version 2.7.6a, using the following non-default parameters: --seedSearchStartLmax 31 --outFilterScoreMinOverLread 0.5 --outFilterMatchNminOverLread 0.5
Multi-mapped reads, which are assigned a map quality score of 5 or lower by STAR, were removed using samtools
Read counts were obtained using the featureCounts function of the Rsubread package version 2.12.2 [63], using the default parameters.
Genome_build: cow, ARS-UCD 2.0
Supplementary_files_format_and_content: comma-delimited text file containing raw counts for each sample generated by featureCounts.
Supplementary_files_format_and_content: tab delimited text file containing normalized abundance measurements (logCPM) for each sample obtained from edgeR after adjustment for batch effects.

organism: Bos taurus

source_name: Longissimus dorsi muscle_QQ

extract_protocol:
Total RNA was extracted using TRIzol® Reagent (Life Technologies) according to the manufacturer’s specifications; 5μg of the total RNA was purified with the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA); quality was assessed with an Agilent Bioanalyzer 2100 (Agilent Technologies); samples with RIN > 7.0 were used to construct sequencing libraries.
1μg  from each sample were used for library construction using the Illumina’s TruSeq Stranded mRNA Library Preparation Kit (Illumina); libraries were indexed, pooled and sequenced for 101 cycles on a HiSeq 2500 (single-end chemistry) using a HiSeq SBS sequencing kit version 4 (four lanes were used).

tag: haplotype

value: QQ

tag: Sex

value: Male

value: post natal day 300

tag: qtl genotype/haplotype

value: Longissimus dorsi muscle


title: QQ rep 4

value: Female


title: QQ rep 5


title: QQ rep 3


title: HiC_WH2_d4-7_DMSO

tag: cell type

tag: treatment


title: HiC_WH2_d4-7_dTAG13


title: qq rep 11

source_name: Longissimus dorsi muscle_qq

value: qq


title: qq rep 12


title: qq rep 8


title: qq rep 7


title: qq rep 9


title: HiC_RG9_d78h_dTAG13


title: qq rep 5


title: HiC_RG9_d78h_DMSO


title: mNET-seq_∆RAD21_UT_rep2

value: FLAG-Halo-CTCF / RAD21-BFP-mAID-V5 / osTir

value: Ethanol, 3 hrs


title: qq rep 2


title: sample1

status: Public on Aug 24 2020

city: Shanghai

first: l

last: y

address: No.138 Xinjun Ring Road, Minhang District

country: China

institute: OE

data_processing:
The Cell Ranger software pipeline (version 3.1.0) provided by 10×Genomics was used to demultiplex cellular barcodes, map reads to the genome and transcriptome using the STAR aligner, and down-sample reads as required to generate normalized aggregate data across samples, producing a matrix of gene counts versus cells
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every cell

source_name: murine embryonic hindlimb tissue

extract_protocol:
Retinas were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: murine embryonic hindlimb tissue


title: 99_Wapl-C20_48h_RNA_rep1

description: Sample 99


title: QQ rep 12


title: 94_Wapl-C20_0h_RNA_rep2

description: Sample 94


title: QQ rep 11


title: QQ rep 10


title: QQ rep 8


title: QQ rep 6


title: QQ rep 1


title: qq rep 10


title: QQ rep 7


title: qq rep 6


title: qq rep 3


title: 47113_K3102G: Epidermis, Ncor1/Ncor2 mutant 4

status: Public on Aug 23 2020

city: New York

first: Sarah

middle: E

last: Millar

state: NY

address: Icahn Building, Floor 13 Room 020C, 1425 Madison Ave

department: Developmental, Cell and Regenerative Biology

institute: Icahn School of Medicine at Mount Sinai

hyb_protocol: Five and a half micrograms of labeled cDNA were added to Affymetrix hybridization cocktails, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC.

scan_protocol: A GeneChip 3000 7G scanner was used to collect fluorescence signal.

