title: Liver-Rag1ko-CBEwt-INT-3C-HTGTS-rep2

city: Guangzhou

first: Hao

last: bingtao

address: ShataiNan Road, Baiyun District, Guangzhou, Guangdong Province

institute: Southern Medical University

data_processing:
Illumina Casava1.7 software used for basecalling.
Pair-wise chromatin interactions for 3C-HTGTS data processing. Paired-end Illumina sequencing fastq data were filtered by removing adapters and low-quality reads with using fastp (v0.20.0). Trimmed reads again were extracted from the sequence file after quality control with Cutadapt (v1.18). Paired-end reads containing nested primer or adapter primer were merged manually using restriction enzyme recognition sequences into single reads with Pear (v0.9.6), then the first digested fragment behind the bait was obtained through spliting the single reads into fragments according to restriction enzyme recognition sequences. The remaining single-end reads were aligned to the enzyme-digested mm10 reference genome with Bowtie2 (v2.4.5, parameter: -p 8 --sensitive), and mouse genome sequence (mm10) was retrieved from the UCSC and we extracted concordantly exactly alignments using SAMtools (v1.9). Self-ligation reads and off-targeted reads were filtered after mapping. For visualization, we converted the final bam files into bedGraph file using Bedtools (v2.29.2). The signal peak bedGraph file was obtained by post-comparison filtering, signal statistics, and standardization. We normalized bedGraph file using CPM (Counts Per Million in cis) normalization method, and visualized on IGV genome browser. Differential pair-wise interactions were identified by the R package R.4Cker (v1.0.0, k=30) with the function nearBaitAnalysis called to define domains of interaction with the bait and DESeq2 (v1.34.0, p < 0.05). Finally, we organized the results report and visualized it with the Bioconductor package ggplot2 (v3.3.6). The analysis of the correlation for experimental repetition used the R package corrplot(v.0.92).
Pair-wise chromatin interactions for 4C data processing. Paired-end reads were obtained through quality filtering and adapters trimming using fastp (v0.20.0). Notablely, 4C reads which contain a secondary restriction enzyme were removed crosslinks after digesting with the primary restriction enzyme MboI and ligating, so more additional fragments from secondary restriction enzyme digestion and ligation should be discarded necessarily. The first digested fragment for the primary restriction enzyme MboI behind the bait was only extracted and mapped to the enzyme-digested mm10 reference genome by Bowtie2 (v2.4.5, parameter: -p 8 --sensitive) in the form of single-end reads, and we extracted concordantly exactly alignments using SAMtools (v1.9). We again filtered the self-ligation reads and off-targeted reads, etc.. For visualization, we converted the final bam files into bedGraph file using Bedtools (v2.29.2). Reads numbers were counted and normalized by CPM (Counts Per Million in cis) normalization method per sample, and then visualized on IGV genome browser. The analysis of the correlation for experimental repetition used the R package corrplot (v.0.92).
Three-way chromatin interactions for 3C-HTGTS data processing. As the 3C-HTGTS analysis of pair-wise chromatin interactions, Paired-end Illumina sequencing reads were filtered by removing adapters and low-quality reads with using fastp (v0.20.0). Trimmed reads again were extracted from the sequence file after quality control with Cutadapt (v1.18). Paired-end reads containing Nested primer or Adapter primer were merged manually using restriction enzyme recognition sequences into single reads with Pear (v0.9.6), then multiple digested fragments for the restriction enzyme MboI behind the bait were splited and individually aligned to the enzyme-digested mm10 reference genome by Bowtie2 (v2.4.5, parameter: -p 8 --sensitive) in the form of single-end reads, and we extracted concordantly exactly alignments using SAMtools (v1.9). We converted the final bam files into bed file using Bedtools (v2.29.2). The self-ligation fragments and off-targeted fragments were filtered after mapping. Subsequently, we put all the digested fragments retrieved from the same read according to the unique ID of each read on one line, then removed the continuous fragments. In order to create contact matrices, we directly extracted the first two digested fragments except for the fragment where bait was located, or a variety of combinations of three fragments were obtained by arranging all fragments from the same read. Raw contact matrices were generated at 3-kb, 5-kb, 10-kb resolutions. For raw contact matrices correction, these interaction counts were normalized for a total of 1,000,000 interactions at the same resolutions. Similar to a Hi-C matrix, coverage was represented in a 2-dimensional matrix where each point represented the number of interactions found between two bins meaning a specific resolution. Visualization of three-way contact matrices was done with the R package GENOVA (v1.0.0) then differential analysis and visualization of local interactions from three-way interactions were obtained using the Bioconductor package DESeq2 (v1.34.0, p < 0.001). Loops seen on IGV genome browser were called using fixed-size bin resolutions from 3-kb to 10-kb. Briefly, interaction loops (contact frequencies >= 20) were identified through using raw contact frequencies. The distribution for the frequencies of unique reads containing the pair-wise and multi-way chromatin interactions was counted after aligning and filtering, and visualized with the Bioconductor package ggplot2 (v3.3.6).
Three-way chromatin interactions for 4C data processing. As the 4C analysis of pair-wise chromatin interactions, adapters of the paired-end reads were trimmed with fastp (v0.20.0). We only retained reads that were separated by sites associated with the first restriction enzyme MboI. In other words, only fragments with two or more primary restriction enzyme sites were considered for identification of multi-way interactions. Multiple digested fragments for the primary restriction enzyme MboI behind the bait were splited and individually aligned to the enzyme-digested mm10 reference genome by Bowtie2 (v2.4.5, parameter: -p 8 --sensitive) in the form of single-end reads, and we extracted concordantly exactly alignments using SAMtools (v1.9). We converted the final bam files into bed file using Bedtools (v2.29.2). We filtered the self-ligation fragments and off-targeted fragments after mapping. Subsequently, we put all the digested fragments retrieved from the same read according to the unique ID of each read on one line, then removed the continuous fragments. In order to create contact matrices, we directly extracted the first two digested fragments except for the fragment where bait was located, or a variety of combinations of three fragments were obtained by arranging all fragments from the same read. Raw contact matrices were generated at 3-kb, 5-kb, 10-kb resolutions. For raw contact matrices correction, these interaction counts were normalized for a total of 1,000,000 interactions at the same resolutions. Also similar to a Hi-C matrix, coverage was represented in a 2-dimensional matrix where each point represented the number of interactions found between two bins meaning a specific resolution. Heatmap of three-way contact matrices was done with the R package GENOVA (v1.0.0) and the distribution for the frequencies of unique reads containing the pair-wise and multi-way chromatin interactions was counted after aligning and filtering, and visualized with the Bioconductor package ggplot2 (v3.3.6).
Assembly: mm10
Supplementary files format and content: bedGraph files were generated using Bedtools (v2.29.2) with normalized values for each sample. we used the custom script to get bedpe files for interaction loops identified by using raw contact frequencies.  The custom utility was used to convert the raw contact frequencies into sparse .matrix format, and  these interaction counts were normalized for a total of 1,000,000 interactions at the same resolutions.
Library strategy: 3C-HTGTS

source_name: Liver

growth_protocol: All experiments involving mice were performed using protocols approved by  Southern Medical University Animal Studies Committee. Mice were on a C57Bl/6 genetic background . Animals were housed and bred in a specific pathogen-free animal facility.

extract_protocol: For 3C-HTGTS, mouse thymus and liver were removed, cells were filtered through nylon mech.10 million cells were cross-linked with 1% formaldehyde. Cell were lysed and digest with 200U MboI overnight at 37°C. The sample was ligated with T4 DNA ligase overnight at 16°C. Crosslinks were reversed by Proteinase K and RNase A prior to DNA extraction with 1:1 phenol-chloroform and precipitation with ethanol. The 10ug 3C DNA was sonicated to 300-500bp and was linearly amplified with a biotinylated primer. The biotin-labeled single-stranded DNA products were enriched with streptavidin C1 beads, and followed by 3’ ends ligation with the bridge adaptor. The adaptor-ligated products were amplified via nested PCR using a nested primer and an adaptor-complementary primer. And a final PCR for another 5-10 cycles with P5 and P7 was performed. After purification of the PCR products, the final libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp or 250 bp paired-end reads.

value: Liver


title: Liver-Rag1ko-CBEwt-TEAp-3C-HTGTS-rep1

status: Public on Nov 28 2022

type: SRA

country: China

library_source: genomic

molecule: genomic DNA

organism: Mus musculus

tag: strain

tag: tissue

tag: cell type

tag: genotype


title: Liver-Rag1ko-CBEwt-Ea-3C-HTGTS-rep2


title: Liver-Rag1ko-CBEwt-Ea-3C-HTGTS-rep1


title: SPL5D

city: Beijing

first: Zefu

last: Lu

state: Haidian

address: No.12 Zhongguancun South St.,Haidian District, Beijing, P.R.China

institute: Institute of Crop Sciences

data_processing:
Library strategy: DAP-seq
Raw reads were trimmed with Trimmomatic v0.36, clean sequencing reads were mapped to the International Wheat Genome Sequencing Consortium (IWGSC) reference genome (version 1.0) using Bowtie2. Peak calling was performed using MACS2. A black list was first generated by using the control samples as input. Raw peaks were first identified with MACS2 with the following parameter with the following parameter “--keep-dup all --nomodel --extsizes 150”; then split into 150bp bins with 50bp overlapping; bins passed filtering using a reads density cutoff were selected and merged with bedtools with “-d 150”; peaks overlapped with the blacklist peaks, or homologous to plant orgenella DNA (NCBI) were discard and the rest were considered as high-quality binding peaks.
Genome_build: IWGSC RefSeq v1.0
Supplementary_files_format_and_content: peak files

organism: Triticum aestivum

source_name: Triticum aestivum 2-weeks-old seedlings

growth_protocol: 16h light  22°C, 8h dark 19°C

extract_protocol: genomic DNA

treatment_protocol: Full length TF was cloned into pIX-Halo vector, the HALO-SPL protein was generated using 500 ng of pIX-HALO-SPL plasmid and the TNT SP6 Coupled Reticulocyte Lysate System (Promega, L4600) according to the manufacturer’s protocol. The reaction was then incubated with 10 μL of Magne-HALO Tag beads (Promega, G7282) for 1 h at 25 °C in 1× PBS with 0.005% Nonidet P-40 (PBST). Bound protein was washed five times with PBST and subsequently treated with DNaseI. The DNA-free HALO-SPL protein was incubated with 500 μg of ultrasonicated genomic DNA library (200~800 bp) for 1 h at 25 °C. The beads were then washed eight times with PBST and incubated with 1μl Tn5 transposomes in 40 μl of tagmentation buffer (10 mM TAPS-NaOH ph 8.0, 5 mM MgCl2) at 55°C for 10 minutes. The DNA were recovered by NEB Monarch™ DNA Cleanup Kit (T1030L) and then amplified using Phusion DNA polymerase for 10-13 cycles. Amplified libraries were purified with AMPure beads to remove primers.

