title: aPDL1_dLN_1

city: Noda

first: Hiroyasu

last: Aoki

address: 2669 Yamazaki

institute: Tokyo University of Science

description: messenger RNA

data_processing:
Data processing of scRNAseq was performed as described in Shichino et al. (Shichino et al., 2021).
For target sequencing data, filtered cell barcode reads were annotated by Python script provided by BD Biosciences with minor modification for compatibility to Python 3.7. Associated cDNA reads were mapped to reference RNA using bowtie2-2.4.2 (Langmead et al., 2012) 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 (https://github.com/pysam-developers/pysam), 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 (Lun et al., 2019) in R-3.6.3. Cells of which total read count was over inflection point were considered as valid cells.
For sample tag data, adapter trimming of sequencing data was performed by using cutadapt 3.4. Filtered reads were chunked to 64 parts for parallel processing by using Seqkit 0.15.0. Filtered cell barcode reads were annotated by Python script provided by BD with minor modification for compatible to Python3.8. Associated sample tag reads were mapped to known barcode fasta by using bowtie2-2.4.2 by the following parameters: -p 2 -D 50 -R 20 -N 0 -L 14 -i S,1,0.75 –norc –seed 656565 –reorder –trim-to 3:40 –score-min L,-9,0 –mp 3,3 –np 3 –rdg 3,3.  Then, cell barcode information of each read were added to the bowtie2-mapped BAM files, and read counts of each Tag in each cell barcode were counted by using mawk. Resulted count data was converted to Tags x cells matrix file by using data.table package in R 3.6.3, and top 1M cell barcodes were extracted. For assignment of each tags to each cell barcodes, read counts of each tag in each valid cell barcode, which defined by the cDNA matrix, were extracted from tag/cell barcode expression matrix. Unassigned cell barcodes were labeled as “not-detected” cells. Then, sum of the total read counts of each tags were normalized to the minimum sum count of each tags, and log2 fold-change between first most tag counts and second most tag counts within each cell barcode. Each cell barcode were ranked by the fold-change ascending order, and top N cells were identified as doublets (N was calculated as theoretically detectable doublets calculated by the Poisson’s distribution based on the number of loaded cells, total Rhapsody well number, and number of tags used). Correspondence table between sample tag and sample sources is summarized in Supplementary Table 1.
Raw data of single-cell TCR sequencing were processed by in-house pipelin. Briefly, sequencing reads were first separated by cell barcode, then adapter trimming and quality filtering of sequencing data were performed by using Cutadapt (Martin et al., 2011). PCR and sequencing error was corrected by lighter (Song et al., 2014). Error-corrected sequence reads were processed by MiXCR (Bolotin et al., 2015) to generate the list of TCRa or TCRb sequences for each cell barcode, using reference mouse TCR V/D/J sequences registered in the international ImMunoGeneTics (IMGT) information system with the parameters summarized in Supplementary Table 3.
Quality filtering of cell barcodes was performed with the following criteria: (1) more than 32 TCR reads were detected. (2) the proportion of the most frequent TCR sequence in cell barcode was over 0.6. (3) the cell barcode was annotated to the T cell cluster in scRNAseq analysis. Finally, the most frequent sequence of TCRa and TCRb was adopted and paired in each cell barcode.
T-cell clones were determined as cells with the same TCRβ, defined by V segment, J segment, and CDR3 nucleotide sequence. Frequency of each clone was calculated as the proportion of cells belonging to the clone in all T cells whose TCRβ was assigned.
Data processing of Bulk TCRseq was performed as described in Aoki et al. (Aoki et al., 2021). Adapter trimming and quality filtering of sequencing data were performed by using Cutadapt-3.2 (Martin et al., 2011) and PRINSEQ-0.20.4 (Schmieder et al., 2011). Sequencing data were processed by MiXCR-3.0.5 (Bolotin et al., 2015). In MiXCR, filtered reads were aligned to reference mouse TCR V/D/J sequences registered in the international IMGT information system: -vParameters.geneFeatureToAlign = VTranscript -vjAlignmentOrder = JThenV, then identical sequences were assembled and grouped in clones with PCR and sequencing error correlation with the following parameters: -badQualityThreshold=10, –separateByV=true, --only-productive=true, –region-of-interest=CDR3. The Variable (V) and Joining (J) segment of TCRs were represented in IMGT gene nomenclature.
List of final clones were analyzed by VDJtools-1.2.1 (Shugay et al., 2015). Then, the sequencing reads of sample was normalized to ten-times of the cell count in each sample by “DownSample” command of VDJtools. T-cell clones were determined as TCR reads with the same TCR V segment, J segment and CDR3 nucleotide sequence.
Assembly: GRCm38
Assembly: IMGT reference sequences of mouse TRA and TRB
Supplementary files format and content: (scRNAseq) tab-delimited text files include raw tag-count values for each sample
Supplementary files format and content: (scTCRseq) correspondence table between cell barcodes and TCR sequences
Supplementary files format and content: (Bulk TCRseq) text files in VDJtools format include read count and frequency, complementarity determining region 3 (CDR3) nucleotide and amino acid sequences, Variable(V)/ Diversity(D)/ Joining(J) segment names, and V, D and J segment boundaries within CDR3 nucleotide sequence of individual clones

source_name: dLN on day 14 after inoculation of B16F10 untreated

extract_protocol:
Intravascular leukocytes were stained by i.v. injection of FITC-conjugated anti-CD45.2 mAb (3 mg/mouse, clone 104, BioLegend) three minutes before sacrifice.
Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.1mg/mL Liberase TM (Roche). The cells were then subjected to 40% Percoll PLUS (Cytiva) and Histopaque-1083 (Sigma-Aldrich) gradient and leukocytes were recovered from the interphase. The cell concentration of the suspensions was determined using Flow-Count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter).
Cells were then stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 93, BioLegend) and fluorophore-conjugated anti-mouse mAbs. Cells were also stained with Sample Tag oligonucleotide-conjugated antibodies in BD® Single-Cell Multiplexing Kit (BD Biosciences) as summarized in Supplementary Table 1.
CD8+ T cells were sorted using FACS Aria II or Aria III (BD Biosciences). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and propidium iodide staining. Intravascular leukocytes were also excluded. Purity of sorted cells was always over 95%.
T cells from the spleen of Pmel-1 transgenic mice which stained with Sample Tag oligonucleotide-conjugated antibodies as summarized in Supplementary Table 1 were spiked in after cell sorting.
Live cells were stained with Calcein AM (Nacalai) and counted using Flow-Count fluorospheres and a Cytoflex flow cytometer.
For single-cell RNA/TCRseq, 18,000-30,000 labeled cells were trapped and reverse-transcribed using the BD RhapsodyTM (BD) according to the manufacturer's instructions. Targeted mRNA, TCRseq and Sample Tag libraries were prepared using according to the manufacturer's instructions (VDJ CDR3 and Sample Tag Library Preparation Protocol) with the following modifications; (1) BD RhapsodyTM Immune Response Panel Mm with supplement panel including Cmpk2, Ifi204, Ifi211, Ifit1, Ifit3, Ifit3b, Ifit3b, Isg15, Mx1, Rsad2, and Usp18 were used for target mRNA amplification. (2) we used original set of primer for TCR amplification as summarized in Supplementary Table 2, and performed additional PCR (3rd PCR, 6 cycles) to introduce unique dual index for sequencing. (3) we used ProNex Size-Selection DNA purification System (Promega) for purification of PCR product. Amplified libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems) and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). Targeted RNAseq and Sample Tag libraries were sequenced on the Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 141 bp read 2) (Illumina) to a depth of approximately 20,000-40,000 reads and 5,000-10,000 reads per cell, respectively. TCRseq libraries were sequenced on the Illumina Novaseq 6000 SP flowcell (67 bp read 1 and 459 or 141 bp read 2) (Illumina) to a depth of approximately 5,000-20,000 reads.
Bulk TCR sequencing library was prepared according to the previous report (Tsunoda et al., 2021). In brief, to perform reverse transcription and template-switching, mRNA-trapped oligo-dT-immobilized Dynabeads M270-streptavidin (Thermo Fisher Scientific) were suspended in 10 µL of RT mix [1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP, 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO], and incubated for 60 min at 42ºC and immediately cooled on ice. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed following the purification of PCR product by an Agencort AM Pure XP kit (Beckman Coulter) at a 0.7:1 ratio of beads to sample and eluted with 20 µL of 10 mM Tris-HCl (pH 8.0). To amplify TCR libraries and add adaptor sequences for the next-generation sequencer, the third PCR was performed. The third-PCR products were purified as second PCR. The products were pooled and then purified and subjected to dual size selection using ProNex size-selective purification system (Promega) and eluted with 25 μL of 10 mM Tris-HCl (pH 8.5). Final TCR libraries, whose lengths were about 600 base pairs were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina). Only read2 contained the sequence regarding the definition of T cell clones.
single-cell targeted RNA-seq / TCR-seq
Bulk TCR-seq

treatment_protocol: B16F10 cells (5 × 10^5 cells) were inoculated subcutaneously (s.c.) into the right flanks of C57BL/6 mice. Anti-PD-L1 mAb (clone 10F.9G2, BioLegend) or Anti-CD4 mAb (clone GK1.5, BioLegend) was injected intraperitoneally (i.p.) at a dose of 200 μg per mouse on days 5 and 9 after tumor inoculation.