data_processing: Cel files were RMA normalized in Transcriptome Analysis Console (TAC, v4.0.2, ThermoFisher)

source_name: E18.5 epidermis

label_protocol: 250ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by poly(T) and random oligomers that incorporated the T7 promoter sequence. Second-strand cDNA synthesis was followed by in vitro transcription with T7 RNA polymerase for linear amplification of each transcript, and the resulting cRNA was converted to cDNA, fragmented, assessed by Bioanalyzer, and biotinylated by terminal transferase end labeling.

growth_protocol: Pregnant dams were placed on 1g/kg doxycycline chow from gestational age E8.5 to E18.5

extract_protocol: Trizol extraction of total RNA was performed using the Qiagen RNeasy kit

treatment_protocol: Dorsal skin was floated dermis side down on dispase solution and incubated at 37ºC until the epidermis could be easily removed. Epidermis was stored in Trizol following the manufacturer's instructions

value: epidermis

tag: experiment

value: Ncor1/ Ncor2 mutant

tag: litter

value: Ncor1/Ncor2 litter 3


title: 47111_K3026A: Epidermis, Ncor1/Ncor2 control 2

tag: tissue

value: control

value: Ncor1/Ncor2 litter 2


title: 47112_K3102B: Epidermis, Ncor1/Ncor2 mutant 3

tag: age


title: 5600_65898_K1812E: Epidermis, Hdac3 control 4

value: Hdac3 litter 2


title: 5600_65899_K1812F: Epidermis, Hdac3 mutant 5

value: Hdac3 mutant


title: 5600_65893_K1674E: Epidermis, Hdac3 control 2

value: Hdac3 litter 1


title: 47115_K3102H: Epidermis, Ncor1/Ncor2 control 4


title: 5600_65897_K1812C: Epidermis, Hdac3 mutant 4


title: QQ rep 2

value: QQ


title: 5600_65894_K1674F: Epidermis, Hdac3 mutant 3


title: 47110_K3026D: Epidermis, Ncor1/Ncor2 mutant 2


title: qq rep 1


title: qq rep 4


title: 47114_K3102F: Epidermis, Ncor1/Ncor2 control 3


title: Epidermis HDAC3_ChIPseq

data_processing:
Illumina’s bcl2fastq version v2.17.1.14 software was used to convert bcl to fastq files
fastq files were aligned to GRCm38.p5 with STAR
peaks were called with homer for experimental sampls vs input
known and denovo motif enricment was calculated with homer
Genome_build: GRCm38
Supplementary_files_format_and_content: homer peak files from findPeaks vs input

source_name: epidermis

growth_protocol: Dorsal epidermis was collected from gestational age E18.5 wild-type mouse embryos

extract_protocol:
Lysates were generated from fixed embryonic epidermis and subjected to sonication. Sonicated chromatin was incubated with anti-HDAC3 or anti-KLF4 antibody. Bound chromatin was isolated and the DNA was purified.
Libraries were prepared following the ChIP Sequencing Sample Preparation Guide by Illumina, using adaptor oligomers and primers from Illumina and enzymes from New England Biolabs. The resulting libraries were purified with the PCR Purification kit (Qiagen) and MinElute kit (Qiagen).

value: wild-type

value: Abcam ab7030


title: CTCF-AID_rna_auxin_48hr_rep2

status: Public on Mar 06 2020

city: La Jolla

first: Bing

last: Ren

address: 9500 Gilman Dr., CMM-East, Admin Area/Rm 2071

institute: University of California, San Diego School of Medicine

data_processing:
fastq: Illumina's HiSeq Control Software
RNA-seq reads were aligned against the mouse mm10 genome assembly using STAR (Dobin et al., 2013). The processed data was created using HTseq (Anders et al., 2015) and used for differential expression analysis by edgeR (Robinson et al., 2010).
Genome_build: mm10
Supplementary_files_format_and_content: The processed file include all sample data of the number of read counts in each gene that were calculated using HTseq (Anders et al., 2015).

source_name: embryonic stem cells

growth_protocol: Cells were grown in DMEM supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, 15% KnockOut Serum Replacement (ThermoFisher, 10828028), Glutamax (Gibco, 35050), 0.1 mM non-essential amino acid, 0.1mM beta-mercaptoethanol, and 500 U/ml LIF at 37°C and 5% CO2

extract_protocol: RNA-seq library sequencing procedures were carried out as previously described according to Illumina HiSeq4000 protocols (Illumina, San Diego, CA).