value: leaves

tag: cultivar

tag: treatment

value: Magne-HALO Tag beads (Promega, G7282)


title: 10LV

city: Chicago

first: Anna

last: Di Rienzo

state: IL

department: Human Genetics

country: USA

institute: University of Chicago

library_source: transcriptomic

data_processing:
Sequence alignment was performed using a standard pipeline. Briefly, reads were aligned using STAR and read counts performed with RSEM using default settings.
Differential expression analyses performed using the r package limma-voom with default settings . All data were processed together, and contrasts made between hypoxia and normoxia for each genotype (WT or KO).
Genome_build: All mouse samples use genome build mm10.
Supplementary_files_format_and_content: rsem expression levels file (text)

molecule: polyA RNA

extract_protocol:
The following morning, both mice were removed together and euthanized one at a time in a randomly selected order via urethane injection. Mice were rapidly dissected and left kidney, right adrenal gland, left lung, left and right atria, and left and right ventricle were all independently flash frozen in either RLT Plus buffer (kidney, adrenal gland, lung) or Trizol (all heart tissues) and stored at -80C degrees. Once all tissues had been collected from all three pairs of mice, tissues were removed from -80C and homogenized. RNA was extracted one tissue at a time using RNeasy Plus Mini kit (Qiagen, 74134) in a Qiagen Qiacube for all samples stored in RLT buffer and by hand using RNeasy plus mini kit with Qiagen Dnase treatment (RNase-Free DNase Set, cat#79254).
Libraries were constructed using the TruSeq RNA Library Preparation Kit v2, Set B (Illumina, RS-122-2002)
All samples for each organ were pooled and each organ run across one lane of a hiSeq4000

treatment_protocol: At age 9 weeks the mice were placed in pairs, one KO and one WT, into a custom hypobaric hypoxia chamber . The pressure was set to 280-290 mmHg which is equivalent to ~8% O2 and the mice were left overnight (~16.5 hours)

value: Left Ventricle

value: WT control mouse

value: WT control mouse, 16.5 hours hypobaric hypoxia, 8% O2


title: Liver-Rag1ko-CBEwt-TEAp-3C-HTGTS-rep2


title: 9LV

value: musENH5 KO mouse

value: musENH5 Knockout, 16.5 hours hypobaric hypoxia, 8% O2


title: Liver-Rag1ko-CBEwt-Trav17-3C-HTGTS-rep2


title: Liver-Rag1ko-CBEwt-INT-3C-HTGTS-rep1


title: DP-Rag1ko-CBEwt-Dd2-3C-HTGTS-rep3

source_name: Thymocytes

value: Thymus

value: DP thymocytes


title: Liver-Rag1ko-CBEwt-Trav17-3C-HTGTS-rep1


title: DP-Rag1ko-CBEwt-Dd2-3C-HTGTS-rep2


title: SPL5B


title: 8LV


title: DP-Rag1ko-CBEwt-Dd2-3C-HTGTS-rep1


title: SPL5A


title: 7LV


title: AvitagRpb1R749H_4 3'READS

city: Aurora

first: David

last: Bentley

state: Colorado

department: BMG

institute: UCD AMC

description: 3' READS mRNAseq

data_processing:
For 3'READS adapters and the initial 4 nt, which consisted of random sequence added during library construction were removed  Reads for which the first non-random nt was not a T, corresponding to an A at the 3’ end of the sequenced mRNA fragment, was discarded. .    The initial Ts were removed and the read truncated to 77nt
Reads were aligned using Bowtie2 version 2.3.2, no mismatches were allowed in the 30 bases upstream of the polyA site
Read coordinates were collapsed to a single base pair coordinate corresponding to the last base before the A''s at the RNA 3’ end
Assembly: GRCh19/hg19
Supplementary files format and content: .txt

organism: Homo sapiens

source_name: HEK293 Flp-in T-REx pcDNA5 B10 Rpb1 Amr R749H

growth_protocol: DMEM 10% FCS, +200 ug/ml hygromycin, +6.5 ug/ml blasticidin, +pen/strep

extract_protocol:
mRNA was Trizol extracted and polyA selected
3'READs Jin et al Curr. Prot. Mol Biol. 110:4231-42317 (2015)

treatment_protocol: Rpb1 R749H expression was induced with 2ug/ml doxycycline for 24 hrs, followed by treatment with a-amanitin (2.5 ug/ml) for 42 hrs.

tag: cell line

tag: growth conditions

value: DMEM 10% FCS, +hygromycin, +blasticidin, +doxycycline

tag: antibody

tag: moleculesubtype

value: mRNA


title: 13RA

value: Right Atrium


title: DP-Rag1ko-CBEko-Ea-3C-HTGTS-rep3


title: AvitagRpb1R749H_3 3'READS

description: 3' READS mRNAseq


title: SPL4D


title: 6LA

value: Left Atrium


title: DP-Rag1ko-CBEko-Ea-3C-HTGTS-rep2


title: 5LA


title: DP-Rag1ko-CBEko-Ea-3C-HTGTS-rep1


title: AvitagRpb1R749H_1 3'READS

description: 3' READS mRNAseq


title: 3LA


title: 4LA


title: SPL3D


title: DP-Rag1ko-CBEwt-Ea-3C-HTGTS-rep2


title: SPL3B


title: DP-Rag1ko-CBEwt-Ea-3C-HTGTS-rep3


title: 2LA


title: RNA-seq_Young, replicate2

city: Brisbane

first: He

last: Huang

state: QUEENSLAND

address: THE UNIVERSITY OF QUEENSLAND, ST LUCIA

country: Australia

institute: The University of Queensland

data_processing:
STAR version 2.7.8a was used for building genome indices and mapping.
Adapter trimming was performed using trimgalore version 0.6.6.
consensusDE version 1.8.0 was used for summarizing read counts and differential expression analysis using R version 4.05.
consensusDE simultaneously conducts statistical analysis using edgeR (version 3.32.1) with the additional option to remove unwanted sources of variation with RUV.
Assembly: mm39

molecule: total RNA

source_name: Hippocampus tissue

extract_protocol:
Total RNA extraction was performed from each individual hippocampal tissue with NucleoZol and NucleoSpin RNA Set (Macherey-Nagel) with rDNase treatment.
libraries were generated using NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB).

value: Hippocampus tissue

tag: Sex

value: Male

tag: age


title: AvitagRpb1WT_4 3'READS

description: 3' READS mRNAseq

source_name: HEK293 Flp-in T-REx pcDNA5 B10 Rpb1 Amr WT

treatment_protocol: WT Rpb1 expression was induced with 2ug/ml doxycycline for 24 hrs, followed by treatment with a-amanitin (2.5 ug/ml) for 42 hrs.


title: 1LA


title: AvitagRpb1WT_3 3'READS

description: 3' READS mRNAseq


title: SPL2D


title: AvitagRpb1WT_5 3'READS

description: 3' READS mRNAseq


title: DP-Rag1ko-CBEko-TEAp-3C-HTGTS-rep3


title: Monkey NP4

city: Suita

first: Noriyuki

last: Tsumaki

state: Select One

address: 2-2 Yamadaoka

department: Department of Biochemistry and Molecular Biology

country: Japan

institute: Osaka University, Graduate School of Medicine

description: sample 2

data_processing:
Pair-end Fastq files (R1: cell barcode reads, R2: RNA reads) were processed as follows. Adapter trimming of sequencing data was performed using cutadapt 3.4. Filtered reads were chunked into 16 parts for parallel processing by using Seqkit 0.14.0. Filtered cell barcode reads were annotated by Python script provided by BD Biosciences with minor modification for compatibility to Python 3.7. Reference RNA sequences were built by concatenating cDNA and ncRNA fasta files of the Ensembl database (build Macaca_fascicularis_6.0 for macaca fascicularis data, and GRCh38 release-101 for human data). Associated cDNA reads were mapped to reference RNA using bowtie2-2.4.2 by the following parameters: -p 2 --very-sensitive-local -N 1 -norc -seed 656565 -reorder. Then, cell barcode information of each read was added to the bowtie2-mapped BAM files by the python script and pysam 0.15.4, and read counts of each gene in each cell barcode were counted using mawk. Resultant count data was converted to a single-cell gene-expression matrix file by using R-3.6.3. The inflection point of the knee-plot (total read count versus the rank of the read count) was detected using DropletUtils package in R-3.6.3. Cells of which total read count was over inflection point were considered as valid cells.
Genome_build: Macaca_fascicularis_6.0 for macaca fascicularis data, and GRCh38 release-101 for human data
Supplementary_files_format_and_content: tab-delimited text files include raw tag-count values for each sample
Supplementary_files_format_and_content: For cell name description, each cell was named as follows: [hashtag ID]_[experiment ID]_[cell barcode].

organism: Macaca fascicularis

source_name: Nucleus pulposus

extract_protocol:
AC, pre-implant hiPS-Cart, and post-implant hiPS-Cart were minced into 1-2 mm pieces. Then the pieces and NP were digested with Liberase solution (for NP, AC, and post-implant hiPS-Cart: RPMI-1640 (Nacalai Tesque) supplemented with 0.2% FBS, 10 mM HEPES pH7.2-7.4, 0.2-0.4 mg/mL Liberase TM (Roche), and 2 kU/mL DNase I (Merck); for pre-implant hiPS-Cart: DMEM with 1% FBS, 1% ITS-X, 50 µg/mL ascorbic acid, 1 mM sodium pyruvate, 1% nonessential amino acids, 1% penicillin streptomycin, 10 ng/mL TGF-β1, 10 ng/mL GDF5, and 10 ng/mL BMP2, 0.2 mg/mL Liberase TM (Roche), and 2 kU/mL DNase I (Merck)) at 37 °C, 5% CO2 for 120-210 minutes with sustained shaking. The cells were agitated 10 times with 1000 μL blue tip and then passed through a 70 μm cell strainer (BD Biosciences), centrifuged at 4°C for 300×g for 5 minutes, and their supernatant was discarded. The cells were resuspended in RPMI-1640 supplemented with 0.2% FBS and 10 mM HEPES.
For cell hashing, we biotinylated cell surface proteins with EZ-Link Sulfo-NHS-Biotin (Thermo Scientific), followed by staining with 0.6 μg/mL of Totalseq (A0951-A0955, and A0436 (BioLegend)) (Details of the method will be described elsewhere.) Cells that were alive and stained with A0951-A0955 were selected by flow cytometry using a FACS Aria II flow cytometer (BD Biosciences) and suspended in Sample Buffer (BD Biosciences).
TAS-Seq libraries were generated by Immunogeneteqs Inc. (Noda city, Chiba, Japan) according to a previous report: S. Shichino, S. Ueha, S. Hashimoto, T. Ogawa, H. Aoki, W. Bin, . . . K. Matsushima, TAS-Seq: a robust and sensitive amplification method for beads-based scRNA-seq, bioRxiv  (2021).
BD Rhapsody scRNA-seq

treatment_protocol: hiPS-Cart was created from human iPS cells (hiPSCs). hiPS-Cart was implanted into intervertebral disks of nude rats for six weeks.

value: 3 years old

value: wild type

value: Nucleus pulposus

tag: samples


title: AvitagRpb1WT_2 3'READS

description: 3' READS mRNAseq


title: SPL2B

value: leaves

value: Magne-HALO Tag beads (Promega, G7282)


title: SPL4B


title: AvitagRpb1R749H_2 3'READS

description: 3' READS mRNAseq


title: SPL2A_rep2


title: DP-Rag1ko-CBEwt-Ea-3C-HTGTS-rep1


title: SPL3A


title: AvitagRpb1WT_1 3'READS

description: 3' READS mRNAseq


title: Monkey NP1,2 and AC1,2

description: sample 1

source_name: Nucleus pulposus (NP), Articular cartilage (AC)

value: NP1, NP2, AC1, AC2


title: DP-Rag1ko-CBEko-TEAp-3C-HTGTS-rep1


title: SPL4A


title: DP-Rag1ko-CBEko-TEAp-3C-HTGTS-rep2


title: AvitagRpb1 R749H_2 NETseq

description: anti-pol II mNET-seq

data_processing:
For eNETseq adapters were trimmed using cutadapt version 2.3
Reads were aligned using Bowtie2 version 2.3.2
PCR duplicates were removed UMI-tools v0.5.4
Read coordinates were collapsed to a single base pair coordinate corresponding to the RNA 3’ end
Assembly: GRCh19/hg19
Supplementary files format and content: BigWig
Library strategy: NET-seq

source_name: HEK293 Flp-in T-REx pcDNA5 Avitag Rpb1 Amr R749H

extract_protocol:
Pol II-associated nascent RNA was isolated by IP
10-20ng of RNA was used with the QIAseq miRNA  kit (#331565)

treatment_protocol: Avi Rpb1 expression was induced with 2ug/ml doxycycline for 24 hrs.