value: Untreated

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+ and CD4- CD8+ T cells

value: 8 weeks

value: female

tag: tumour cell line

value: B16F10 melanoma

tag: organ

value: dLN


title: Control_aPDL1_Tumor_1 Tag

status: Public on May 11 2023

type: SRA

state: Chiba

department: Research Institute for Biomedical Sciences

country: Japan

description: messenger RNA

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

source_name: tumor on day 14 after inoculation of B16F10 untreated or anti-PDL1-mAb-treatment

tag: strain

tag: treatment

value: anti-PDL1 mAb or Untreated

tag: cell type

tag: age

tag: Sex

value: Tumor


title: Control_aPDL1_Tumor_1 Target

description: messenger RNA


title: Control_aPDL1_Tumor_1 scTCR

description: messenger RNA


title: Control_dLN_1

description: messenger RNA

source_name: dLN on day 14 after inoculation of B16F10 with anti-PDL1 mAb treatment

value: anti-PDL1 mAb


title: WG_RAD.replicate_25.H3K27ac

city: Chengdu

first: jin

last: long

address: Hui ming

country: China

institute: Sichuan Agricultural University

description: WG_group5_RAD_merge_AllEnhancers.table.txt

library_source: genomic

data_processing:
H3K27ac ChIP-seq reads were mapped to the pig reference genome (Sscrofa 11.1) using BWA (version 0.7.15)
PCR duplicates were removed using Samtools (version 1.3.1).
Peaks were called using the SICER tool
Enhancers and super-enhancers were identified by ROSE algorithm
AssemblySusscrofa11.1
RE and SE files

molecule: genomic DNA

organism: Sus scrofa

source_name: retroperitoneal adipose

extract_protocol:
Adipose tissue samples were fixed with 1% formaldehyde. Then samples were lysed and chromatins were obtained on ice. Chromatins were sonicated to get soluble sheared chromatin with average DNA length of 200–500 bp.
Half of the soluble chromatin was stored at -20°C for sequencing as input DNA, while the remaining was used for immunoprecipitation reacted with H3K27ac (ab4729, Abcam) antibodies. For both input DNA and immunoprecipitated DNA, each ChIP-seq library was sequenced on Illumina HiSeq X Ten platform to generate 150 bp paired-end reads.

tag: tissue

value: retroperitoneal adipose

tag: chip antibody

value: H3K27ac (Abcam, #ab4729)


title: WG_RAD.replicate_34.hic

description: whole.samples.ABindex.results.txt.gz

data_processing:
Contacts matrix were generated by using juicer tools.
TADs were identified using deDoc.
Compartment A/B were defined by ABindex.
Susscrofa11.1
TAD files
ABindex files

extract_protocol:
1 g adipose tissue were pulverized, and 37% formaldehyde was added to obtain a final concentration of 4% for chromatin cross-linking. Mixture were incubated at room temperature (20–25℃) for 30 minutes, then glycine was added to obtain a final concentration of 0.25 mol/L to quench the formaldehyde. The mixture was then centrifuge at 1,500×g for 10 min at room temperature, and sediment was added the lysis buffer and homogenized. Homogenate was centrifuge at 5,000×g for adipocytes sediment. Nuclei of formaldehyde fixed adipose tissue were permeabilized and DNA was digested with 200 units of MboI (a 4-cutter restriction enzyme) for one hour at 37℃. The restriction fragment overhangs were filled and labbled by biotinylated nucleotides, then ligated in a small volume.
After crosslink. reversal, ligated DNA was purified and sheared to a length of 300–500 bp, at which point ligation junctions were pulled down with streptavidin beads and prepped for Illumina NovaSeq 6000 sequencing.


title: MEF, WT, ctrl, rep2

city: Grand Rapids

first: Russell

last: Jones

state: MI

address: 333 Bostwick Ave. NE

country: USA

institute: Van Andel Institute

data_processing:
vari-bbc/rnaseq_workflow (https://github.com/vari-bbc/rnaseq_workflow)
Sequence reads were trimmed using Trim Galore 0.6.0 (-q 20)
Trimmed sequence reads were mapped to GrCM38/mm10 using STAR version 2.7.8a (parameters- --twopassMode Basic, --outSAMtype BAM SortedByCoordinate, --quantMode GeneCounts)
Indexing was done using samtools version 1.9 (parameters- --twopassMode Basic, --outSAMtype BAM SortedByCoordinate, --quantMode GeneCounts)
Read count extraction was done using salmon version 1.5.2 (parameters - -l A, --validateMappings)
Read count normalization was performed DESeq2
Assembly: mm10
Supplementary files format and content: tab-delimited text files include gene counts values for each Sample

source_name: mouse embryonic fibroblast

extract_protocol:
RNA was harvested using Rneasy mini kit (Qiagen). 1 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.

value: mouse embryonic fibroblast

tag: genotype

value: WT

value: control


title: WG_RAD.replicate_21.H3K27ac


title: B16_CD8_Tumor_Tim3_PDL1_3

data_processing:
Data processing of Bulk TCRseq was performed as described in Aoki et al. (Aoki et al., 2021).
For some files, fastq file obtained from Novaseq 6000 S4 and SP were concatanated. Adapter trimming and quality filtering of sequencing data were performed by using Cutadapt-3.2  (Martin et al., 2011) and PRINSEQ-0.20.4 (Schmieder et al., 2011). Sequencing data were processed by MiXCR-3.0.5 (Bolotin et al., 2015). In MiXCR, filtered reads were aligned to reference mouse TCR V/D/J sequences registered in the international ImMunoGeneTics (IMGT) information system: -vParameters.geneFeatureToAlign = VTranscript -vjAlignmentOrder = JThenV, then identical sequences were assembled and grouped in clones with PCR and sequencing error correlation with the following parameters: -badQualityThreshold=10, –separateByV=true, --only-productive=true, –region-of-interest=CDR3. The Variable (V) and Joining (J) segment of TCRs were represented in IMGT gene nomenclature.
List of final clones were analyzed by VDJtools-1.2.1 (Shugay et al., 2015). Then, the sequencing reads of sample was normalized to nine-times of the cell count in each sample by “DownSample” command of VDJtools. T-cell clones were determined as TCR reads with the same TCR V segment, J segment and CDR3 nucleotide sequence.
Assembly: IMGT reference sequences of mouse TRA and TRB
Supplementary files format and content: Text files in VDJtools format include read count and frequency, complementarity determining region 3 (CDR3) nucleotide and amino acid sequences, Variable(V)/ Diversity(D)/ Joining(J) segment names, and V, D and J segment boundaries within CDR3 nucleotide sequence of individual clones

source_name: tumor on day 14 after innoculation of B16

extract_protocol:
Intravascular leukocytes were stained by i.v. injection of FITC-conjugated anti-CD45.2 mAb (3 mg/mouse, clone 104, BioLegend) three minutes before sacrifice.
dLN cells were prepared by standard method. Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.1% collagenase (FUJIFILM Wako), then subjected to 40% Percoll PLUS (Cytiva) and Histopaque-1083 (Sigma-Aldrich) gradient, and leukocytes were recovered from the interphase. The cell concentration of the suspensions was determined using Flow-Count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter).
Cells were stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 93, BioLegend) and fluorophore-conjugated anti-mouse mAbs. For dLN samples, Lineage+ (CD11b, B220, NK1.1, Ter119) cells were depleted using BD IMag Streptavidin Particles Plus-DM (BD Biosciences). For tumor samples, TCRb+ T cells were enriched magnetically using Anti-APC MicroBeads (Miltenyi) and LS columns (Miltenyi).
CD8+, CD8+ PD1-, CD8+ PD1+ Ly108+ Tim3-, CD8+ PD1+ Ly108+ Tim3+, and CD8+ PD1+ Ly108- Tim3+ T cells from tumor, and CD8+ CD44high T cells from dLN were sorted using FACS Aria II or Aria III (BD Biosciences). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and propidium iodide staining. Intravascular leukocytes were also excluded. Purity of sorted cells was always over 95%.
Bulk TCR sequencing library was prepared according to the previous report (Tsunoda et al., 2021). In brief, to perform reverse transcription and template-switching, mRNA-trapped oligo-dT-immobilized Dynabeads M270-streptavidin (Thermo Fisher Scientific) were suspended in 10 µL of RT mix [1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP, 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO], and incubated for 60 min at 42ºC and immediately cooled on ice. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed following the purification of PCR product by an Agencort AM Pure XP kit (Beckman Coulter) at a 0.7:1 ratio of beads to sample and eluted with 20 µL of 10 mM Tris-HCl (pH 8.0). To amplify TCR libraries and add adaptor sequences for the next-generation sequencer, the third PCR was performed. The third-PCR products were purified as second PCR. The products were pooled and then purified and subjected to dual size selection using ProNex size-selective purification system (Promega) and eluted with 25 μL of 10 mM Tris-HCl (pH 8.5). Final TCR libraries, whose lengths were about 600 base pairs were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina). Only read2 contained the sequence regarding the definition of T cell clones.

treatment_protocol: B16F10 or LLC cells (5 × 105 cells) were inoculated subcutaneously (s.c.) into the right flanks of C57BL/6 mice. Anti-PD-L1 mAb (clone 10F.9G2, BioLegend) or Anti-CD4 mAb (clone GK1.5, BioLegend) was injected intraperitoneally (i.p.) at a dose of 200 μg per mouse on days 5 and 9 after tumor inoculation.

value: anti-PD-L1 mAb

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+, PD1+, and Ly108- Tim3+ T cells

tag: date


title: WG_RAD.replicate_15.H3K27ac


title: WG_RAD.replicate_33.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_RAD.replicate_37.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_Tim3_PDL1_2


title: B16_CD8_Tumor_Tim3_PDL1_5


title: B16_CD8_Tumor_Tim3_PDL1_4


title: WG_RAD.replicate_31.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_Tim3_Control_5


title: WG_RAD.replicate_32.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_Tim3_PDL1_1


title: WG_RAD.replicate_14.H3K27ac


title: B16_CD8_Tumor_Tim3_Control_4


title: WG_MAD.replicate_7.H3K27ac

description: WG_group5_MAD_merge_AllEnhancers.table.txt

source_name: mesenteric adipose

value: mesenteric adipose


title: B16_CD8_Tumor_Tim3_Control_3

value: Untreated


title: WG_RAD.replicate_30.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_MAD.replicate_46.H3K27ac

description: WG_group2_MAD_merge_AllEnhancers.table.txt


title: B16_CD8_Tumor_Tim3_Control_1


title: WG_RAD.replicate_28.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_Tim3_Control_2


title: B16_CD8_Tumor_Tim3_CD4PDL1_5

value: combination of anti-CD4 and anti-PD-L1 mAbs


title: WG_MAD.replicate_45.H3K27ac


title: B16_CD8_Tumor_Tim3_CD4PDL1_4

value: 8 weeks


title: WG_MAD.replicate_44.H3K27ac


title: B16_CD8_Tumor_Tim3_CD4PDL1_2

value: female


title: B16_CD8_Tumor_Tim3_CD4PDL1_3

value: B16F10 melanoma


title: B16_CD8_Tumor_Tim3_CD4PDL1_1


title: B16_CD8_Tumor_Tim3_aCD4_4

value: anti-CD4 mAb


title: WG_RAD.replicate_21.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_RAD.replicate_26.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_MAD.replicate_42.H3K27ac


title: B16_CD8_Tumor_Tim3_aCD4_1


title: WG_RAD.replicate_20.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_RAD.replicate_25.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_PD1nega_PDL1_4