treatment_protocol: To deplete CTCF, 1 ul 500 mM auxin per 1 ml medium was added, and medium with auxin was changed every 24 hours. Cells were harvested 24, 48 or 96 hours after stating auxin treatment. Neural progenitor cell differentiation treatment was performed with or without auxin treatment, and harvested on day 2, 4, 6 and 2 days after washing out auxin from day 4 and day 6 samples.

value: F123 mouse ES cell


title: 47108_K2304F: Epidermis, Ncor1/Ncor2 mutant 1

value: Ncor1/Ncor2 litter 1


title: 5600_65900_K1812G: Epidermis, Hdac3 control 5


title: Meis_IP_FL

status: Public on May 25 2020

city: Madrid

first: Manuel

middle: J

last: Gomez

state: Madrid

address: Melchor Fernández Almagro, 3

country: Spain

institute: CNIC

description: ChIP sample

data_processing:
Fastq files containing reads for each library were extracted and demultiplexed using Casava v1.8.2.
Illumina adapter contaminations were removed from reads using Cutadapt v1.3.
Processed reads were mapped against mm9 whole genome using bwa aln / bwa samse; samtools was used to generate output files in bam format.
Alignments were filtered with samtools to retain only properly mapped, non duplicate reads, with mapping quality above 0, not aligning to unlocalized or unassembled contigs, the mitochondrial genome, or chromosome Y.
ChIPSeq peaks were called against input using MACS2.
Genome_build: mm9
Supplementary_files_format_and_content: ENCODE narrow peak files (extended bed format) including coordinates, score and qvalue for ChIPSeq peaks called for each sample against input, using MACS2.

source_name: Forelimb

extract_protocol:
Around 500 E10.5 (36so) wild-type FLs and 500 E10.5 wild-type HLs were fixed according to standard procedures for ChIP experiments. For the IP, we used ChIP-IT High Sensitivity Columns (Active Motif). Two anti-Meis antibodies were used simultaneously, one recognizing Meis1a and Meis2a,c and the other recognizing all Meis1 isoforms.
ChIP-seq libraries were generated using the NEBNext Ultra DNA Library Prep Kit for Illumina and sequenced (single-end mode and length of 50 bp) on a HiSeq 2500 (Illumina)

value: Forelimb

tag: developmental stage


title: 5600_65895_K1812A: Epidermis, Hdac3 control 3


title: Meis_Input_FL

description: Input sample


title: CTCF-AID_rna_auxin_48hr_rep1

tag: cell line


title: 47109_K2304B: Epidermis, Ncor1/Ncor2 control 1

tag: genotype


title: KLF4 E18.5 CONT 3: Epidermis, control 5

value: litter 2


title: KLF4 E18.5 CONT 2: Epidermis, control 4


title: CTCF-AID_rna_auxin_24hr_rep1


title: 5080_55671_KLF4 E18.5 CONT 14: Epidermis, control 2

value: litter 1


title: 5600_65892_K1674D: Epidermis, Hdac3 mutant 2


title: 5600_65889_K1674A: Epidermis, Hdac3 control 1


title: 5600_65891_K1674C: Epidermis, Hdac3 mutant 1


title: 5080_55668_KLF4 E18.5 MUT 11: Epidermis, Klf4 mutant 1

value: Klf4 mutant


title: KLF4 E18.5 MUT 3: Epidermis, Klf4 mutant 5

value: epidermis


title: KLF4 E18.5 MUT 2: Epidermis, Klf4 mutant 4


title: Epidermis KLF4_ChIPseq

tag: chip antibody


title: KLF4 E18.5 MUT 1: Epidermis, Klf4 mutant 3


title: CTCF-AID_rna_auxin_96hr_rep2


title: Epidermis input DNA


title: CTCF-AID_rna_auxin_96hr_rep1


title: CTCF-AID_rna_auxin_0hr_rep1


title: CTCF-AID_rna_auxin_24hr_rep2