value: Rabbit anti-avi-tag (GenScript A00674)

value: Nascent RNA


title: RBC,  45 min- 3

city: Odense M

first: Jens

middle: Sivkær

last: Pettersen

state: Danmark

address: Campusvej 55

country: Denmark

institute: University of Southern Denmark

data_processing:
Raw reads from the fastq files were mapped to the genome with bowtie2 using local alignment. Samtools was used for SAM-to-BAM conversion and sorting.
Raw reads were counted at a feature level with featureCounts. Multimapping reads were counted and fractionally assigned to their features. Reads overlapping several features were assigned by largest overlap.
Parameters used with featureCounts (example): featureCounts -p -M --fraction -O --largestOverlap -t gene -f -g  Name  --extraAttributes locus_tag,gene_biotype -a CP027540.1.gff3 -s 2 -o featureCounts_report FILENAMES
Assembly: CP027540.1 (several sRNA features identified in D39W were manually added to the General Feature Format (GFF)-file).
Supplementary files format and content: File contains read counts for all features of S. pneumoniae D39V in all samples (file format: csv). In addition to feature coordinates, the file also contains locus_tags, names, gene_biotypes.

organism: Streptococcus pneumoniae

source_name: Bacterial cell

growth_protocol: Streptococcus pneumoniae D39V was grown in a chemically-defined medium (CDM) supplemented with 0.5 % glucose prior to inoculation of CDM mixed with 20 % of the respective human biological samples.

extract_protocol:
RNA was extracted from Streptococcus pneumoniae by a phenol-chloroform extraction.
1 µg RNA was depleted for ribosomal RNA using the NEBNext® rRNA Depletion Kit (Bacteria). RNA libraries were then prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit.

value: Bacterial cell

value: CDM with 20 % human RBC suspension

tag: time

value: 45 min


title: RBC,  45 min - 2


title: SPL2A_rep1


title: DP-Rag1ko-CBEwt-TEAp-3C-HTGTS-rep3

value: Thymus

value: DP thymocytes


title: AvitagRpb1 R749H_1 NETseq

description: anti-pol II mNET-seq

value: DMEM 10% FCS, +hygromycin, +blasticidin, +doxycycline


title: SPL1D_rep2


title: DP-Rag1ko-CBEwt-TEAp-3C-HTGTS-rep2


title: RBC,  45 min - 1


title: SPL1D_rep1


title: AvitagRpb1WT_2 NETseq

description:
anti-pol II mNET-seq
GSE202749, GSM6132197

source_name: HEK293 Flp-in T-REx pcDNA5 Avitag Rpb1 Amr WT


title: AvitagRpb1WT_1 NETseq

description:
anti-pol II mNET-seq
GSE202749, GSM6132196


title: HIP, 45 min- 3

value: CDM with 20 % human heat-inactivated plasma


title: AK58_Axillary bud_ATAC-seq_rep2

data_processing:
Raw reads were trimmed with Trimmomatic v0.36, clean sequencing reads were mapped to the International Wheat Genome Sequencing Consortium (IWGSC) reference genome (version 1.0) using Bowtie2. Peak calling was performed using MACS2. A black list was first generated by using the control samples as input.The raw peaks were first called with MACS2 with the following parameter “--keep-dup all --nomodel --extsizes 150 --shift -75”; then split into 150bp bins with 50bp overlapping; bins passed filtering by a Tn5 integration site density cutoff were selected and merged with bedtools with “-d 150”; peaks overlapped with the blacklist peaks, or homologous to plant orgenella DNA (NCBI) were discard  and the rest  were considered as high-quality ACRs.
Genome_build: IWGSC RefSeq v1.0
Supplementary_files_format_and_content: peak files

source_name: Triticum aestivum 6-weeks-old seedlings

extract_protocol: ATAC-seq libraries were prepared following the ATAC-seq protocol as previously described in (Lu et al. 2016)

treatment_protocol: The three tissues of wheat, including leaves, roots and shoot apices, were obtained after 2 weeks of growth. The 2-week-old seeding were vernalized at 4°C under 16h light/8h dark conditions for 30 days and 1.0 - 2.0 cm axillary buds were obtained from these vernalized seedings.

value: axillary buds

value: vernalized at 4°C under for 30 days


title: DP-Rag1ko-CBEwt-TEAp-3C-HTGTS-rep1


title: DP-Rag1ko-CBEwt-INT-3C-HTGTS-rep3


title: HIP,  45 min - 1


title: DP-Rag1ko-CBEwt-INT-3C-HTGTS-rep1


title: HIP,  45 min - 2


title: C283T_transcriptome_2

city: Rockville

first: Jiyeon

last: Choi

state: Maryland

address: 9615 Medical Center Drive, Rm 3116

institute: NCI

description: Column headings of the supplementary file: NCBI_Entrez_Gene, Gene_symbol, Melanoma_1_raw_count, Melanoma_2_raw_count, Melanoma_3_raw_count, Melanocyte_1_raw_count, Melanocyte_2_raw_count, Melanocyte_3_raw_count

data_processing:
Illumina Casava1.7 software was used for basecalling and the BCL (base calls) binary is converted into FASTQ utilizing illumina package bcl2fastq
FASTQ files were used as input to Rsubread program
Rsubread was used for alignment and quantification of transcript expression using RefSeq gene annotation for human genome GRCh38/hg38 (build 38.2)
Assembly: GRCh38
Supplementary files format and content: Tab-delimited text files of Rsubread quantificiation include Entrez gene ID (first column) and corresponding raw read count values (second column) for each Sample

extract_protocol:
Total RNA was isolated using Qiagen RNeasy kit, and mRNA was subsequently isolated using PolyA purist MAG kit.
Illumina Truseq stranded mRNA library prep kit was used following manufacturer's protocols

value: Immortalized melanocyte


title: C283T_transcriptome_1


title: DP-Rag1ko-CBEwt-INT-3C-HTGTS-rep2


title: Plasma, 45min- 3

value: wild type

value: CDM with 20 % human plasma


title: AK58_Shoot apex_ATAC-seq_rep1

description: Ta-VY.final.bed

value: shoot apcies


title: C283T_transcriptome_3


title: UACC903_transcriptome_3

value: Melanoma cell line


title: AK58_Shoot apex_ATAC-seq_rep2

description: Ta-VY.final.bed


title: Plasma, 45 min - 2


title: AK58_Root_ATAC-seq_rep2

description: Ta-R.final.bed

value: roots


title: DP-Rag1ko-CBEko-Trav17-3C-HTGTS-rep3


title: UACC903_transcriptome_2


title: Plasma, 45 min - 1


title: AK58_Root_ATAC-seq_rep1

description: Ta-R.final.bed


title: CSF, 45 min- 3

value: CDM with 20 % human CSF


title: UACC903_transcriptome_1


title: DP-Rag1ko-CBEko-Trav17-3C-HTGTS-rep1


title: DP-Rag1ko-CBEwt-Trav17-3C-HTGTS-rep3


title: AK58_Leaf_ATAC-seq_rep2

description: Ta-L.final.bed

value: leaves


title: AK58_Leaf_ATAC-seq_rep1

description: Ta-L.final.bed


title: MPRA_Input_2

data_processing:
Illumina Casava1.7 software was used for basecalling and the BCL (base calls) binary is converted into FASTQ utilizing illumina package bcl2fastq
The number of reads (R1) completely matching 12bp barcode sequences (tag counts) plus 2bp adjacent spacer sequences were counted
Tag counts were converted to tag counts per million reads (TPM) for each sample and DNA input
A pseudo count of 1 was added to TPM of each tag (adjusted TPM). Adjusted TPM of each tag from each sample was normalized to adjusted TPM from DNA input to obtain normalized expression levels of each tag
Melanoma_T1, T2, and T3 were normalized to Input_1; Melanoma_T4, T5, T6, T7,T8, and Melanocyte_T1 were normalized to Input_2; Melanocyte_T2,T3,T4,and T5 were normalized to Input_3
Supplementary files format and content: Tag abundance measurements are included in *_count_TPM.txt files with column headings of Tag:14bp tag sequence, strand: minus strand, Depth: count of the reads matching the 14bp tag sequence, TotalCount: total read counds of the sample matching any tag sequence, TPM: Depth normalized over the TotalCount times one million

source_name: DNA construct

extract_protocol:
Total RNA was isolated using Qiagen RNeasy kit, and mRNA was subsequently isolated using PolyA purist MAG kit.
cDNA was synthesized from mRNA using Superscript III reverse transcriptase, from which short sequences encompassing 12 bp unique tags were amplified using Q5 high-fidelity polymerase and primers introducing Illumina TruSeq adapter sequences.

value: DNA construct


title: DP-Rag1ko-CBEwt-Trav17-3C-HTGTS-rep2


title: MPRA_Input_3


title: CSF, 45 min - 1

value: Bacterial cell


title: MPRA_Input_1


title: OA3.Ts cells, mock-infected [miRNA 3]

city: Kunming

first: Zhuoran

last: Li

state: Yunnan

address: Fengyu Road

department: Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory

institute: Yunnan Animal Science and Veterinary Institute

data_processing:
The miRNA raw data were processed through an in-house program, ACGTA101-miR (v4.2) (LC Science, USA) to remove adapter dimers, junk, low complexities, common RNA families (rRNA, tRNA, snRNA, and snoRNA), repeats, and sequences <18 nt or >26 nt in length were filtered out using Rfam (v13.0) and Repbase Update.
Subsequently, the unique sequences with a length of 18⁓26 nt were mapped to miRNA sequences in miRBase (v22.0).
Mapping was also performed on pre-miRNA against Ovis aries genomic data (GCA_000298735.1). The unique sequences that aligned to the known miRNA sequences in miRBase (v22.0) were identified as known miRNAs.
Secondary structure of pre-miRNAs was presented as a hairpin, including 5p- and 3p- derived miRNA. The unique sequences mapping to the other arm of the pre-miRNA sequences, which were not annotated in the miRBase (v22.0), were considered to be 5p- or 3p- derived miRNA candidates.
The remaining unmapped sequences were matched to the Ovis aries genomic sequences (GCA_000298735.1) in search of candidate novel miRNAs. To identify the results of putative miRNAs of Ovis aries, all the obtained miRNAs were used to predict the secondary structures using RNAfold software.
Assembly: Ovis aries (GCA_000298735.1)
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

organism: Ovis aries

source_name: embryonic testicular

growth_protocol: OA3.Ts cells were maintained in F12: DMEM =1: 1 medium supplemented with 10% fetal bovine serum (FBS) in humidified atmosphere with 5% CO2 at 37℃.

extract_protocol:
Total RNA from each sample was extracted with TRIzol reagent (Invitrogen, USA). Approximately 1 μg of total RNA were used to prepare miRNA library according to protocol of TruSeq small RNA Sample Prep Kits (Illumina, USA).
Briefly, the miRNA molecules were ligated to a 5’ adaptor and a 3’ adaptor by T4 RNA ligase (Promega, USA). Subsequently, the adaptor-ligated miRNAs were reverse transcribed into cDNA and PCR amplified. PCR products were purified and DNA fragments of 150±10 bp (the length of small RNA inserts plus the 5’ and 3’ adaptors) were quantified following the Solexa sequencing protocol (Illumina).

treatment_protocol: OA3.Ts cells were infected with BTV-1 Y863 strain with a multiplicity of infection (MOI) of 1. After 1 h of incubation, the supernatants were discarded. OA3.Ts cells were then maintained with F12: DMEM =1: 1 medium supplemented with 1% FBS for 24 h. The uninfected OA3.Ts cells served as the mock-infected controls.

value: embryonic testicular

value: embryonic testicular cells

value: mock-infected


title: CSF, 45 min - 2

value: 45 min


title: DP-Rag1ko-CBEwt-Trav17-3C-HTGTS-rep1

value: Thymus

value: DP thymocytes


title: DP-Rag1ko-CBEko-Trav17-3C-HTGTS-rep2


title: OA3.Ts cells, mock-infected [miRNA 2]


title: OA3.Ts cells, mock-infected [miRNA 1]


title: hKO1(-)_rep2

first: Kit-yeng

last: Sheng

address: Yamadaoka 2-2

institute: Osaka University

data_processing:
Illumina NovaSeq 6000 platform in 101-base single-end mode
Sequence mapped using TopHat software (v 2.0.13) combined with Bowtie2 (v 2.2.3) and SAM tools (v 0.1.19).
FPKM calculated using Cufflinks software (v 2.2.1)
Genome_build: mm10
Supplementary_files_format_and_content: count, fpkm

source_name: Neural cells

extract_protocol:
hKO1(+) brain regions (hyphothalamus, midbrain, hindbrain) were micro-dissected and sorted by FACS. hKO1(+) and hKO1(-) cells were collected in TRIzol-LS. Total RNA extraction done using Direct-zol RNA microprep kit (Zymo Research) and eluted in 8uL for library construction.
Library preparation was prepared using SMARTer Ultra-Low RNA Kit (Clontech) and cDNA amplified according to manufacturer's protocol.