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+ and PD1- T cells


title: WG_MAD.replicate_33.H3K27ac


title: WG_RAD.replicate_19.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_PD1nega_PDL1_3


title: B16_CD8_Tumor_PD1nega_PDL1_5


title: WG_RAD.replicate_27.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_Tim3_aCD4_3


title: WG_MAD.replicate_30.H3K27ac


title: B16_CD8_Tumor_PD1nega_PDL1_2


title: B16_CD8_Tumor_PD1nega_PDL1_1


title: WG_MAD.replicate_28.H3K27ac


title: B16_CD8_Tumor_PD1nega_Control_4


title: WG_RAD.replicate_18.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_MAD.replicate_25.H3K27ac


title: WG_RAD.replicate_16.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_PD1nega_Control_5


title: WG_MAD.replicate_43.H3K27ac

description: WG_group4_MAD_merge_AllEnhancers.table.txt


title: B16_CD8_Tumor_Tim3_aCD4_5


title: B16_CD8_Tumor_Tim3_aCD4_2


title: B16_CD8_Tumor_PD1nega_Control_2


title: WG_MAD.replicate_41.H3K27ac


title: WG_MAD.replicate_21.H3K27ac


title: B16_CD8_Tumor_PD1nega_Control_1


title: B16_CD8_Tumor_PD1nega_CD4PDL1_5


title: B16_CD8_Tumor_PD1nega_CD4PDL1_4


title: WG_MAD.replicate_20.H3K27ac


title: WG_RAD.replicate_13.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_RAD.replicate_14.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_PD1nega_CD4PDL1_2


title: WG_MAD.replicate_14.H3K27ac


title: B16_CD8_Tumor_PD1nega_Control_3


title: WG_RAD.replicate_11.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_PD1nega_CD4PDL1_3


title: B16_CD8_Tumor_PD1nega_CD4PDL1_1


title: WG_RAD.replicate_10.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_46.H3K27ac

description: WG_group5_GOM_merge_AllEnhancers.table.txt

source_name: greater omentum fat

value: greater omentum fat


title: WG_GOM.replicate_7.H3K27ac

description: WG_group1_GOM_merge_AllEnhancers.table.txt


title: B16_CD8_Tumor_PD1nega_aCD4_4


title: WG_RAD.replicate_9.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_PD1nega_aCD4_3


title: B16_CD8_Tumor_Ly108_PDL1_5

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+, PD1+, and Ly108+ Tim3- T cells


title: B16_CD8_Tumor_PD1nega_aCD4_1


title: B16_CD8_Tumor_PD1nega_aCD4_2


title: WG_RAD.replicate_8.hic

description: whole.samples.ABindex.results.txt.gz

value: retroperitoneal adipose


title: WG_RAD.replicate_15.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_45.H3K27ac

description: WG_group3_GOM_merge_AllEnhancers.table.txt

value: H3K27ac (Abcam, #ab4729)


title: B16_CD8_Tumor_Ly108_PDL1_4


title: WG_GOM.replicate_43.H3K27ac


title: B16_CD8_Tumor_Ly108_PDL1_3


title: B16_CD8_Tumor_Ly108_PDL1_2


title: B16_CD8_Tumor_PD1nega_aCD4_5


title: WG_GOM.replicate_42.H3K27ac


title: B16_CD8_Tumor_Ly108_PDL1_1


title: B16_CD8_Tumor_Ly108_Control_5


title: WG_GOM.replicate_41.H3K27ac


title: WG_RAD.replicate_6.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_RAD.replicate_5.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_38.H3K27ac


title: B16_CD8_Tumor_Ly108_Control_2


title: B16_CD8_Tumor_Ly108_Control_1


title: WG_GOM.replicate_33.H3K27ac


title: B16_CD8_Tumor_Ly108_Control_3


title: WG_GOM.replicate_30.H3K27ac


title: B16_CD8_Tumor_Ly108_CD4PDL1_3


title: WG_RAD.replicate_4.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_RAD.replicate_2.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_RAD.replicate_3.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_28.H3K27ac


title: B16_CD8_Tumor_Ly108_CD4PDL1_5


title: WG_RAD.replicate_1.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_Ly108_CD4PDL1_1


title: WG_GOM.replicate_25.H3K27ac


title: WG_GOM.replicate_46.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_RAD.replicate_7.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_Ly108_Control_4


title: B16_CD8_Tumor_Ly108_aCD4_5


title: B16_CD8_Tumor_Ly108_aCD4_4


title: B16_CD8_Tumor_Ly108_aCD4_3


title: WG_GOM.replicate_21.H3K27ac


title: WG_GOM.replicate_45.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_Ly108_aCD4_1


title: WG_GOM.replicate_20.H3K27ac


title: B16_CD8_Tumor_Ly108_CD4PDL1_4


title: B16_CD8_Tumor_DP_PDL1_5

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+, PD1+, and Ly108+ Tim3+ T cells


title: WG_GOM.replicate_44.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_Ly108_CD4PDL1_2


title: B16_CD8_Tumor_Ly108_aCD4_2


title: B16_CD8_Tumor_DP_PDL1_4


title: MEF, KO, A485+IL1β, rep2

value: mouse embryonic fibroblast

value: LKB1 knockout


title: WG_GOM.replicate_14.H3K27ac


title: WG_GOM.replicate_43.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_DP_PDL1_2

value: anti-PD-L1 mAb


title: NC_ULB.replicate_7.H3K27ac

source_name: upper layer of backfat

value: upper layer of backfat


title: WG_GOM.replicate_41.hic

description: whole.samples.ABindex.results.txt.gz


title: VORA_8

city: Boston

first: Ania

last: Tassinari

state: MA

address: 200 Pier 4 Blvd

department: Data Science

institute: Servier Pharmaceuticals

data_processing:
Transcriptional profiling (RNA-seq) of frozen surgical samples was conducted using the ACE Research Transcriptome assay (Personalis).
Paired-end reads in FASTQ format were aligned to the human genome (GRCh38, release 85) with HISAT, v.2.0.5.
SAM-to-BAM conversion and sorting were performed using Samtools v.1.4
Transcript assembly with RefSeq annotation in GTF format and gene abundance estimation were carried out using StringTie v.1.3.3b and the built-in prep_DE.py Python script, producing gene-level raw count expression values as well as transcripts per million (that is, counts corrected for gene length and sequencing depth)
Assembly: GRCh38, release 85
Supplementary files format and content: tab-delimited gene_abundances text files include TPM, FPKM and coverage values for each Sample; tab-delimited gene_count_matrix includes raw counts for all Samples

organism: Homo sapiens

source_name: VORA

extract_protocol: none provided

value: vorasidenib


title: B16_CD8_Tumor_DP_Control_5

value: Untreated


title: NC_ULB.replicate_3.H3K27ac


title: B16_CD8_Tumor_DP_Control_4


title: NC_ULB.replicate_10.H3K27ac


title: WG_GOM.replicate_40.hic

description: whole.samples.ABindex.results.txt.gz


title: NC_RAD.replicate_3.H3K27ac


title: VORA_4


title: VORA_6


title: VORA_23


title: B16_CD8_Tumor_DP_PDL1_3


title: WG_GOM.replicate_39.hic

description: whole.samples.ABindex.results.txt.gz


title: VORA_22


title: NC_RAD.replicate_10.H3K27ac


title: WG_GOM.replicate_15.H3K27ac


title: WG_GOM.replicate_38.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_DP_CD4PDL1_5

value: combination of anti-CD4 and anti-PD-L1 mAbs


title: B16_CD8_Tumor_DP_Control_2

value: 8 weeks


title: WG_GOM.replicate_36.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_42.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_37.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_DP_PDL1_1

value: female


title: B16_CD8_Tumor_DP_CD4PDL1_4

value: B16F10 melanoma


title: NC_GOM.replicate_3.H3K27ac


title: VORA_9


title: VORA_21


title: VORA_24


title: NC_GOM.replicate_10.H3K27ac


title: B16_CD8_Tumor_DP_CD4PDL1_3


title: VORA_7


title: WG_GOM.replicate_34.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_DP_Control_3


title: NC_GOM.replicate_7.H3K27ac


title: NC_MAD.replicate_3.H3K27ac

value: mesenteric adipose


title: B16_CD8_Tumor_DP_Control_1


title: WG_GOM.replicate_35.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_33.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_DP_CD4PDL1_2


title: VORA_2


title: WG_GOM.replicate_32.hic

description: whole.samples.ABindex.results.txt.gz


title: WL_ULB.replicate_5.input


title: VORA_19


title: WL_RAD.replicate_7.input


title: B16_CD8_Tumor_DP_aCD4_5

value: anti-CD4 mAb


title: B16_CD8_Tumor_DP_CD4PDL1_1


title: VORA_18


title: WG_GOM.replicate_31.hic

description: whole.samples.ABindex.results.txt.gz

value: greater omentum fat


title: VORA_17


title: WG_GOM.replicate_29.hic

description: whole.samples.ABindex.results.txt.gz


title: WL_RAD.replicate_5.input


title: B16_CD8_Tumor_DP_aCD4_4


title: B16_CD8_Tumor_DP_aCD4_3


title: WG_GOM.replicate_30.hic

description: whole.samples.ABindex.results.txt.gz


title: WL_RAD.replicate_1.input


title: WG_GOM.replicate_28.hic

description: whole.samples.ABindex.results.txt.gz


title: WL_MAD.replicate_5.input


title: VORA_16


title: B16_CD8_Tumor_DP_aCD4_2


title: VORA_15


title: WL_GOM.replicate_4.input


title: WG_GOM.replicate_27.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_Tumor_DP_aCD4_1