value: post natal 3 months

value: brain

tag: cell population


title: C283T_MPRA_T5

value: Immortalized melanocyte


title: CDM, 45 min - 2

value: wild type

value: CDM


title: hKO1(-)_rep1


title: OA3.Ts cells, BTV-1 Y863 strain infected for 24 h [miRNA 3]

value: BTV-1 Y863 strain infected for 24 h


title: CDM, 45 min- 3


title: CDM, 45 min - 1


title: hKO1(+)_rep2


title: hKO1(+)_rep1


title: C283T_MPRA_T4


title: OA3.Ts cells, BTV-1 Y863 strain infected for 24 h [miRNA 2]


title: RBC, 15 min- 3

value: CDM with 20 % human RBC suspension

value: 15 min


title: AK58_Axillary bud_ATAC-seq_rep1

value: axillary buds

value: vernalized at 4°C under for 30 days


title: C283T_MPRA_T2


title: R342X Wild Type 4

city: Ottawa

first: Dave

last: Picketts

state: Ontario

address: 501 Smyth Road

department: Regenerative Medicine

country: Canada

institute: OHRI

data_processing:
Quantified transcripts from GENCODE vM19 with salmon v0.12.0 using --gcBias flag
Performed fold change analysis using DESeq2 as follows
Read transcript quantification data with tximport library and summarized to gene level quantification
Filtered to retain genes with at least 5 reads assigned in at least two samples
Constructed DESeqDataSet object with eight samples (four replicates of two conditions from two litter), using the model ~litter + condition (condition is cell type/treatment)
Calculated fold changes using the lfcShrink() function with the apeglm method.
Genome_build: GRCm38
Supplementary_files_format_and_content: salmon transcript quantification: Quantifrication of GENCODE vM19 transcripts using salmon v0.12.0
Supplementary_files_format_and_content: DESeq2 fold change results: Results from the DESeq2 lfcShrink() function comparing two conditions, including results for all genes retained after filtering

source_name: Mouse brain cortex tissue

extract_protocol:
RNA was extracted from flash frozen mouse cortex, according to the TRIzol Reagent protocol (Thermo Fisher Scientific)
mRNA libraries were prepared using NEBNext dual adapters (NEBNext multiplex oligos for Illumina (Dual index primers set 1)).

tag: background

value: FVB/N

value: Mouse brain cortex

tag: genotype/variation


title: C283T_MPRA_T1


title: RBC, 15 min - 1

tag: strain

value: Bacterial cell


title: R342X Wild Type 3

tag: tissue


title: UACC903_MPRA_T8

tag: cell type

value: Melanoma cell line


title: HIP, 15 min- 3

tag: genotype

value: CDM with 20 % human heat-inactivated plasma


title: HIP, 15 min - 2


title: HIP, 15 min - 1


title: R342X Wild Type 2


title: C283T_MPRA_T3


title: R342X Wild Type 1


title: UACC903_MPRA_T7


title: Plasma, 15 min- 3

value: CDM with 20 % human plasma


title: R342X Mutant 4

value: mutant


title: UACC903_MPRA_T5


title: UACC903_MPRA_T4


title: Plasma, 15 min - 2


title: Ventral Tegmental Area Projecting VP Biol rep 3

city: Baltimore

first: Mahashweta

last: Basu

state: MD

address: 670 W. Baltimore Street

institute: Institute for Genome Sciences

data_processing: none provided by the submitter

source_name: ventral pallidum

extract_protocol: RNA was extracted with a DNase step (Qiagen) using the RNeasy mini kit (Qiagen #74104) by adding lysis buffer supplemented with beta mercaptoethanol.

treatment_protocol: Polyribosomes were immunoprecipitated from VP of NPAS-RiboTag and RiboTag mice with AAV-Cre infused in the VP. 2 15-gauge NAc tissue punches per mouse (5 mice pooled/sample) three weeks following AAVinfusions. Tissue punches were homogenized by douncing in 1 mL homogenization buffer, and the supernatant was incubated with 5 µl anti-HA antibody (Covance #MMS-101 R) at 4ºC overnight with constant rotation. Samples were then incubated with 400 uL of protein G magnetic beads (Life Technologies #100.09D) overnight at 4ºC with constant rotation. Beads were washed in a magnetic rack with high-salt buffer.

value: ventral pallidum

value: male

value: VTA Proj VP

value: AAV-Rg-Cre and Ribotag Mouse Line


title: Ventral Tegmental Area Projecting VP Biol rep 2


title: Ventral Tegmental Area Projecting VP Biol rep 1


title: R342X Mutant 3


title: RBC, 15 min - 2

value: wild type


title: Medial Dorsal Thalamus Projecting VP Biol rep 4

value: MDT Proj VP


title: Plasma, 15 min - 1


title: OA3.Ts cells, BTV-1 Y863 strain infected for 24 h [miRNA 1]

value: embryonic testicular

value: embryonic testicular cells

value: BTV-1 Y863 strain infected for 24 h


title: Ventral Tegmental Area Projecting VP Biol rep 4


title: R342X Mutant 2


title: UACC903_MPRA_T3


title: Medial Dorsal Thalamus Projecting VP Biol rep 2


title: CSF, 15 min- 3

value: CDM with 20 % human CSF

value: 15 min


title: Low n-6/n-3 postnatal PUFA exposure

city: Oklahoma City

first: Michael

middle: C.

last: Rudolph

state: OK

address: 975 NE 10th St. BRC North rm 364

department: Biochemistry and Physiology

institute: OUHSC

description: adipose tissue was enzymatically digested producing a stromal vasular pellet tha was flow sorted for adipocyte precursor cells

data_processing:
10x Genomics CellRanger Count function was used on primary FastQ files for initial QC and reads alignment
CellRanger Aggregate function was applied to coalesce outputs from the Count Function as a single output
Seurat version 3.0.1 was used for filtering, normalization, scaling, and analyses
Genome_build: Mm10
Supplementary_files_format_and_content: Matrix, features, and barcode files

source_name: Flow-sorted adipocyte precursor cells

growth_protocol: WT pups were crossfostered onto dams producing high or low n-6/n-3 PUFA ratio milk fat

extract_protocol:
3000 fresh, living, sorted adipocyte precursor cells (per condition) were isolated from whole subcutaneous adipose tissue were input directly to 10x Genomics cell capture.
10x Genomics 3prime V2

treatment_protocol: Cells were pelleted, washed, and cryopreserved in FBS +10% DMSO. After thawing at 37°C, permeable cells were removed after staining with a fixable permeability dye.

value: 12-day old cross-fostered

value: subcutaneous adipose tissue

value: Flow-sorted adipocyte precursor cells

tag: pufa ratio

value: low n-6/n-3


title: UACC903_MPRA_T2

value: Melanoma cell line


title: R342X Mutant 1

value: FVB/N

value: Mouse brain cortex

value: mutant


title: CSF, 15 min - 2

value: Bacterial cell

tag: treatment


title: CSF, 15 min - 1


title: WT6.H

description: Sustain Hypoxia RNA-seq (teloHAECs): For sustained hypoxia experiments, three independent clonal isolates of ENH-5 KO and WT control teloHAECs were plated at very low confluence in two sets of 6-well plates and allowed to adhere overnight. The following day, one set of plates were placed in a humidified incubator contained within the box, and the entire box was maintained at 37C, 5% CO2, 1%O2. The cells were fed every two days for 14 days and then harvested. RNA-seq library prep and sequencing were performed at the University of Chicago Genomics core and run together on the Illumina NovaSEQ (PE 100bp).

data_processing:
sequence alignment and gene expression value estimation was performed using Sequence alignment was performed using a standard pipeline. Briefly, reads were aligned using STAR and read counts performed with RSEM using default settings.
Differential expression analysis was performed with the R packages limma-voom and edgeR with standard settings. All data were assessed in a model including an interaction term (~genotype+condition+genotype:condition)
Genome_build: All human samples use genome build hg19
Supplementary_files_format_and_content: rsem expression levels file (text)

source_name: teloHAEC

extract_protocol:
RNA was Harvested using RLT plus buffer (qiagen, Rneasy Plus mini kit)
RNA-seq library prep and sequencing were performed at the University of Chicago Genomics core and run together on the Illumina NovaSEQ (PE 100bp).
all samples were pooled and run together.

value: telomerase-immortalized aortic endothelium, WT control

value: Aortic endothelium

value: Hypoxia 14 days


title: Lateral Hypothalamus Projecting VP Biol rep 4

value: ventral pallidum

value: male

value: LH Proj VP

value: AAV-Rg-Cre and Ribotag Mouse Line


title: Medial Dorsal Thalamus Projecting VP Biol rep 1

value: MDT Proj VP


title: UACC903_MPRA_T1

tag: cell line


title: Medial Dorsal Thalamus Projecting VP Biol rep 3


title: High n-6/n-3 postnatal PUFA exposure

value: 12-day old cross-fostered

value: High n-6/n-3


title: CDM, 15 min- 3

value: wild type

value: CDM

value: 15 min


title: 119Minus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_preadipocytes.xlsx
119minus

data_processing:
BBDuk (version 38.86) trim adapter sequences
salmon (version 0.14.2) quantify trimmed reads at the transcript level
R programming environment (version 4.0.2)
DESeq2 (version 1.28.1)
Genome_build: mm10; Mus_musculus.GRCm38.79
Supplementary_files_format_and_content: excel spreadsheet containing gene level quantification for all genes from all samples

source_name: preadipocyte APC High n6/n3

growth_protocol: Cross-fostered wildtype pups were exposed postnatally to milk containing either High or Low n6/n3 PUFA ratios during initial 12-days of life

extract_protocol:
RNA was isolated using Duet Kit according to Zymogen Research protocol
RNA libraries were prepared for sequencing using standard Illumina protocols
NuGEN universal Plus mRNA SEQ protocol part no. 9133

treatment_protocol: Adipocyte precursor cells were isolated by flow cytometry. CD24+ and CD24- ("plus" = progenitors and "minus" = preadipocytes) were sequenced following 10 days of early life high or low n6/n3 exposure.

value: subcutaneous adipose tissue

value: preadipocytes

value: Low n6/n3


title: Lateral Hypothalamus Projecting VP Biol rep 3


title: CDM, 15 min - 2

value: Bacterial cell


title: Lateral Hypothalamus Projecting VP Biol rep 1


title: WT4.H

growth_protocol: For sustained hypoxia experiments, three independent clonal isolates of ENH-5 KO and WT control teloHAECs were plated at very low confluence in two sets of 6-well plates and allowed to adhere overnight.

treatment_protocol: The following day, one set of plates were placed in a humidified incubator contained within the box, and the entire box was maintained at 37C, 5% CO2, 1%O2. The cells were fed every two days for 14 days

value: telomerase-immortalized aortic endothelium, WT control


title: CDM, 15 min - 1


title: 114Minus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_preadipocytes.xlsx
114minus


title: Lateral Hypothalamus Projecting VP Biol rep 2


title: WT5.H

value: Aortic endothelium


title: 133Minus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_preadipocytes.xlsx
133minus


title: KO4.H

value: telomerase-immortalized aortic endothelium, crispr-modified KO

value: Hypoxia 14 days


title: UACC903_MPRA_T6

value: Melanoma cell line


title: Habneula Projecting VP Biol rep 4

value: ventral pallidum

value: male

value: Hb Proj VP


title: T48HKm-3_L3_712E35

city: Lanzhou

first: jinqiang

last: huang

address: Yintanlu Stress

institute: Gansu Agricultural University

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

organism: Oncorhynchus mykiss

source_name: head kidney tissues

extract_protocol:
The samples were collected immediately, then flash-frozen in liquid nitrogen,and total RNA was extracted using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: head kidney tissues

value: virus challenge

tag: phenotype

value: wild-type


title: 108Minus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_preadipocytes.xlsx
108minus


title: Habneula Projecting VP Biol rep 3

value: AAV-Rg-Cre and Ribotag Mouse Line


title: KO3.H


title: T48HKm-2_L3_711E35


title: T48HKm-1_L3_710E35


title: Habneula Projecting VP Biol rep 2


title: WT6.N

value: Normoxia control 14 days


title: 1015Minus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_preadipocytes.xlsx
1015minus

value: subcutaneous adipose tissue

value: High n6/n3


title: WT5.N


title: C48HKm-3_L3_S20E35

value: control


title: 133Plus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_progenitors.xlsx
133Plus

source_name: progenitor APC Low n6/n3

value: progenitors

value: Low n6/n3


title: KO5.H

value: Aortic endothelium


title: Habneula Projecting VP Biol rep 1


title: Control VP biol rep 4

value: Bulk VP:Control VP


title: WT4.N

value: telomerase-immortalized aortic endothelium, WT control


title: C48HKm-1_L3_S16E35


title: 114Plus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_progenitors.xlsx
114Plus


title: 119Plus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_progenitors.xlsx
119Plus


title: Control VP biol rep 2


title: KO5.N

value: telomerase-immortalized aortic endothelium, crispr-modified KO


title: Control VP biol rep 3

value: ventral pallidum


title: KO4.N


title: 1019Minus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_preadipocytes.xlsx
1019minus

value: preadipocytes


title: KO3.N


title: hiPS cells

description: sample 5

source_name: human iPS cells

value: wild type

value: human iPS cells

value: hiPSC.1, hiPSC.2


title: 1017Minus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_preadipocytes.xlsx
1017minus


title: Control VP biol rep 1

value: male


title: anti PD-1 antibody and dasatiinib treatment, day14 rep2

city: Hiroshima

first: RYO

last: YUGE

state: Hiroshima

address: 1-2-3, Kasumi Minamiku

institute: Hiroshima University Hospital

data_processing:
The sequencing kit: DNBSEQ-G400RS High-throughput Sequencing kit (FCL PE100)
The loading concentration of the final library: 8-20 ng/ul measured by Qubit™ ssDNA Assay Kit (Catalog number:  Q10212) and Qubit 4 Fluorometer
The software used to analyze the data: Filter：【 SOAPnuke-1.5.6 】, Alignment_hisat：【 hisat2-2.1.0 】, Alignment_bowtie：【 bowtie2-2.3.4.3 】
Method used to verify the quality/integrity of the processed samples：Concentration is measured using ExKubit dsDNA HS Assay Kits（NGS00-3012) and BMG Fluostar Omega Microplate Reader. Fragment size is detected by Agilent DNA 1000 Kit (Part Number:5067-1504) and 2100 Bioanalyzer Instrument
Assembly: GRCm38  (Version: GCF_000001635.26_GRCm38.p6)
Supplementary files format and content: xlsx files include FPKM values for each Sample

source_name: Liver

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 3 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

treatment_protocol: The CT26 mouse colon cancer cell line were transplanted into the spleen of BALB/c mice to produce experimental liver metastases. Mice were divided into two groups: one group received anti-PD-1 antibody 1, 7, and 13 days after transplantation, and the other group received dasatinib daily from 1 to 13 days after transplantation in addition to anti-PD-1 antibody. On day 14 after transplantation, liver metastatic tumors from each group were excised for  gene expression profiling analysis .

value: Liver

value: colon cancer cells

value: WT

value: anti PD-1 antibody and dasatiinib


title: anti PD-1 antibody and dasatiinib treatment, day14 rep3


title: anti PD-1 antibody and dasatiinib treatment, day14 rep1


title: 1018Plus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_progenitors.xlsx
1018Plus

source_name: progenitor APC High n6/n3

value: subcutaneous adipose tissue

value: High n6/n3


title: Post-implant hiPS-Cart

description: sample 4

source_name: hiPSC-derived cartilage (hiPS-Cart) after implantation

value: disk.imp.3, disk.imp.4, disk.imp.6


title: NPAS Positive biol rep 3

value: NPAS+ VP

value: Npas-Cre::Ribotag Mouse Line


title: NPAS Positive biol rep 2


title: C48HKm-2_L3_S17E35

value: head kidney tissues

value: control

value: wild-type


title: 1017Plus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_progenitors.xlsx
1017Plus

value: progenitors


title: Pre-implant hiPS-Cart

description: sample 3

source_name: hiPSC-derived cartilage (hiPS-Cart) before implantation

value: wild type

value: hiPS.C.Pa.14w.2, hiPS.C.Pa.14w.3


title: 1019Plus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_progenitors.xlsx
1019Plus


title: anti PD-1 antibody treatment, day14 rep1

value: anti PD-1 antibody


title: 108Plus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_progenitors.xlsx
108Plus

value: Low n6/n3


title: NPAS Positive biol rep 1

value: ventral pallidum

value: male


title: Hemin (50 μM)_rep2 [Hemin3]

status: Public on Nov 27 2022

city: beijing

first: chao

last: wei

address: No. 10, Xitiao, Youanmenwai, Fengtai District, Beijing

institute: capital medical university

data_processing:
After filtering, the “clean reads” were aligned to the reference sequences with Bowtie2 (Version: v2.2.5)
Gene expression level was calculated with RSEM (Version: v1.2.12).
Genome_build: mm8
Supplementary_files_format_and_content: FPKM values for each Sample

source_name: mouse primary cortical neurons

extract_protocol:
Primary cortical neurons were treated with vehicle (DMSO), 10 μM 4-OI, 50 μM Hemin or Hemin+4-OI for 24 h, and then collected for RNA-seq analysis (Beijing igeneCode Biotech Co. Ltd, Beijing, China).
RNA libraries were prepared for sequencing using standard Illumina protocols

value: mouse primary cortical neurons

value: Hemin (50 μM) for 24 h


title: DMSO_rep2 [Veh3]

value: DMSO for 24 h


title: Hemin (50 μM)_rep1 [Hemin2]


title: NPAS Positive biol rep 4


title: 1015Plus

description:
Flow-sorted APCs from subcutaneous adipose tissue
All_genes_progenitors.xlsx
1015Plus

value: subcutaneous adipose tissue

value: High n6/n3


title: 4-OI (10 μM)_rep1 [4-OI2]

value: 4-OI (10 μM) for 24 h


title: 4-OI (10 μM)_rep2 [4-OI3]


title: DMSO_rep1 [Veh2]


title: NR

city: Chongqing

first: Zongren

last: Xu

state: Chongqing

address: 1 Youyi Road, Yuzhong District, Chongqing, China

department: ophthalmology department

institute: The First Affiliated Hospital of Chongqing Medical University

description: one sample from human eye tissue

data_processing:
Single cells were analyzed by 10× Chromium system (10× Genomics) and V3 single-cell reagent kits. After the library was built, the reads were aligned to the GRch38 human genome using the 10× analysis tool Cellranger Toolkit (Version 3.1.0), and a unique molecular identifier (UMI) was used to quantify gene expression. Cellranger Count was used to preprocess and preliminarily filter the data. Generate three files matrix.mtx.gz,features.tsv.gz and barcodes.tsv.gz.
Assembly: GRCh38
Supplementary files format and content: Matrix table with raw gene counts for every gene and every cell

source_name: Eye

extract_protocol:
Eyeballs from six donors were divided into five tissue regions within 32 hours including the iris, pars plicata, pars plana, RPE, and neuroepithelial layer (Donors 1-6, Table S1). The samples were finely minced with scalpels and dissociated in buffer containing papain (20 U/ml, LS003119, Worthington) supplemented with DNase-I (2000 U/ml, 10104159001, Roche) in Earle's Balanced Salt Solution at 37°C for 30 min. The cell suspensions were resuspended in Earle's Balanced Salt Solution and passed through a 30-µm cell strainer. Then, the viability of the samples was evaluated with AO/PI prior to loading on the single-cell platform. The samples displayed 90% viability on average. Finally, single-cell samples were subjected to scRNA-seq analysis with a high-throughput droplet-mediated scRNA-seq platform (10× Genomics Chromium).
The appropriate volume of each sample was diluted to the appropriate numbers of tissue cells (pars plicata, pars plana, RPE, Iris and NR). Subsequently, the single-cell suspension, gel beads and oils were all added to the 10× Genomics single-cell A chip. After droplet generation, samples were transferred into 200 μl PCR tubes, and reverse transcription was performed using a T100 Thermal Cycler (Bio-Rad, California, USA). After reverse transcription, cDNA was recovered using a recovery agent provided by 10× Genomics. SPRIselect beads were used for cDNA clean-up, and then the cDNA was amplified for 10 cycles. The concentration of cDNA was detected by a Qubit2.0 fluorometer (Invitrogen, Canada). Single-cell cDNA libraries were prepared according to the Chromium Single Cell 3’ Reagent Kit v3 user guidance.

value: NR

value: adult


title: Iris

description: one sample from human eye tissue

value: Iris


title: Pars plana

description: one sample from human eye tissue

value: Pars plana


title: Human Chondrocytes 7 48hr HU308 R

city: jerusalem

first: lital

last: zecharyahu

address: Campus Ein Kerem

country: Israel

institute: hebrew university

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using Trimmomatic (Quality threshold=30).
Trimmed sequence reads were mapped to hg38 using STAR with standard presets except for intron size, which was set between 30 and 30000 bp (–alignIntronMin 30 and –alignIntronMax 30000). Uniquely mapping reads is accounting for ∼90% of total mapped reads in each sample.
Uniquely mapping reads were count using HT-Seq.
Assembly: hg38
Supplementary files format and content: tab-delimited count files include HT-Seqq results for each Sample

source_name: Articular cartilage

growth_protocol: Human chondrocytes were isolated from cartilage samples obtained from human patients undergoing total koint replacment surgery. Cartilage samples were treated overnight with collagenase II (cat# LS004177, Worthington Biochemical Corporation, NJ) solution (1 mg/mL in DMEM) at 37°C overnight, followed by tissue filtration in a 40um mesh filter and cultured in growth media until reaching passage 2.

extract_protocol:
Chondrocytes were harvasted and RNA was extracted 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 was tested for quality EMBL guidelines using a Tapstation Analysis Software (A.02.02(SR1)) .
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Human derived chondrocytes were treated (passage 2) with HU308 100nM  for 4, 48hr or untreated (20v/v%DMSO; untreated).

value: Articular cartilage

value: Knee OA dervied chondrocytes

value: Chondrocytes

tag: gender

value: Female

value: 48hr HU308 (100 nM in 20v/v%DMSO)

tag: batch

value: Batch B


title: anti PD-1 antibody treatment, day14 rep2

value: Liver

value: colon cancer cells

value: WT

value: anti PD-1 antibody


title: Hemin (50 μM) +4-OI (10 μM)_rep1 [Hemin_4-OI2]

value: mouse primary cortical neurons

value: Hemin (50 μM) +4-OI (10 μM) for 24 h


title: Human Chondrocytes 7 4hr HU308 R

value: 4hr HU308 (100 nM in 20v/v%DMSO)


title: Pars plicata

description: one sample from human eye tissue

value: Pars plicata


title: Human Chondrocytes 6 48hr HU308 R

value: Male


title: Human Chondrocytes 6 4hr HU308 R


title: Wildtype colon - mouse 8

first: Cambrian

middle: Yangshao

last: Liu

address: 5841 S Maryland Ave

department: Department of Medicine

institute: The University of Chicago

data_processing:
Illumina software was used for basecalling.
Transcript abundances were obtained from cellranger 3.0.2 using default options.
Downstream analyses were primarily performed using monocle3. Count matrices were loaded from the cellranger quantification. Cells with fewer than 100 UMI counts were filtered out. All samples were merged into a CellDataSet object.
The count matrix was normalized by the per-cell count total and subsequently log-transformed (preprocess_cds() and reduce_dimension() function defaults). The top 50 principal components were used for dimensional reduction and uniform manifold approximation and projection (UMAP). Clustering of cells was performed using the leiden algorithm with automatic detection of the resolution (cluster_cells() function defaults). Trajectories were computed using the defaults of the learn_graph() function.
Genome_build: mm10
Supplementary_files_format_and_content: Sparse Matrix (mtx) file of merged UMI counts with each row representing a gene and each column a cell barcode, can be read into R with readMM function in the Matrix package
Supplementary_files_format_and_content: Tab-delimited text (txt) files providing gene names (row data) or cell clustering and sample information (column data), matched to the sparse Matrix file

growth_protocol: Sprouty2-floxxed mice were mated to VillinCre mice to induce intestinal epithelial-specific knockout of the Sprouty2 gene. These conditional knockout mice and their littermate controls (Sprouty2-floxxed mice without Cre expression) were raised until 8 wks of age. Mice were euthanized for dissection.