title: VORA_20


title: VORA_13


title: WL_ULB.replicate_7.input


title: WG_GOM.replicate_26.hic

description: whole.samples.ABindex.results.txt.gz


title: VORA_11


title: WG_GOM.replicate_25.hic

description: whole.samples.ABindex.results.txt.gz


title: WL_GOM.replicate_1.input


title: B16_CD8_dLN_PDL1_4

source_name: dLN on day 14 after innoculation of B16

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+ and CD44high T cells


title: WG_ULB.replicate_45.input

description: WG_group6_ULB_merge_AllEnhancers.table.txt


title: VORA_10


title: WG_GOM.replicate_24.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_dLN_PDL1_3


title: VORA_1


title: B16_CD8_dLN_PDL1_2


title: IVO_9

source_name: IVO

value: ivosidenib


title: WG_GOM.replicate_22.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_dLN_PDL1_1


title: WG_ULB.replicate_15.input

description: WG_group5_ULB_merge_AllEnhancers.table.txt


title: WG_ULB.replicate_25.input


title: IVO_6


title: B16_CD8_dLN_Control_5


title: WG_ULB.replicate_44.input


title: WG_RAD.replicate_46.input

value: retroperitoneal adipose


title: IVO_5


title: WG_GOM.replicate_21.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_20.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_dLN_Control_4


title: WG_RAD.replicate_45.input


title: IVO_4


title: WG_RAD.replicate_42.input

description: WG_group6_RAD_merge_AllEnhancers.table.txt


title: WG_GOM.replicate_19.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_dLN_PDL1_5


title: B16_CD8_dLN_Control_3


title: IVO_3


title: B16_CD8_dLN_Control_2


title: IVO_25


title: WG_GOM.replicate_23.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_18.hic

description: whole.samples.ABindex.results.txt.gz


title: IVO_24


title: WG_RAD.replicate_30.input


title: B16_CD8_dLN_Control_1


title: WG_GOM.replicate_17.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_dLN_CD4PDL1_5


title: IVO_23


title: WG_GOM.replicate_15.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_16.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_RAD.replicate_21.input


title: WG_GOM.replicate_14.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_RAD.replicate_28.input


title: IVO_22


title: WG_RAD.replicate_25.input


title: IVO_21


title: B16_CD8_dLN_CD4PDL1_3


title: B16_CD8_dLN_CD4PDL1_4


title: WG_MAD.replicate_46.input


title: WG_MAD.replicate_45.input


title: WG_GOM.replicate_13.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_CD8_dLN_CD4PDL1_1


title: B16_CD8_dLN_CD4PDL1_2


title: IVO_20


title: WG_GOM.replicate_12.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_MAD.replicate_43.input


title: IVO_2


title: WG_GOM.replicate_11.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_MAD.replicate_42.input


title: B16_CD8_dLN_aCD4_5


title: IVO_19


title: WG_GOM.replicate_10.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_MAD.replicate_38.input


title: B16_CD8_dLN_aCD4_4


title: IVO_18


title: WG_GOM.replicate_9.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_MAD.replicate_28.input


title: WG_GOM.replicate_8.hic

description: whole.samples.ABindex.results.txt.gz


title: IVO_17


title: B16_CD8_dLN_aCD4_3


title: WG_MAD.replicate_30.input


title: IVO_16


title: B16_CD8_dLN_aCD4_2


title: B16_CD8_dLN_aCD4_1


title: WG_GOM.replicate_7.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_6.hic

description: whole.samples.ABindex.results.txt.gz


title: IVO_15


title: WG_MAD.replicate_25.input


title: WG_MAD.replicate_20.input


title: WG_MAD.replicate_15.input


title: WG_GOM.replicate_5.hic

description: whole.samples.ABindex.results.txt.gz


title: MEF, DKO_A2, IL1β, rep3

data_processing:
Reads were trimmed with TrimGalore v0.6.0.
Using STAR v2.7.8a, trimmed reads were aligned to mm10 GENCODE vM24 to obtain read counts per gene.
Assembly: mm10
Supplementary files format and content: tab-delimited file containing read counts per gene.

value: LKB1/CRTC2 knockout clone A2


title: IVO_12


title: WG_GOM.replicate_4.hic

description: whole.samples.ABindex.results.txt.gz


title: IVO_13


title: MEF, DKO_A1, IL1β, rep3

value: mouse embryonic fibroblast

value: LKB1/CRTC2 knockout clone A1


title: MEF, DKO_A2, ctrl, rep3

value: control


title: WG_GOM.replicate_45.input


title: WG_GOM.replicate_7.input


title: IVO_11


title: MEF, DKO_A1, ctrl, rep3


title: WG_GOM.replicate_3.hic

description: whole.samples.ABindex.results.txt.gz


title: MEF, KO, IL1β, rep3

value: LKB1 knockout


title: WG_GOM.replicate_2.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_41.input


title: CTRL9II cells, D28, biol rep 3

city: Stockholm

first: Michelle

last: Watts

address: Solnavägen 30

country: Sweden

institute: Karolinska Institutet

data_processing:
Raw data was trimmed of adapters using cutadapt v2.3 with command line parameters: -j 10 -m 20 -e 0.1 -q 20 -O 1.
Back-spliced junctions were detected from trimmed paired end reads using three separate programs; CIRCexplorer2 v2.3.8 (Zhang et al, 2016), MapSplice2 v2.1.8 (Wang et al, 2010) and CIRI2 v2.0.6 (Gao et al, 2018). For CIRCexplorer2, modules were run with default options, reads were pre-aligned with TopHat v2.0.9 tophat_fusion (with Bowtie v1.1.2 and Samtools v0.1.19) using –fusion-min-dist 200. MapSplice was run using python v2.7.6 and options –fusion-non-canonical –min-fusion-dist 200. CIRI2 was run using Perl v5.26.2 with default settings after aligning reads with BWA v0.7.17 (with Samtools v1.9). The R package circRNAprofiler v1.4.2 (Aufiero et al, 2020) was used as described in the manual to merge and filter back-spliced junction reads from all three detection programs as well as for circRNA annotation and extraction of circRNA sequences.
For linear RNA gene expression and slpicing analysis trimmed read data was aligned to the genome with STAR 2.6.1a with default settings. Count files were generated using featureCounts v1.6.3.
Analysis of differential exon usage was performed using the DEXSeq package (v1.40.0) (Anders et al, 2012) using provided python scripts to generate a flattened annotation file of exon bins, with aggregation disabled, and to count reads within exon bins.
Assembly: hg37
Supplementary files format and content: The tab-delimited text file "circRNA_counts.txt" contains count data for each sample - output from circRNAprofiler after merging raw count data as described above.
Supplementary files format and content: The tab-delimited text file "linearRNA_counts.txt" contains count data for each sample - output from featureCounts.
Supplementary files format and content: The tab-delimited text file "exon_counts.txt" contains count data for each sample - output from DEXSeq.
Supplementary files format and content: The BAM files provided for each file ("sampledescription.bam") contain reads aligned with TopHat v2.0.9 tophat_fusion (with Bowtie v1.1.2 and Samtools v0.1.19) using –fusion-min-dist 200. This data has been anonymised by removal of variant information using BAMboozle v05.0 (Ziegenhain & Sandberg, 2021).

source_name: CTRL-9-II; RRID:CVCL_JL84

growth_protocol: NES were seeded on tissue culture flasks coated with 20 μg/ml poly-L-ornithine (Sigma-Aldrich P3655), and 1 μg/ml laminin (Sigma-Aldrich L2020). Cells were grown in DMEM/F12+GlutaMAX medium (ThermoFisher 31331093) supplemented with 0.05X B27 (ThermoFisher 17504044), 1X N2 (ThermoFisher 17502001), 10 ng/ml bFGF (fisher scientific CTP0261), 10 ng/ml EGF (PeproTech AF-100-15) and 10 U/ml penicillin/streptomycin (ThermoFisher 15140122). Medium was exchanged 50% daily and cells maintained in 5% CO2 at 37ºC, passaging once 100% confluent and seeding at a density of 5x104 cells/cm2.

extract_protocol:
Cells were lysed in TRIzol reagent (ThermoFischer 15596026) before separating with chloroform and mixing the aqueous phase with isopropanol as per manufacturer directions. RNA was then isolated from the isopropanol/chloroform solution using the ReliaPrep RNA Cell Miniprep kit (Promega Z6010).
Libraries were prepared with Illumina Truseq Stranded total RNA RiboZero GOLD kit.

treatment_protocol: Neural differentiation was induced by growth factor withdrawal the day after plating with media B27 concentration increased to 0.5X. Media was exchanged 50% every second day up until D15, after which media was supplemented with 0.4 ug/ml laminin and exchanged 50% every three days.

tag: cell line

value: CTRL-9-II; RRID:CVCL_JL84

value: iPSC-derived neuroepithelial stem cells

value: neurotypical control

value: male

value: neuronal differentiation

tag: time

value: Day 28


title: WG_GOM.replicate_1.hic

description: whole.samples.ABindex.results.txt.gz


title: NC_ULB.replicate_12.hic

description: whole.samples.ABindex.results.txt.gz

value: upper layer of backfat


title: WG_GOM.replicate_38.input


title: MEF, WT, IL1β, rep3

value: WT


title: CTRL9II cells, D28, biol rep 1

source_name: CTRL-9-II; RRID:CVCL_JL82

value: CTRL-9-II; RRID:CVCL_JL82


title: WG_GOM.replicate_33.input

value: greater omentum fat


title: CTRL9II cells, D5, biol rep 4

source_name: CTRL-9-II; RRID:CVCL_JL81

value: CTRL-9-II; RRID:CVCL_JL81

value: Day 5


title: MEF, WT, ctrl, rep3


title: CTRL9II cells, D28, biol rep 2

source_name: CTRL-9-II; RRID:CVCL_JL83

value: CTRL-9-II; RRID:CVCL_JL83


title: CTRL9II cells, D5, biol rep 3

source_name: CTRL-9-II; RRID:CVCL_JL80

value: CTRL-9-II; RRID:CVCL_JL80


title: NC_ULB.replicate_9.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_30.input


title: WG_GOM.replicate_28.input


title: MEF, DKO_A2, ctrl, rep2

value: mouse embryonic fibroblast

value: LKB1/CRTC2 knockout clone A2


title: CTRL9II cells, D5, biol rep 2

source_name: CTRL-9-II; RRID:CVCL_JL79

value: CTRL-9-II; RRID:CVCL_JL79


title: NC_ULB.replicate_5.hic

description: whole.samples.ABindex.results.txt.gz


title: MEF, DKO_A2, IL1β, rep2


title: WG_GOM.replicate_25.input


title: NC_ULB.replicate_7.hic

description: whole.samples.ABindex.results.txt.gz


title: CTRL9II cells, D5, biol rep 1

source_name: CTRL-9-II; RRID:CVCL_JL78

value: CTRL-9-II; RRID:CVCL_JL78


title: NC_ULB.replicate_2.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_21.input


title: MEF, DKO_A1, IL1β, rep2

value: LKB1/CRTC2 knockout clone A1


title: NC_MAD.replicate_12.hic

description: whole.samples.ABindex.results.txt.gz

value: mesenteric adipose


title: IVO_1

value: ivosidenib


title: MEF, KO, ctrl, rep3

value: LKB1 knockout

value: control


title: MEF, DKO_A1, ctrl, rep2


title: CTRL9II cells, NES, biol rep 4

source_name: CTRL-9-II; RRID:CVCL_JL77

value: CTRL-9-II; RRID:CVCL_JL77

value: iPSC-derived neuroepithelial stem cells

value: neurotypical control

value: male

value: neuronal differentiation

value: Day 0


title: NC_MAD.replicate_11.hic

description: whole.samples.ABindex.results.txt.gz


title: CTRL9II cells, NES, biol rep 3

source_name: CTRL-9-II; RRID:CVCL_JL76

value: CTRL-9-II; RRID:CVCL_JL76


title: WG_GOM.replicate_15.input

value: greater omentum fat


title: WG_GOM.replicate_14.input


title: MEF, KO, ctrl, rep2

source_name: mouse embryonic fibroblast

value: mouse embryonic fibroblast


title: MEF, KO, IL1β, rep2


title: NC_MAD.replicate_10.hic

description: whole.samples.ABindex.results.txt.gz


title: CTRL9II cells, NES, biol rep 2

source_name: CTRL-9-II; RRID:CVCL_JL75

value: CTRL-9-II; RRID:CVCL_JL75


title: NC_MAD.replicate_9.hic

description: whole.samples.ABindex.results.txt.gz

source_name: mesenteric adipose


title: MEF, WT, IL1β, rep2

value: WT


title: NC_MAD.replicate_8.hic

description: whole.samples.ABindex.results.txt.gz


title: MEF, WT, ctrl, rep2


title: NC_ULB.replicate_3.input

value: upper layer of backfat


title: MEF, DKO_A2, IL1β, rep1

value: LKB1/CRTC2 knockout clone A2


title: NC_ULB.replicate_10.input


title: CTRL9II cells, NES, biol rep 1

source_name: CTRL-9-II; RRID:CVCL_JL74

value: CTRL-9-II; RRID:CVCL_JL74


title: NC_ULB.replicate_7.input

source_name: upper layer of backfat


title: NC_MAD.replicate_7.hic

description: whole.samples.ABindex.results.txt.gz

value: mesenteric adipose


title: NC_RAD.replicate_3.input

source_name: retroperitoneal adipose

value: retroperitoneal adipose


title: NC_MAD.replicate_6.hic

description: whole.samples.ABindex.results.txt.gz


title: MEF, DKO_A2, ctrl, rep1

value: control


title: NC_MAD.replicate_5.hic

description: whole.samples.ABindex.results.txt.gz


title: WG_GOM.replicate_20.input

source_name: greater omentum fat


title: MEF, DKO_A1, IL1β, rep1

value: LKB1/CRTC2 knockout clone A1


title: NC_RAD.replicate_10.input


title: SKM-1, VEM+BOR, SAMPLE4

city: Chongqing

first: Qiao

last: Zhou

state: State...

address: 1 Youyi Road, Yuzhong, Chongqing,China

institute: The First Affiliated Hospital of Chongqing Medical University

data_processing:
The image data measured by the high-throughput sequencer are converted into sequence data (reads) by CASAVA base recognition. Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing N base and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 (v2.0.5) and paired-end clean reads were aligned to the reference genome using Hisat2 (v2.0.5). We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
featureCounts (v1.5.0-p3) was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels
(For DESeq2 with biological replicate) Differential expression analysis of two  conditions/groups (two biological replicates per condition) was performed using the  DESeq2 R package (1.20.0). DESeq2 provide statistical routines for determining  differential expression in digital gene expression data using a model based on the  negative binomial distribution. The resulting P-values were adjusted using the  Benjamini and Hochberg’s approach for controlling the false discovery rate . padj<=0.05 and |log2(foldchange)| >= 1 were set as the threshold for significantly  differential expression.
(For edgeR without biological replicates) Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the edgeR R package (3.22.5). The P values were adjusted using the Benjamini & Hochberg method. padj<=0.05 and |log2(foldchange)| >= 1 were set as the threshold for significantly differential expression.
Assembly: HISAT2
Supplementary files format and content: group2vsgroup1_deg.txt
Supplementary files format and content: group3vsgroup1_deg.txt
Supplementary files format and content: group4vsgroup1_deg.txt

source_name: cells

extract_protocol:
Total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA)
Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR amplification, the PCR product was purified by AMPure XP beads, and the library was finally obtained. In order to ensure the quality of the library, the library needs to be tested. After the construction of the library, the library was initially quantified by Qubit2.0 Fluorometer, then diluted to 1.5ng/ul, and the insert size of the library is detected by Agilent 2100 bioanalyzer. After insert size meets the expectation, qRT-PCR is used to accurately quantify the effective concentration of the library (the effective concentration of the library is higher than that of 2nM) to ensure the quality of the library
After the library is qualified, the different libraries are pooling according to the effective concentration and the target amount of data off the machine, then being sequenced by the Illumina NovaSeq 6000. The end reading of 150bp pairing is generated. The basic principle of sequencing is to synthesize and sequence at the same time (Sequencing by Synthesis). Four fluorescent labeled dNTP, DNA polymerase and splice primers were added to the sequenced flowcell and amplified. When the sequence cluster extends the complementary chain, each dNTP labeled by fluorescence can release the corresponding fluorescence. The sequencer captures the fluorescence signal and converts the optical signal into the sequencing peak by computer software, so as to obtain the sequence information of the fragment to be tested.

value: cells

value: AML cell lines

tag: group

value: VEM+BOR


title: SKM-1, BOR, SAMPLE6

value: BOR


title: SKM-1, None, SAMPLE5


title: SKM-1, VEM, SAMPLE3

value: VEM


title: NC_MAD.replicate_4.hic

description: whole.samples.ABindex.results.txt.gz


title: NC_MAD.replicate_3.input


title: NC_MAD.replicate_3.hic

description: whole.samples.ABindex.results.txt.gz


title: NC_GOM.replicate_7.input


title: NC_MAD.replicate_2.hic

description: whole.samples.ABindex.results.txt.gz


title: SKM-1, None, SAMPLE1


title: NC_GOM.replicate_3.input


title: NC_MAD.replicate_1.hic

description: whole.samples.ABindex.results.txt.gz


title: NC_GOM.replicate_10.input


title: NC_RAD.replicate_12.hic

description: whole.samples.ABindex.results.txt.gz


title: MEF, WT, IL1β, rep1


title: H. pylori_3_IP

city: Changsha

first: Huan

last: Li

state: Hunan

address: 138 Tongzipo Road, Yuelu district

department: Gastroenterology department

institute: The Third Xiangya Hospital of Central South University

data_processing:
Base calling software: BCL convert; Data filter: fastp(0.19.4)，remove adapters, repeat sequences and low quality sequences; reads alignment : HISAT2(2.0.4); Peak calling diffpeak : R package: exomePeak (1.5.0); Peak annotation : ANNOVAR; Transcript assembly and quantification : StringTie (2.1.2); Differential Expression Analysis :R package: edgeR(4.1); Motif: HOMER(4.10)"
Assembly: Homo_sapiens.GRCh38.Ensembl_V101

growth_protocol: GES-1 cells were maintained in 1640 supplemented with 10% fetal bovine serum (FBS)  in humidified atmosphere with 5% CO2 at 37C

extract_protocol: Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel. Poly (A) RNA is purified from 50μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 86℃ 7min. Then the cleaved RNA fragments were incubated for 2h at 4℃ with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). Then the IP RNA was reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs withE. coliDNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA）. An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol

treatment_protocol: Co-culture with H. pylori for 48h

value: Helicobacter pylori


title: MEF, WT, ctrl, rep1


title: H. pylori_3_input


title: NC_RAD.replicate_11.hic

description: whole.samples.ABindex.results.txt.gz


title: H. pylori_2_IP


title: MEF, DKO, IL-1β, Input

data_processing:
Reads were trimmed with TrimGalore v0.6.0
Trimmed reads were aligned with bwa mem v0.7.17 and duplicates were marked using SAMBLASTER v0.1.24.
BigWig files were generated using bamCoverage in DeepTools v with the parameters, '--binSize 10 --extendReads --normalizeUsing "CPM" --samFlagExclude 1024 --samFlagInclude 64' and '--blackListFileName' to specify the ENCODE blacklist v2.
Assembly: mm10
Supplementary files format and content: Bigwig

extract_protocol:
Cells were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin was sonicated using a E220 focused-ultrasonicator (Covaris).
ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.