extract_protocol:
Colons were dissected, manually stripped of myenteric layers, and enzymatically digested in Liberase TM/DNAse I. Dissociated tissue was strained through 70 and 40-micron filters, and the resulting single-cell suspension was treated with a red blood cell lysis buffer. Dead cells were removed using a magnetic separation kit with antibodies targeted against annexin V. The purified cellular suspension was submitted for microfluidic processing.
All samples were processed on the same day, representing a single batch run. Cells were washed with PBS and pre-loading viability was confirmed to exceed 75%. Cells were run on the Chromium (10X Genomics) platform for single-cell lysis, reverse transcription, barcoding, and library preparation. Paired-end sequencing of 3' transcript ends was performed on a HiSeq machine (Illumina).

value: Distal colonic mucosa


title: Knockout colon - mouse 7

value: KO


title: Hemin (50 μM) +4-OI (10 μM)_rep2 [Hemin_4-OI3]


title: Human Chondrocytes 6 unt R

value: Articular cartilage

value: unt (20v/v%DMSO)


title: Human Chondrocytes 5 48hr HU308 R

value: Knee OA dervied chondrocytes

value: Chondrocytes

value: Batch A


title: Human Chondrocytes 5 4hr HU308 R

value: Female


title: Wildtype colon - mouse 5


title: Wildtype colon - mouse 4


title: Human Chondrocytes 5 unt R


title: Knockout colon - mouse 6


title: Knockout colon - mouse 2


title: T7

city: Wuhan

first: Dong

last: Chen

state: Hubei

address: 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone

department: Center for Genome Analysis

institute: ABLife, Inc.

data_processing:
Illumina bcl2fastq software used for basecalling.
The raw reads were trimmed of low-quality bases using a FASTX-Toolkit (v.0.0.13;   http://hannonlab.cshl.edu/fastx_toolkit/).  Then the clean reads were evaluated using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc).
Clean reads were aligned to rat_NCBI_v7.2 genome by tophat2 (Kim, Pertea et al. 2013) allowing 4 mismatches.
Uniquely mapped reads were ultimately used to calculate read number and fragments per kilobase of exon model per pillion mapped fragments (FPKM) for each gene. The expression levels of genes were evaluated using FPKM.
The software edgeR (Robinson, McCarthy et al. 2010) , which is specifically used to analyze the differential expression of genes, was applied to screen the RNA-seq data for DEGs. The results were analyzed based on the fold change (FC≥1.5 or ≤0.37) and p-value (p-value ≤ 0.01) to determine whether a gene was differentially expressed.
Assembly: rat_NCBI_v7.2
Supplementary files format and content: tab-delimited text files include reads counts for each Sample
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

organism: Rattus norvegicus

source_name: skin

extract_protocol:
Total RNA was extracted using TRIzol reagent (Invitrogen, cat. NO 15596026). RNA quality was determined by examining the A260/A280 ratio using a Nanodrop One spectrophotometer (Thermo Fisher Scientific Inc., USA). Qualified RNAs were quantified using Qubit3.0 with a Qubit RNA Broad Range Assay kit (Thermo Fisher Scientific Inc., USA).
Two micrograms of total RNA were used for stranded RNA sequencing library preparation using the Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Catalog No. MRZG12324, Illumina, USA) and the KCTM Stranded mRNA Library Prep Kit for Illumina (catalog no. DR08402, Wuhan SeqHealth Co., Ltd. China), according to the manufacturer’s instructions. The library products corresponding to 200–500 bp were enriched, quantified, and sequenced on a NovaSeq 6000 sequencer (Illumina, USA) with the PE150 model.

value: skin

value: GAG(DAY 14)


title: RPE

description: one sample from human eye tissue

value: RPE

value: adult


title: Human Chondrocytes 7 unt R

value: Batch B


title: Knockout colon - mouse 3


title: T4

value: GAG(DAY 7)


title: Human Chondrocytes 3 4hr HU308 NR

value: 4hr HU308 (100 nM in 20v/v%DMSO)


title: Human Chondrocytes 2 48hr HU308 NR

value: 48hr HU308 (100 nM in 20v/v%DMSO)


title: Human Chondrocytes 2 unt NR


title: Human Chondrocytes 2 4hr HU308 NR


title: Human Chondrocytes 3 unt NR

value: Male


title: Wildtype colon - mouse 1

value: Distal colonic mucosa

value: WT


title: Human Chondrocytes 4 48hr HU308 NR

value: Articular cartilage


title: Human Chondrocytes 4 4hr HU308 NR


title: H7

value: Alg (DAY 14)


title: N4

value: Control(DAY 7)


title: Human Chondrocytes 4 unt NR

value: Knee OA dervied chondrocytes

value: Chondrocytes

value: unt (20v/v%DMSO)


title: N1

value: skin

value: Control(DAY 3)


title: T1

value: GAG(DAY 3)


title: Human Chondrocytes 1 4hr HU308 NR

value: Female

value: Batch A


title: Human Chondrocytes 3 48hr HU308 NR

value: Batch B


title: Human Chondrocytes 1 unt NR


title: B1

value: d-SMG(DAY 3)


title: Human Chondrocytes 1 48hr HU308 NR

value: 48hr HU308 (100 nM in 20v/v%DMSO)


title: B7

value: d-SMG(DAY 14)


title: H1

value: Alg DAY 3)


title: Dyrk1a_cKO_LZ_4

city: Rehovot

first: Ziv

last: Shulman

address: Herzl St 234

department: Department of Immunology

institute: Weizmann Institute of Science

data_processing:
Reads were trimmed using Cutadapt
Reads were aligned to the genome using STAR
Quantification was done using htseq-count as previously described in R. Kohen, BMC Bioinformatics, 2019.
Assembly: mm10
Supplementary files format and content: Excel file with Deseq2 output

source_name: B cells

extract_protocol:
mRNA was extracted from sorted cells using mRNA direct kit (Invitrogen) and mRNA from each sample was barcoded during reverse transcription and pooled.
Libraries were prepared using a bulk adaptation of the previously described protocol (Keren-Shaul et al. Nat. Protoc, 2019 and Jaitin et al. Science, 2014).Following Agencourt Ampure XP bead cleanup (Beckman Coulter), the pooled samples underwent second strand synthesis and were linearly amplified by T7 in vitro transcription. The resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with Illumina sequences during ligation, RT, and PCR.  Libraries were quantified by Qubit and TapeStation as well as by qPCR for the ACTB gene as previously described. Sequencing was done using a Nextseq 75 cycle high output kit (Illumina; paired end sequencing).

value: Germinal center light  zone B cells

value: CD23 Cre Dyrk1a fl/fl


title: H4

value: Alg (DAY 7)


title: N7

value: Control(DAY 14)


title: Dyrk1a_cKO_LZ_2


title: mir-126-KD_AC_29492

city: Denver

first: William

last: Janssen

state: CO

address: 1400 Jackson St

institute: National Jewish Health

data_processing:
FASTQ files were generated from BCL files using Illumina bcl2fastq converter (version 2.17) without adapter trimming.
Illumina TruSeq adapters were trimmed with skewer (version 0.2.2).
Trimmed reads were mapped with the STAR aligner (version 2.5.1b) to the hg19 assembly of the human genome using gene annotations from Ensembl version 75.
Reads mapping to each gene of the Ensembl 75 annotation were counted with the featureCounts program from the Subread software package (v1.6.2).
Genome_build: hg19
Supplementary_files_format_and_content: counts per gene (Ensembl 75 human annotation) for each sample

source_name: HLMVEC_miR-126 knockdown

growth_protocol: HLMVEC were cultured in EGM-2 MV media supplemented with 5% FBS and growth factors (CC-3202, LONZA) .

extract_protocol:
Total RNA (including miRNA and mRNA) were isolated using the miRNeasy mini kit (Qiagen, Germantown, MD), following the manufacturer’s instructions.
The library construction started from isolation of total RNA species, followed by mRNA (poly-A) isolation, 1st and 2nd strand cDNA synthesis, adaptor ligation, amplification, and cluster generation. Once validated, the libraries were sequenced as barcoded-pooled samples and run on the HiSeq 2500

treatment_protocol: HLMVEC were transfected with non-targeting scramble controls, synthetic miR-126-3p mimics for overexpression or miR-126-3p hairpin inhibitors for knockdown (KD) at 5nM respectively for 16hrs using lipofectamine RNAiMAX (Fisher Scientific).

tag: donor id

value: primary human lung microvascular endothelial cells (HLMVEC)

value: miR-126 knockdown

value: Air control

tag: mir-126 copy number


title: mir-126-KD_AC_29055


title: Dyrk1a_cKO_LZ_1


title: mir-126-KD_AC_31653


title: WT_LZ_3

value: CD23 Cre Dyrk1a +/+


title: WT_LZ_2


title: Scramble_AC_29492

source_name: HLMVEC_Scramble control

value: Scramble control


title: B4

value: skin

value: d-SMG(DAY 7)


title: Scramble_AC_29055


title: mir-126_AC_29492

source_name: HLMVEC_miR-126 over-expression

value: miR-126 over-expression


title: Spinal cord tissue, Ctrl, 1

city: Shanghai

first: Runzhi

last: Huang

state: Shanghai

address: No 289 Xincun Road

institute: Department of Orthopaedics, Tongji Hospital, Tongji University School of Medicine, Shanghai, 200065, China

data_processing:
Sample preparation and spatial transcriptomics and scRNA-seq were performed following the 10X Visium procedure (https://support.10xgenomics.com/spatial-gene-expression/sample-prep/doc/demonstrated-protocol-visium-spatial-protocols-tissue-preparation-guide). Briefly, samples were embedded in precooled optical coherence tomography (OCT) medium, and frozen sections of 10 µm were carefully attached to Visium slides. The Visium Spatial Tissue Optimization Kit (10X Genomics) was used to determine the optimal permeability time, which was 6 min for sham samples and 12 min for contusion spinal cord samples. Then, the Visium Spatial Gene Expression Slide & Reagent kit (10X Genomics) was used to generate sequencing libraries, and the libraries were sequenced on a NextSeq (Illumina). Following demultiplexing of the bcl ﬁles, the sequencing results were first separated into two fastq files of paired-ended reads, which were trimmed to remove the polyA tail sequence and the template switch oligo (TSO) sequence. Then, the clean reads were aligned to the GRCm38 (Version: 100) genome assembly and quantified by Space Ranger Software (Version 1.0.0) (http://10xgenomics.com/).The quantitative gene expression matrices obtained from seven libraries were processed with the Seurat pipeline (Version: 3.2.2) for subsequent analysis. Only cells with more than 100,000 transcripts expressed and less than 10% mitochondrial genes mapped were included in further analysis, and only genes expressed in at least 3 individual cells were considered for further analysis. After the quality control (QC) processes were applied, all samples were merged with the “IntegrateData” function into one Seurat object, and the merged Seurat object was scaled and normalized with the “ScaleData” function. The “vst” method was used to identify the top 1,500 variable genes. For dimensionality reduction, principal component analysis (PCA) was performed, and the ﬁrst 20 PCs were integrated as input for uniform manifold approximation and projection for dimension reduction (UMAP) analysis. The same series of analyses was applied to all independent cross sections and coronal section samples. Additionally, to elucidate the temporal and spatial changes in cells and molecules induced by solu-medrol therapy, single cells from the sham control coronal sections, all 7 dpi coronal sections, all 14 dpi coronal sections, and the 7 dpi and 14 dpi coronal sections with solu-medrol therapy at 6 dpi were merged into individual single Seurat objects for spatial transcriptomics analysis.
Genome_build: mm10
Supplementary_files_format_and_content: The results of processed data including spatial figures (.png, .jpg) and sparse matrixes (barcodes.tsv, features.tsv, matrix.mtx) for each sample.