value: LKB1/CRTC2 knockout


title: NC_RAD.replicate_10.hic

description: whole.samples.ABindex.results.txt.gz


title: SAMP8 Male HxTX1[R14A] R.92

city: Melbourne

first: Deanna

middle: S

last: Deveson Lucas

state: Victoria

address: 15 Innovation walk, Monash University, Clayton

country: Australia

institute: Monash University

data_processing:
Raw fastq files have been analysed with nfcore/RNAseq pipeline using STAR  aligner (Dobin et al., 2013) mapping raw reads to the mus musculus GRCm38-mm10 (GCF_000001635.20, NCBI) reference genome, reads were quantified with salmon.
Raw counts were then analysed with Degust (Powell 2015) web tool to do differential expression analysis to produce list of differentially expressed genes and several quality plots including classical multidimensional scaling (MDS) and MA plots
In this analysis limma voom (Law et al. 2014) was used for differential expression analysis. Degust (Powell 2015) largely follows limma voom workflow with typical counts per million (CPM) library size normalisation and trimmed mean of M values (TMM) normalisation (Robinson and Oshlack 2010) for RNA composition normalisation.
Assembly: GRCm38-mm10 (GCF_000001635.20, NCBI)
Supplementary files format and content: raw gene counts, text file, tab separated, includes all samples

extract_protocol:
RNA was harvested using RNeasy Lipid Tissue RNeasy Lipid Tissue Mini Kit (Qiagen). 1.3 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.

value: brain

value: Male

value: HsTX1[R14A] 1mg/kg


title: MEF, DKO_A1, ctrl, rep1

value: mouse embryonic fibroblast


title: H. pylori_2_input


title: NC_RAD.replicate_9.hic

description: whole.samples.ABindex.results.txt.gz


title: MEF, DKO, IL-1β, H3K27ac

tag: antibody


title: SAMP8 Male HxTX1[R14A] R.89


title: H. pylori_1_IP


title: MEF, KO, IL1β, rep1

value: LKB1 knockout


title: SKM-1, BOR, SAMPLE2


title: MEF, KO, ctrl, rep1


title: H. pylori_1_input


title: NC_RAD.replicate_8.hic

description: whole.samples.ABindex.results.txt.gz


title: Cortical inhibitory neurons, replicate 2

first: Christopher

last: Harvey

address: 210 Longwood Ave

department: Neurobiology

institute: Harvard Medical School

description: 10x Genomics

library_source: genomic single cell

data_processing:
Reads were mapped to the mouse genome (mm10), and cell barcodes were processed using the cellranger pipeline (cellranger-ATAC version 1.1.0, 10x Genomics) using default parameters. SnapATAC (version 1.0.0) was used to process the data into a snap object.
Assembly: mm10
Supplementary files format and content: Comma separated files, snap objects.

source_name: Cerebral cortex

extract_protocol:
Posterior cortex (roughly 3 mm by 3 mm) was dissected in ice-cold choline solution (2.1g/L NaHCO3, 2.16 g/L glucose, 0.172 g/L NaH2PO4·H2O, 7.5 mM MgCl2·6H2O, 2.5 mM KCl, 10 mM HEPES, 15.36 g/L choline chloride, 2.3 g/L ascorbic acid, 0.34 g/L pyruvic acid), and transferred to a Dounce containing homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, protease inhibitors). The tissue was dounced with a tight pestle until it was well homogenized (10-15 strokes). IGEPAL (final 0.15%) was added, followed by 5-10 more strokes. The homogenate was passed through a 40 µm filter. Tween-20 (final 0.1%) and BSA (final 1%) were added to the filtrate. Nuclei were centrifuged at 500g for 5 min at 4º C and resuspended in 0.5 mL Wash Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween-20). Nuclei from each sample were sorted by FACS (Sony SH800Z) based on the Sun1-GFP signal into two wells in a cooled 96-well plate coated with Wash Buffer.
We followed the manufacturer’s protocol for single cell ATAC (10x Genomics CG000168 Rev A). We used one Chromium Controller (10x Genomics) lane per sample for cell barcoding. Libraries were sequenced on a NextSeq 500 DNA sequencer (Illumina).

treatment_protocol: Mice were taken from their home cage without treatment.

value: Cerebral cortex

value: Inhibitory neurons

value: Gad2-Cre+/-; Sun1-GFP+/-

value: home cage


title: SAMP8 Male HxTX1[R14A] R.81


title: NC_RAD.replicate_7.hic

description: whole.samples.ABindex.results.txt.gz

value: retroperitoneal adipose


title: Control_3_IP

value: PBS


title: MEF, KO, IL-1β, H3K27ac


title: SAMP8 Male HxTX1[R14A] R.93


title: Control_3_input


title: NC_RAD.replicate_6.hic

description: whole.samples.ABindex.results.txt.gz


title: SAMP8 Male HxTX1[R14A] R.88


title: MEF, KO, IL-1β, Input


title: Control_2_IP


title: Cortical inhibitory neurons, replicate 1

description: 10x Genomics


title: NC_RAD.replicate_5.hic

description: whole.samples.ABindex.results.txt.gz


title: MEF, KO, ctrl, H3K27ac

value: control


title: MEF, DKO, ctrl, H3K27ac


title: SAMP8 Male HxTX1[R14A] R.74


title: NC_RAD.replicate_4.hic

description: whole.samples.ABindex.results.txt.gz


title: Control_2_input


title: SAMP8 Female HxTX1[R14A] R.72

value: Female


title: MEF, WT, IL-1β, H3K27ac

value: WT


title: Control_1_IP


title: MEF, DKO, ctrl, Input


title: Control_1_input


title: NC_RAD.replicate_2.hic

description: whole.samples.ABindex.results.txt.gz


title: SAMP8 Female HxTX1[R14A] R.71


title: MEF, WT, IL-1β, Input


title: SAMP8 Female HxTX1[R14A] R.98


title: MEF, KO, ctrl, Input


title: MEF, WT, ctrl, H3K27ac


title: NC_RAD.replicate_1.hic

description: whole.samples.ABindex.results.txt.gz


title: SAMP8 Male PBS R.91


title: hCD4 STARR Donor IV

city: Cincinnati

first: Kurtis

last: Stefan

state: OH

address: 3333 Burnet Ave

department: Dept Allergy & Immunology

institute: University of Cincinnati

data_processing:
Fastq are concatenated for each biologic donor before alignment.
Trimming the adapters with TrimGalore. This step is particularly important when the reads are long and the fragments are short-resulting in sequencing adapters at the end of read. If adapter is not removed the read will not map. TrimGalore can recognize standard adapters, such as Illumina or Nexterra/Tn5 adapters.
Mapping reads with BowTie. Only uniquely mapped reads with less than 3 mismatches are used in the downstream analysis. Results are saved as a .bam file. (Bam files are downloaded for further processing)
The per duplication count of each unique read for each biologic donor was fitted to the negative binomial distribution for each technical replicate (input plasmid library). Duplicate reads exceeding the 75th percentile were removed from each sample by MACS2 to reduce false discovery of enhancers resulting from PCR duplication. Enhancers were called using MACS2 in which the plasmid reads were used as a control, [macs2 callpeak -f BEDPE --keep-dup all -g 2.7E9], and with -log10(P-value) greater than 75. NRE were also detected with MACS2, using the plasmid reads as treatment and the STARR RNA as control [macs2 callpeak -f BEDPE --keep-dup all -g 2.7E9], with logP greater than 30. Both enhancers and silencers are retained only if intersecting an ATAC-Seq narrowPeak called using MACS2 from the plasmid reads alone.
Assembly: hg19
Library strategy: Lenti-STARR-seq

source_name: Peripheral Blood

growth_protocol: PBMC were isolated from Lymphocyte Reduction Filters obtained from male donors at Hoxworth Blood Bank at the University of Cincinnati. CD4+ T cells were isolated by magnetic negative selection using Untouched Human CD4+ Isolation Kit (Invitrogen 11346D) per manufacture’s recommendations. CD4+ T cells are cultured in Optimizer CTS (Gibco #A1048501) with 2mM L-Glutamine (Sigma # G7513) and 1x Penicillin-Streptomycin (Sigma # P4333) in incubators maintained at 37°C with 5% CO2.

extract_protocol:
HEK-293T Cells were grown in 15cm2 dishes in DMEM (Gibco #11995-065) with 10% FBS (R&D Systems #S11150). Cells were grown to 70% confluency, then DMEM was replaced two hours before transfection. Each 15cm2 plate is transfected with a total of 21ug plasmid DNA (1:2:1 ratio psPAX2-D64V:pATAC-STARR:pLAI-Env) [psPAX-D64V (Addgene #63586), and pLAI-HIV (Addgene #133996)]. DNA was mixed with 4.25x PEI (Polysciences #23966) in at total of 2mL DPBS per plate, let rest for complex formation for 10 minutes at 22°C, then added dropwise to the cell culture while swirling. 10 hours after transfection the  cells were washed once with DPBS, then cultured in DMEM with 10% FBS and 2mM Caffeine (Millipore #205548). 48 hours after transfection the media was collected and stored at 4°C. 72 hours after transfection the media is collected and pooled. The viral supernatant was sterile filtered through a 0.45uM PES filter, and loaded into Ultracentrifuge Tubes (Beckman #344058), and spun for 2.5 hours, 4°C, at 35,000g. Each pellet was dissolved in 200uL DPBS, which was left overnight at 4°C to resuspend. Concentrated Lentivirus was filtered through 0.45uM PES filter, and snap frozen in an ethanol/dry-ice bath at -80°C until use.
Lenti-STARR Transduction: Freshly isolated hCD4 T Cells were suspended to 6.6E6 cells per mL in CTS Optimizer, then combined with 0.33mL concentrated lentivirus per mL cells. Cells were spinfected in low-bind 6-well plates (Corning #3471) with 7ug/mL polybrene (MilliporeSigma #TR1003G) at 950g, 32°C, for 2 hours. Immediately following spinfection, cells were gently resuspended and cultured for 2 hours at 37°C, 5% CO2. Cells were collected and centrifuged and suspended cultured for 24 hours. Cells are treated with 100U DNaseI (NEB M0303S) for 30 minutes at 37°C.
Total RNA was purified from transduced cells (Qiagen #75144) and concentrated 7.5x using RNA Cleanup (NEB #T2040L). RNA was Reverse Transcribed according to manufacturer guidelines for STARR-Seq transcript specific primers (Supplemental File 1) with SuperScript IV (Invitrogen #18090010), using twice the recommended RNA input. cDNA was purified using a Reaction Cleanup Kit (Qiagen #28206). cDNA was amplified using a custom STARR-Seq transcript specific primer (Supplemental File 1) as previously described.2 Diluted Lenti-STARR-Seq plasmid ‘input’ control was amplified using an input concentration which yielded the same amplification cycle number as the STARR library for hCD4+ Donor I. PCR amplified library was purified using PCR cleanup columns (Qiagen #28206), and quantified for PE-150bp sequencing (Novogene).