extract_protocol:
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
scRNA-Seq

value: Spinal cord tissue

value: Sham/Central/coronal & cross sections/Control


title: Spinal cord tissue, solu, medrol, 6D

value: SCI/Central/coronal sections/7&14dpi


title: Dyrk1a_cKO_DZ_4

value: Germinal center dark zone B cells


title: Spinal cord tissue, Ctrl

source_name: Ctrl

value: Sham/Central/cross sections/Control


title: mir-126_AC_31653


title: Scramble_AC_31653


title: WT_LZ_1

value: Germinal center light  zone B cells


title: Dyrk1a_cKO_DZ_1

value: CD23 Cre Dyrk1a fl/fl


title: Spinal cord tissue, C, 3

value: SCI/Caudal/cross sections/7&14dpi


title: WT_DZ_3


title: Spinal cord tissue, B, 3

value: SCI/Central/cross sections/7&14dpi


title: Spinal cord tissue, C, 9

value: SCI/Central/coronal sections/14dpi


title: Spinal cord tissue, B, 1

value: SCI/Central/coronal sections/7dpi


title: Dyrk1a_cKO_LZ_3


title: mir-126_AC_29055

value: primary human lung microvascular endothelial cells (HLMVEC)

value: Air control


title: Dyrk1a_cKO_DZ_3


title: WT_DZ_1

value: CD23 Cre Dyrk1a +/+


title: WT_DZ_2


title: testicular cells - cKO

city: Yayoi, Bunkyo-ku

first: Yoshiakira

last: Kanai

state: Tokyo

institute: The University of Tokyo

data_processing:
The Cell Ranger Single-Cell Software Suite (v6.0.2) was used to perform sample demultiplexing, barcode processing, and single-cell 3' gene counting. The cDNA insert was aligned to the mm10/GRCm38 reference genome by using STAR.
Data integratioin of cKO mice and the controls were conducted by using the Cell Ranger aggr program.
Genome_build: mm10/GRCm38
Supplementary_files_format_and_content: Three files were generated using the Cell Ranger Single-Cell Software per sample. matrix.mtx: This file contains the gene-barcode matrix. barcodes.tsv: This file contains the barcodes represented in the mtx file. genes.tsv: This file contains all annotated genes.

source_name: Testis

extract_protocol:
Each sample analyzed by scRNAseq consisted of testicular cells pooled from three pups. Testes from P14 cKO and the control mice were collected, and the region containing the rete tesis and adjacent seminiferous tubule were dispesed into single cell and then loaded on the Chromium Controller (10x Genomics Inc.) for 5000 cells per sample.
Cells were subjected to scRNA seq library construction on the 10X Genomics platform using Chromium™ Single Cell 3' v3 Reagent Kits and a Chromium instrument according to the manufacturer’s protocol.

value: Testis


title: Spinal cord tissue, A, 3

value: SCI/Rostral/cross sections/7&14dpi


title: testicular cells - cKO 2

library_source: transcriptomic single cell

data_processing:
The Cell Ranger Single-Cell Software Suite (v6.0.2) was used to perform sample demultiplexing, barcode processing, and single-cell 3' gene counting. The cDNA insert was aligned to the mm10/GRCm38 reference genome by using STAR.
Assembly: mm10/GRCm38
Supplementary files format and content: Three files were generated using the Cell Ranger Single-Cell Software per sample. matrix.mtx: This file contains the gene-barcode matrix. barcodes.tsv: This file contains the barcodes represented in the mtx file. genes.tsv: This file contains all annotated genes.


title: water7d1

status: Public on Nov 26 2022

city: shanghai

first: li

last: zihao

address: dongchuan road 800

institute: shanghai jiao tong university

data_processing:
DNBs were transformed to single end 50 bases reads and sequencing on BGISEQ-500 platform (BGI-Shenzhen, China).
We used SOAPnuke (v1.5.2) to filter reads and generate FASTQ format
Then aligned clean reads to the reference mouse genome using the Bowtie2 (v2.2.5) with default parameters
calculated gene expression level with RSEM (v1.2.12)
In GO analysis and pathway analysis, DEGs were classified according to official classification with the GO or KEGG annotation results and phyper (a function of R) was performed in GO and pathway functional enrichment.
Genome_build: mm9
Supplementary_files_format_and_content: gene_expression.CSV reports FPKMs for all samples

source_name: colorectum

growth_protocol: no

extract_protocol:
Colon samples 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

treatment_protocol: Colitis mice model was induced by DSS treatment for 7d, while BBR was given via gavage administration for 6d

value: colorectum

value: water 7d

tag: disease state

value: control

tag: group

value: normal mice


title: water7d3


title: testicular cells - control


title: DSS7d4

value: DSS 7d

value: colitis

value: colitis mice


title: DSS7d2


title: DSS_BBR7d3

value: DSS+BBR 7d

value: colitis mice receiving berberine


title: water7d4


title: BBR7d3

value: BBR 7d

value: normal mice receiving berberine


title: DSS7d1


title: testicular cells - control 2


title: DSS_BBR7d2


title: Experiment I DA 1

city: Cambridge

first: Sophia

middle: Alisa

last: Wild

state: Cambridgshire

address: Robinson Way

department: School of Clinical Medicine

country: United Kingdom

institute: University of Cambridge and Cancer Research UK Cambridge Institute

data_processing:
10X Cell Ranger v3 was used to align transcriptomes to a custom index, including the mm9 mouse genome index and the barcode transcript
Sparse expression matrices for transcriptomes were produced using 10X Cell Ranger v3
Clonal annotation was produced by extracting barcodes from 10X data and enrichment PCRs using custom scripts
Assembly: Custom mm9 index with barcoding transcript.
Supplementary files format and content: .tsv files are tab seperated txt files and .mtx files are sparse expression matrices
Supplementary files format and content: .rds files are Seurat objects containing barcode annotation in metadata

source_name: Tumor tissue

extract_protocol:
Tumours were harvested, dissociated into single cells using the tumor dissociation kit and the gentleMACS Octo dissociator from Miltenyi, and sorted for live-dead with propidium iodide. Sorted cells were resuspended in PBS + 0.04% BSA prior to counting with a hemocytometer. The final single cell suspension was diluted as required and prepared for sequencing using the Gene Expression 3' v3.1 dual index kit of 10X Genomics.
Libraries were prepared according to 10X Genomics instructions. After cdDNA amplification, 10 µL cDNA was split off for barcode enrichment PCRs. Transcriptimic library construction contiunued as in the 10X user guide instructions. Barcode enrichment PCRs were prepared using a custom targeted PCR with Illumina-indexed primers. Barcode libraries were purified using gel purification.

treatment_protocol: Seven days post implantation, tumor were treated with either DTX treatment 12.5 mg/kg for 3 times a week for two weeks) and JQ1 treatment at 75 mg/kg (5 days on, 2 days off until endpoint)

value: Balb/C

value: 12.5 mg/kg DTX + 60 U L-Asparaginase treated


title: Experiment I D 1

value: 12.5 mg/kg DTX treated


title: Experiment I D 2


title: Experiment I DA 2


title: Experiment I DA 3


title: Experiment I D 3


title: Experiment I DV 3

value: DTX vehicle treated


title: Experiment I DV 2


title: Experiment I DV 1


title: DSS_BBR7d4


title: Experiment H 4T1 in vitro

source_name: Cell line


title: BBR7d4


title: BBR7d1


title: Experiment G NSG5

value: NSG


title: Experiment G NSG4


title: Experiment G NSG6


title: Dyrk1a_cKO_DZ_2

value: Germinal center dark zone B cells

value: CD23 Cre Dyrk1a fl/fl


title: Experiment E D3


title: Experiment F NSG2


title: Experiment F NSG1


title: Experiment E DV1


title: Experiment D D4


title: Experiment F NSG3


title: Experiment E D1


title: Experiment E D2


title: Experiment D D5


title: Experiment E DV2


title: Experiment D DV6


title: Experiment E DV3


title: Experiment D DV5


title: Experiment C D2


title: Experiment D DV4


title: Experiment C DV2


title: Experiment C D3


title: Experiment C DV1


title: Experiment C DV3


title: Experiment B J6

value: 75 mg/kg JQ1 treated


title: Experiment B J5


title: Experiment D D6


title: Experiment B JV6

value: JQ1 vehicle treated


title: Experiment B J4


title: MDAMB231 cells, siRNF31-1

city: xinxing

first: huijie

last: yang

address: Hongqi District, Xinxiang City

institute: Xinxiang Medical University

data_processing:
Using BGISEQ-500 sequencing platform
The sequencing data was filtered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Assembly: not provided

source_name: Breast cancer

growth_protocol: MDAMB231 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
Lysis of cells with Trizol regent and harvesting of RNA.
RNA libraries for RNA-seq were prepared using BGISEQ-500 protocols.

treatment_protocol: MDAMB231 cells were transfected with siControl and siRNF31 for 48 h.

value: Breast cancer

value: Triple negative breast cancer cell


title: MDAMB231 cells, siControl-3

value: siControl


title: MDAMB231 cells, siRNF31-2


title: MDAMB231 cells, siRNF31-3


title: MDAMB231 cells, siControl-2


title: MDAMB231 cells, siControl-1


title: Experiment B JV4


title: Experiment C D1


title: Experiment A J3


title: Experiment A J2


title: Experiment A JV3


title: Experiment A JV2


title: Experiment A J1


title: direct-RNA-pulldown_rep1

city: Chicago

first: Jon

last: Staley

state: IL

institute: University of Chicago

data_processing:
Data were processed as follows: (1) 6 nt UMIs were trimmed from reads and attached to read names to maintain their association with reads using fastp (Chen et al., 2018a), (2) adaptors were trimmed using Cutadapt. Reads shorter than 15 nts were discarded, (3) the remaining reads were aligned to the human reference genome (hg38) with GENCODE transcriptome annotation (v26) using the STAR software (Dobin et al., 2013) with the following settings: --outSAMattributes NH HI AS nM NM MD jM jI XS --outFilterMultimapNmax 50 --outFilterMismatchNmax 15 --outSJfilterDistToOtherSJmin 10 0 5 10 --alignIntronMin 15 --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMatchNmin 15 --outSAMtype BAM SortedByCoordinate --twopassMode Basic, (4) aligned read pairs were further filtered for unique mapping using samtools, (5) to remove RT mis-primed reads, raw reads were separately mapped without UMI trimming. If the UMI portion of a read was mapped to the genome without any mismatch, it is considered as a mis-primed read and removed from the reads that were mapped with UMI trimming. These RT mis-primed reads were removed using samtools and pysam, (6) PCR duplicates were removed using UMI-tools(Smith et al., 2017), (7) reads overlapping with ncRNAs (snRNAs, snoRNAs, miRNAs, rRNAs) were removed using samtools based on the GENCODE reference (v26). The remaining reads were used for downstream analyses.
Adaptors were removed by Cutadapt default setting. When applicable, UMIs are processed by fastp default setting.
Filtered reads were aligned using STAR.
Mapped reads were filtered for unique-mapping reads using samtools
Unique-mapping reads are deduplicated using either samtools or umi-tools when reads have UMIs
Bed files were generated using bedtools
Assembly: hg38
Supplementary files format and content: BED format; ELI and NLI reads identified in each dataset
Supplementary files format and content: tsv format; read abundance calculated by kallisto
Library strategy: RNA pulldown

growth_protocol: K562 cells were maintained with in RPMI + GlutaMAX (Life Technologies) supplemented with 10% (v/v) fetal bovine serum (Life Technologies). Cells were cultured at 37 C with 5% CO2