treatment_protocol: STARR-Seq is preformed according to ATAC-STARR-Seq.2 Briefly, OMNI ATAC-Seq was performed as previously described with 160,000 human CD4+ cells (in total), in batches of 100,000 cells per reaction.3 ATAC-Seq reaction products were cleaned up using the Qiagen Reaction Cleanup Kit (Qiagen #28206) and eluted in 12uL per 100K cells transposed. Each resultant elution was amplified in 50ul with custom primers (Supplemental File 1) for 10 total cycles, as described previously.2 The amplified library was purified using a PCR Cleanup Kit (Qiagen #28104), and size selected for 150-500bp fragments on a 2% agarose gel, using 300mg per column (NEB #T1020L). The eluted ATAC-Seq library was cloned into AgeI and SalI digested pLenti-STARR using a 3:1 molar ratio (insert:backbone) in a total reaction volume of 100uL (NEB #E5520S). The reaction was concentrated to 20uL in a Reaction Cleanup (Qiagen #28206). The cloned ATAC-Lenti-STARR was electroporated into MegaX DH10B cells (Invitrogen #C6400003) using 10uL library per 100uL cells 2.0 kV, 200 Ω, 25 µF, in 20uL electroporation reactions in 0.1cm cuvettes (BioRad #165-2089) on a Harvard Apparatus (ECM Model 630). 950uL prewarmed recovery media was added and grown for one hour at 37°C. All electroporation reactions were pooled to 1L sterile Luria Broth (Fisher #BP1426), and grown in 0.2L per 2L flask, for 8hrs at 37°C, 300RPM. Each 200mL of culture was purified in EndoFree MaxiPrep Reactions (Zymo #D4203) and eluted in 0.4mL per column.

value: Peripheral Blood

tag: sample type

value: Donor IV

tag: protocol

value: STARR-Seq (ATAC)


title: SAMP8 Male PBS R.90

value: brain


title: MEF, WT, ctrl, Input


title: hCD4 STARR Donor II

value: Donor II


title: aCD4_dLN_1

data_processing:
Data processing of scRNAseq was performed as described in Shichino et al. (Shichino et al., 2021).
For target sequencing data, filtered cell barcode reads were annotated by Python script provided by BD Biosciences with minor modification for compatibility to Python 3.7. Associated cDNA reads were mapped to reference RNA using bowtie2-2.4.2 (Langmead et al., 2012) 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 (https://github.com/pysam-developers/pysam), 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 (Lun et al., 2019) in R-3.6.3. Cells of which total read count was over inflection point were considered as valid cells.
For sample tag data, adapter trimming of sequencing data was performed by using cutadapt 3.4. Filtered reads were chunked to 64 parts for parallel processing by using Seqkit 0.15.0. Filtered cell barcode reads were annotated by Python script provided by BD with minor modification for compatible to Python3.8. Associated sample tag reads were mapped to known barcode fasta by using bowtie2-2.4.2 by the following parameters: -p 2 -D 50 -R 20 -N 0 -L 14 -i S,1,0.75 –norc –seed 656565 –reorder –trim-to 3:40 –score-min L,-9,0 –mp 3,3 –np 3 –rdg 3,3.  Then, cell barcode information of each read were added to the bowtie2-mapped BAM files, and read counts of each Tag in each cell barcode were counted by using mawk. Resulted count data was converted to Tags x cells matrix file by using data.table package in R 3.6.3, and top 1M cell barcodes were extracted. For assignment of each tags to each cell barcodes, read counts of each tag in each valid cell barcode, which defined by the cDNA matrix, were extracted from tag/cell barcode expression matrix. Unassigned cell barcodes were labeled as “not-detected” cells. Then, sum of the total read counts of each tags were normalized to the minimum sum count of each tags, and log2 fold-change between first most tag counts and second most tag counts within each cell barcode. Each cell barcode were ranked by the fold-change ascending order, and top N cells were identified as doublets (N was calculated as theoretically detectable doublets calculated by the Poisson’s distribution based on the number of loaded cells, total Rhapsody well number, and number of tags used). Correspondence table between sample tag and sample sources is summarized in Supplementary Table 1.
Raw data of single-cell TCR sequencing were processed by in-house pipelin. Briefly, sequencing reads were first separated by cell barcode, then adapter trimming and quality filtering of sequencing data were performed by using Cutadapt (Martin et al., 2011). PCR and sequencing error was corrected by lighter (Song et al., 2014). Error-corrected sequence reads were processed by MiXCR (Bolotin et al., 2015) to generate the list of TCRa or TCRb sequences for each cell barcode, using reference mouse TCR V/D/J sequences registered in the international ImMunoGeneTics (IMGT) information system with the parameters summarized in Supplementary Table 3.
Quality filtering of cell barcodes was performed with the following criteria: (1) more than 32 TCR reads were detected. (2) the proportion of the most frequent TCR sequence in cell barcode was over 0.6. (3) the cell barcode was annotated to the T cell cluster in scRNAseq analysis. Finally, the most frequent sequence of TCRa and TCRb was adopted and paired in each cell barcode.
T-cell clones were determined as cells with the same TCRβ, defined by V segment, J segment, and CDR3 nucleotide sequence. Frequency of each clone was calculated as the proportion of cells belonging to the clone in all T cells whose TCRβ was assigned.
Data processing of Bulk TCRseq was performed as described in Aoki et al. (Aoki et al., 2021). Adapter trimming and quality filtering of sequencing data were performed by using Cutadapt-3.2  (Martin et al., 2011) and PRINSEQ-0.20.4 (Schmieder et al., 2011). Sequencing data were processed by MiXCR-3.0.5 (Bolotin et al., 2015). In MiXCR, filtered reads were aligned to reference mouse TCR V/D/J sequences registered in the international IMGT information system: -vParameters.geneFeatureToAlign = VTranscript -vjAlignmentOrder = JThenV, then identical sequences were assembled and grouped in clones with PCR and sequencing error correlation with the following parameters: -badQualityThreshold=10, –separateByV=true, --only-productive=true, –region-of-interest=CDR3. The Variable (V) and Joining (J) segment of TCRs were represented in IMGT gene nomenclature.
List of final clones were analyzed by VDJtools-1.2.1 (Shugay et al., 2015). Then, the sequencing reads of sample was normalized to ten-times of the cell count in each sample by “DownSample” command of VDJtools. T-cell clones were determined as TCR reads with the same TCR V segment, J segment and CDR3 nucleotide sequence.
Assembly: GRCm38
Assembly: IMGT reference sequences of mouse TRA and TRB
Supplementary files format and content: (scRNAseq) tab-delimited text files include raw tag-count values for each sample
Supplementary files format and content: (scTCRseq) correspondence table between cell barcodes and TCR sequences
Supplementary files format and content: (Bulk TCRseq) text files in VDJtools format include read count and frequency, complementarity determining region 3 (CDR3) nucleotide and amino acid sequences, Variable(V)/ Diversity(D)/ Joining(J) segment names, and V, D and J segment boundaries within CDR3 nucleotide sequence of individual clones

source_name: dLN on day 14 after innoculation of B16F10 with anti-CD4 mAb treatment