extract_protocol:
Total RNA were extracted from K562 cells using Trizol (Invitrogen) followed by a second extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and isopropanol precipitation. The isolated total RNA was furthered treated with Turbo DNase (Invitrogen) to remove residual genomic DNA, followed by extraction with phenol:chloroform:isoamyl alcohol (25:24:1). Then, 1 mg of total RNA was incubated with three sets of biotinylated anti-sense oligos (ASOs), which target intron 16 of HNRNPK,  intron 1 of EIF, and intron 6 of PPP2CA (1 µM per oligo; Table S7), in a solution containing 2x SSC, 5 mM EDTA, and 10% Formamide. The resulting hybridization reaction was put through an oligo annealing cycle (5 min at 80 ˚C, cool down to 22˚C at 0.1 °C/sec), after which RNA bound biotinylated ASOs were pulled down using Dynabeads™ MyOne™ Streptavidin C1 beads (Invitrogen), according to the manufacturer’s manual. The ASOs were digested with DNas I and the pulled-down RNA was extracted using Trizol and precipitated by ethanol.
The 3’ ends of RNA were ligated to a pre-adenylated 3’ end adapter (table S5), which includes a 6 nt UMI (unique molecule identifier) at the 5’ end to allow correction for PCR duplication and RT mis-priming. The ligation reaction contained 17.5% PEG-8000 (w/v), 1X T4 RNA ligase buffer (NEB), 1 µM of the pre-adenylated 3’ end adaptor, and 10 units/µL T4 RNA Ligase 2 (truncated K227Q, NEB). The ligation reaction was incubated for 3 hours at 22 ˚C. After the reaction excess adapters were removed by size selection using SPRI paramagnetic beads (Beckman Coulter) with a beads to sample ratio of 1.6:1. Then, the adapter-ligated RNA was used for cDNA synthesis using 0.25 µM of RT primer (table S5) and 10 units/µL of Superscript III (Invitrogen) at 50 ˚C for 1 hour, according to the manufacturer’s manual. Afterwards, to degrade excessive RT primer, 3.5 µL of ExoSAP-IT was added to the cDNA synthesis reaction and incubated for another 30 min at 37 ˚C. The reaction was stopped by adding 10 µL of 0.5M EDTA and RNA was degraded by adding 10 µL of 1 N NaOH. The resulting cDNA was cleaned up using Oligo Clean & Concentrator columns (Zymo Research) and was circularized with Circligase (Lucigen), according to the manufacturer’s manual. Final library amplification was performed on the circularized cDNA using Phusion polymerase (Thermo Fisher Scientific) with primers that add sequencing adaptor sequences and specific sequencing indices, which are located in the reverse primers (table S5). The number of PCR cycles was between 15 and 18 cycles. The amplified product was run on a 6% TBE gel and DNA fragments between 175 and 750 bps were gel purified and 3 nM of the library was sequenced in a 100 bp paired-end mode on an Illumina HiSeq 4000 (Genomics Facility at University of Chicago).

tag: protocol

value: RNA 3' end sequencing

tag: molecule subtype

value: Targeted RNA pull down from total RNA


title: ESC_Day6_rep1_HiChIP

city: Salt Lake City

first: Dean

last: Tantin

state: UT

address: 15 North Medical Drive East

department: Department of Pathology

institute: University of Utah

data_processing:
paired-end Illumina sequencing reads were aligned to the mouse Mm10 reference genome using HiC-Pro pipeline.
Hi-ChIP was analyzed using Hichipper to perform restriction site bias-aware modeling of the output from HiC-Pro and to call interaction loops.
Assembly: mm10
Supplementary files format and content: Paired end bed  (.bedpe) containing discovered loops
Library strategy: Hi-ChIP

source_name: ESC

extract_protocol:
Chromatin was crosslinked with 1% formaldehyde in growth media and isolated using Hi-C lysis buffer supplemented with protease inhibitor.
Hi-ChIP as previously described by M. R. Mumbach et al.,.Nat Methods 13, 919-922 (2016).

value: ESC

value: Parental

tag: time

value: day 6


title: Experiment A JV1

tag: strain

value: Balb/C


title: ESC_Day6_rep2_HiChIP


title: direct-RNA-pulldown_rep2


title: lariat-seq_rep1

data_processing:
Data were processed according to previously described methods (Mercer et al., 2015; Pineda and Bradley, 2018).
Adaptors were removed by Cutadapt default setting. When applicable, UMIs are processed by fastp default setting.
Filtered reads were aligned using STAR.
Mapped reads were filtered for unique-mapping reads using samtools
Unique-mapping reads are deduplicated using either samtools or umi-tools when reads have UMIs
Bed files were generated using bedtools
Assembly: hg38
Supplementary files format and content: BED format; ELI and NLI reads identified in each dataset
Supplementary files format and content: tsv format; read abundance calculated by kallisto
Library strategy: Lariat-seq

extract_protocol:
Chromatin RNA was isolated from K562 cells similar to previously described methods (Pandya-Jones and Black, 2009; Wuarin and Schibler, 1994; Werner and Ruthenburg, 2015), with modifications noted below. All the steps were performed with pre-chilled, ice-cold, freshly prepared RNase-free buffers using RNase-free equipment on ice or at 4 ˚C. Roughly 5% of subcellular fractions were saved for quality control (western and RNA fragment size analysis). Fifty million K562 cells were spun down at 100 x g for 3 minutes and washed twice with 15 mL of ice-cold 1X PBS (phosphate-buffered saline) and once with 1 mL of ice-cold 1X PBS. Washed cells were then gently resuspended in 1mL of 0.1% (v/v) Triton X-100-containing buffer A (10 mM HEPES (pH 7.5), 10 mM KCl, 10% glycerol, 0.116 g/mL sucrose, 1 mM DTT, 1x protease inhibitor, 2 mM EDTA) and lysed on ice for 9 min. After incubation, the lysed cells were centrifuged at 800 x g for 5 minutes at 4 ˚C. The supernatant corresponding to the cytoplasmic fraction was carefully removed. The remaining nuclei pellet was gently resuspended in 250 µL of NRB buffer (20 mM HEPES (pH 7.5), 75 mM NaCl, 50% glycerol, 0.5 mM EDTA, 1 mM DTT, 1x protease inhibitors). Then, 250 µL of NUN buffer (50 mM HEPES (pH 7.5), 300 mM NaCl, 0.2 mM EDTA, 1 M urea, 1 mM DTT, 1% NP-40) was slowly added and mixed by inversion. The resulting nuclei suspension was incubated on ice for 5 minutes and centrifuged at 800 x g for 5 minutes at 4 ˚C. The supernatant corresponding to the nucleoplasmic fraction was carefully removed. The chromatin pellet was washed once with 500 µL of premixed NRB/NUN buffer (1:1) and centrifuged at 800 x g for 5 minutes at 4 ˚C. The supernatant was carefully removed. The washed chromatin pellet was gently resuspended in 200 µL of NRB buffer. The chromatin pellet were extracted by Trizol (Invitrogen) followed by a second extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and isopropanol precipitation. The isolated RNA was furthered treated with Turbo DNase (Invitrogen) to remove residual genomic DNA, followed by extraction with phenol:chloroform:isoamyl alcohol (25:24:1).
10 µg chromatin-associated RNAs were depleted of ribosomal RNA depletion using Ribo-Zero Magnetic Gold Kit (human/mouse/rat; Illumina), according to the manufacturer’s instruction. To further enrich lariat RNA species, RNase R (Lucigen) digestion was performed to degrade linear RNA, according to the manufacturer’s instruction; treatment also trims tails of lariat RNA species. The resulting RNA was used to prepare an RNA-seq library using NEBNext Ultra™ Directional RNA Library Prep Kit from Illumina, according to the manufacturer’s instruction except for the RT step, during which 1 mM MnCl2 was included to facilitate reverse transcriptase go through the BP. The final library was sequenced in a 100 bp paired-end mode on an Illumina Hiseq 4000.

value: Lariat-seq

value: Rnase R treated Chromatin-associated RNA


title: lariat-seq_rep2


title: chromation-associated-RNA-seq_rep2

data_processing:
Data were pre-processed as follows: (1) adaptors were trimmed using Cutadapt (Martin, 2011), with default parameters except discarding reads shorter than 15 nts, (2) remaining reads were aligned to the human reference genome (hg38) with GENCODE transcriptome annotation (v26) using the STAR software package (Dobin et al., 2013) with the following settings: --outSAMattributes NH HI AS nM NM MD jM jI XS, --outFilterMultimapNmax 50, --outFilterMismatchNmax 15, --outSJfilterDistToOtherSJmin 10 0 5 10 --alignIntronMin 15, --outSAMtype BAM SortedByCoordinate, --twopassMode Basic, (3) aligned reads were further filtered for unique mapping and proper alignment using samtools (Li et al., 2009). The resulting reads were used for downstream analyses. Separately, after the adapter removal step, reads were used for gene expression quantitation using Kallisto with default settings (Bray et al., 2016).
Adaptors were removed by Cutadapt default setting. When applicable, UMIs are processed by fastp default setting.
Filtered reads were aligned using STAR.
Mapped reads were filtered for unique-mapping reads using samtools
Unique-mapping reads are deduplicated using either samtools or umi-tools when reads have UMIs
Bed files were generated using bedtools
Assembly: hg38
Supplementary files format and content: BED format; ELI and NLI reads identified in each dataset
Supplementary files format and content: tsv format; read abundance calculated by kallisto

extract_protocol:
Chromatin RNA was isolated from K562 cells similar to previously described methods (Pandya-Jones and Black, 2009; Wuarin and Schibler, 1994; Werner and Ruthenburg, 2015), with modifications noted below. All the steps were performed with pre-chilled, ice-cold, freshly prepared RNase-free buffers using RNase-free equipment on ice or at 4 ˚C. Roughly 5% of subcellular fractions were saved for quality control (western and RNA fragment size analysis). Fifty million K562 cells were spun down at 100 x g for 3 minutes and washed twice with 15 mL of ice-cold 1X PBS (phosphate-buffered saline) and once with 1 mL of ice-cold 1X PBS. Washed cells were then gently resuspended in 1mL of 0.1% (v/v) Triton X-100-containing buffer A (10 mM HEPES (pH 7.5), 10 mM KCl, 10% glycerol, 0.116 g/mL sucrose, 1 mM DTT, 1x protease inhibitor, 2 mM EDTA) and lysed on ice for 9 min. After incubation, the lysed cells were centrifuged at 800 x g for 5 minutes at 4 ˚C. The supernatant corresponding to the cytoplasmic fraction was carefully removed. The remaining nuclei pellet was gently resuspended in 250 µL of NRB buffer (20 mM HEPES (pH 7.5), 75 mM NaCl, 50% glycerol, 0.5 mM EDTA, 1 mM DTT, 1x protease inhibitors). Then, 250 µL of NUN buffer (50 mM HEPES (pH 7.5), 300 mM NaCl, 0.2 mM EDTA, 1 M urea, 1 mM DTT, 1% NP-40) was slowly added and mixed by inversion. The resulting nuclei suspension was incubated on ice for 5 minutes and centrifuged at 800 x g for 5 minutes at 4 ˚C. The supernatant corresponding to the nucleoplasmic fraction was carefully removed. The chromatin pellet was washed once with 500 µL of premixed NRB/NUN buffer (1:1) and centrifuged at 800 x g for 5 minutes at 4 ˚C. The supernatant was carefully removed. The washed chromatin pellet was gently resuspended in 200 µL of NRB buffer. The chromatin pellet were extracted by Trizol (Invitrogen) followed by a second extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and isopropanol precipitation. The isolated RNA was furthered treated with Turbo DNase (Invitrogen) to remove residual genomic DNA, followed by extraction with phenol:chloroform:isoamyl alcohol (25:24:1).
For each library, 10 µg chromatin-associated RNAs were depleted of ribosomal RNA depletion using Ribo-Zero Magnetic Gold Kit (human/mouse/rat; Illumina), according to the manufacturer’s instruction. Abundant ncRNAs were depleted as described in the section of enrichment for lariat RNA species (see methods). Then, 100 ng rRNA/ncRNA-depleted RNA was used to prepare an RNA-seq library using NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina, according to the manufacturer’s instruction. The final library was sequenced in a 100 bp paired-end mode on an Illumina HiSeq 4000.

value: RNA-seq

value: Chromatin-associated RNA