extract_protocol:
Intravascular leukocytes were stained by i.v. injection of FITC-conjugated anti-CD45.2 mAb (3 mg/mouse, clone 104, BioLegend) three minutes before sacrifice.
Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.1mg/mL Liberase TM (Roche). The cells were then subjected to 40% Percoll PLUS (Cytiva) and Histopaque-1083 (Sigma-Aldrich) gradient and leukocytes were recovered from the interphase. The cell concentration of the suspensions was determined using Flow-Count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter).
Cells were then stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 93, BioLegend) and fluorophore-conjugated anti-mouse mAbs. Cells were also stained with Sample Tag oligonucleotide-conjugated antibodies in BD® Single-Cell Multiplexing Kit (BD Biosciences) as summarized in Supplementary Table 1.
CD8+ T cells were sorted using FACS Aria II or Aria III (BD Biosciences). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and propidium iodide staining. Intravascular leukocytes were also excluded. Purity of sorted cells was always over 95%.
T cells from the spleen of Pmel-1 transgenic mice which stained with Sample Tag oligonucleotide-conjugated antibodies as summarized in Supplementary Table 1 were spiked in after cell sorting.
Live cells were stained with Calcein AM (Nacalai) and counted using Flow-Count fluorospheres and a Cytoflex flow cytometer.
For single-cell RNA/TCRseq, 18,000-30,000 labeled cells were trapped and reverse-transcribed using the BD RhapsodyTM (BD) according to the manufacturer's instructions. Targeted mRNA, TCRseq and Sample Tag libraries were prepared using according to the manufacturer's instructions (VDJ CDR3 and Sample Tag Library Preparation Protocol) with the following modifications; (1) BD RhapsodyTM Immune Response Panel Mm with supplement panel including Cmpk2, Ifi204, Ifi211, Ifit1, Ifit3, Ifit3b, Ifit3b, Isg15, Mx1, Rsad2, and Usp18 were used for target mRNA amplification. (2) we used original set of primer for TCR amplification as summarized in Supplementary Table 2, and performed additional PCR (3rd PCR, 6 cycles) to introduce unique dual index for sequencing. (3) we used ProNex Size-Selection DNA purification System (Promega) for purification of PCR product. Amplified libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems) and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). Targeted RNAseq and Sample Tag libraries were sequenced on the Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 141 bp read 2) (Illumina) to a depth of approximately 20,000-40,000 reads and 5,000-10,000 reads per cell, respectively. TCRseq libraries were sequenced on the Illumina Novaseq 6000 SP flowcell (67 bp read 1 and 459 or 141 bp read 2) (Illumina) to a depth of approximately 5,000-20,000 reads.
Bulk TCR sequencing library was prepared according to the previous report (Tsunoda et al., 2021). In brief, to perform reverse transcription and template-switching, mRNA-trapped oligo-dT-immobilized Dynabeads M270-streptavidin (Thermo Fisher Scientific) were suspended in 10 µL of RT mix [1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP, 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO], and incubated for 60 min at 42ºC and immediately cooled on ice. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed following the purification of PCR product by an Agencort AM Pure XP kit (Beckman Coulter) at a 0.7:1 ratio of beads to sample and eluted with 20 µL of 10 mM Tris-HCl (pH 8.0). To amplify TCR libraries and add adaptor sequences for the next-generation sequencer, the third PCR was performed. The third-PCR products were purified as second PCR. The products were pooled and then purified and subjected to dual size selection using ProNex size-selective purification system (Promega) and eluted with 25 μL of 10 mM Tris-HCl (pH 8.5). Final TCR libraries, whose lengths were about 600 base pairs were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 141 bp read 2) (Illumina) to a depth of approximately 20,000-40,000 reads and 5,000-10,000 reads per cell, respectively. TCRseq libraries were sequenced on the Illumina Novaseq 6000 SP flowcell (67 bp read 1 and 459 or 141 bp read 2) (Illumina) to a depth of approximately 5,000-20,000 reads.(67 bp read 1 and 140 bp read 2) (Illumina). Only read2 contained the sequence regarding the definition of T cell clones.
single-cell targeted RNA-seq / TCR-seq
Bulk TCR-seq

value: anti-CD4 mAb

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+ and CD4- CD8+ T cells

value: 8 weeks

value: female

tag: tumour cell line

value: B16F10 melanoma


title: SAMP8 Male PBS R.83

value: Male


title: NC_GOM.replicate_9.hic

description: whole.samples.ABindex.results.txt.gz

value: greater omentum fat


title: hCD4 STARR Donor I

value: Donor I


title: NC_GOM.replicate_8.hic

description: whole.samples.ABindex.results.txt.gz


title: aCD4_Tumor_1 Target

source_name: tumor on day 14 after innoculation of B16F10 with anti-CD4 mAb treatment

tag: organ


title: SAMP8 Male PBS R.82

value: PBS


title: NC_RAD.replicate_3.hic

description: whole.samples.ABindex.results.txt.gz

value: retroperitoneal adipose


title: SAMP8 Male PBS R.101


title: aCD4_Tumor_1 Tag

tag: strain


title: NC_GOM.replicate_7.hic

description: whole.samples.ABindex.results.txt.gz


title: SAMP8 Male PBS R.102

tag: treatment


title: Plasmid Control pLenti-STARR

tag: cell type

value: Plasmid


title: aCD4_Tumor_1 scTCR

tag: age


title: NC_GOM.replicate_6.hic

description: whole.samples.ABindex.results.txt.gz


title: hCD4 STARR Donor III

value: Donor III


title: LNA-unrelated_rep1

city: Beijing

first: Yuanchao

last: Xue

address: Datun Road #15

department: Institute of Biophysics

institute: Chinese Academy of Sciences

description:
Strand specific RNA-seq (paired-end sequencing).
Chromatin associated RNA.
Replicates were merged for processing.

data_processing:
Basecalls were performed using bcl2fastq (v2.16) for Novaseq output.
Raw sequence reads were trimmed for adaptor sequence/low-quality sequence using Trimmomatic (v0.36).
Trimmed sequence reads were mapped to human rRNA sequence, and unmapped reads were aligned to human genome (hg19) using HISAT2 (v2.1.0).
Strand-specific alignments were separated by samtools (v1.3.1) view with specific flags 99, 147, 83 and 163.
Genome coverage bigWig files were generated by deeptools (v3.5.1) bamCoverage with the parameter ‘–normalizeUsing RPKM–binSize 1’.
Assembly: hg19
Supplementary files format and content: Genome coverage bigWig file for each sample.

source_name: HeLa

growth_protocol: Human HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin in humidified atmosphere with 5% CO2 at 37°C.

extract_protocol:
Chromatin fraction of HeLa cells treated with LNA oligonucleotides were harvested, chromatin associated RNA was extracted with TRIzol LS and used for the construction of sequencing libraries.
rRNA was depleted with specific probes, and strand-specific RNA library was constructed as standard procedure.

treatment_protocol: LNA ASOs were transfected into HeLa cells using Lipofectamine RNAiMAX for 24 h.

value: HeLa

value: Cervical cancer cells

value: MISP knockdown

value: LNA transfection

value: 24 hours


title: NC_GOM.replicate_10.hic

description: whole.samples.ABindex.results.txt.gz


title: NC_GOM.replicate_5.hic

description: whole.samples.ABindex.results.txt.gz


title: B16_dLN_aPDL1_UT_CD8_4

description:
The Fscn1 knockin (Fscn1-KI) mouse (Accession No. CDB0154E:  http://www2.clst.riken.jp/arg/mutant%20mice%20list.html) was generated by CRISPR/Cas9-mediated knockin in zygotes as previously described (Abe et al., 2020). For the homologous recombination-mediated knockin, the donor vector consisting of homology arms and P2A-DTR-P2A-Cre (insert gene) was generated to insert the P2A-DTR-P2A-Cre cassette at the 3-base upstream of the PAM sequence. For microinjection, the mixture of crRNA (CRISPR RNA) (50ng/ul), tracrRNA (trans-activating crRNA) (100ng/ul), donor vector (10ng/ul) and Cas9 protein (100ng/ul) were injected into the pronucleus of one-cell stage zygote. From 233 zygotes, 57 F0 mice were obtained and 3 of them were knockin mice confirmed by PCR, in which the PCR fragments of KI allele with 5’FW and 5’REV primers (1301 bp), and 3’FW and 3’REV primers (587 bp) were detected. The germline transmissions of knockin F0 mice were confirmed by genotyping of F1 mice. gRNA target sequences and primers used for genotyping are shown in Supplementary Table 1.
Recipient B6 mice were lethally irradiated (8.5 Gy, split into 2 doses given 3 hours apart) the day before transplantation with 3 × 106 Fscn1-KI BM cells. Mice were used for the experiments at least 8 weeks after transplantation.

data_processing:
Data processing of Bulk TCRseq was performed as described in Aoki et al. (Aoki et al., 2021).
Adapter trimming and quality filtering of sequencing data were performed by using Cutadapt-3.2  (Martin et al., 2011) and PRINSEQ-0.20.4 (Schmieder et al., 2011). Sequencing data were processed by MiXCR-3.0.5 (Bolotin et al., 2015). In MiXCR, filtered reads were aligned to reference mouse TCR V/D/J sequences registered in the international ImMunoGeneTics (IMGT) information system: -vParameters.geneFeatureToAlign = VTranscript -vjAlignmentOrder = JThenV, then identical sequences were assembled and grouped in clones with PCR and sequencing error correlation with the following parameters: -badQualityThreshold=10, –separateByV=true, --only-productive=true, –region-of-interest=CDR3. The Variable (V) and Joining (J) segment of TCRs were represented in IMGT gene nomenclature.
List of final clones were analyzed by VDJtools-1.2.1 (Shugay et al., 2015). Then, the sequencing reads of sample was normalized to the cell count in each sample by “DownSample” command of VDJtools. T-cell clones were determined as TCR reads with the same TCR V segment, J segment and CDR3 nucleotide sequence.
Assembly: IMGT reference sequences of mouse TRA and TRB
Supplementary files format and content: Text files in VDJtools format include read count and frequency, complementarity determining region 3 (CDR3) nucleotide and amino acid sequences, Variable(V)/ Diversity(D)/ Joining(J) segment names, and V, D and J segment boundaries within CDR3 nucleotide sequence of individual clones

source_name: dLN on day 14 after innoculation of B16

extract_protocol:
Intravascular leukocytes were stained by i.v. injection of FITC-conjugated anti-CD45.2 mAb (3 mg/mouse, clone 104, BioLegend) three minutes before sacrifice.
dLN was digested for 45 minutes at 37°C with 0.1% collagenase (FUJIFILM Wako) then subjected to 25% Percoll PLUS (Cytiva) gradient and leukocytes were recovered from the pellet.
Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.1% collagenase (FUJIFILM Wako), then subjected to 40% Percoll PLUS (Cytiva) and Histopaque-1083 (Sigma-Aldrich) gradient, and leukocytes were recovered from the interphase. The cell concentration of the suspensions was determined using Flow-Count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter).
Cells were stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 93, BioLegend) and fluorophore-conjugated anti-mouse mAbs. For dLN samples, Lineage+ (CD11b, B220, NK1.1, Ter119) cells were depleted using BD IMag Streptavidin Particles Plus-DM (BD Biosciences). For tumor samples, TCRb+ T cells were enriched magnetically using Anti-APC MicroBeads (Miltenyi) and LS columns (Miltenyi).
CD8+ T cells from tumor, and CD8+ CD44high T cells from dLN were sorted using FACS Aria II or Aria III (BD Biosciences). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and propidium iodide staining. Intravascular leukocytes were also excluded. Purity of sorted cells was always over 95%.
Bulk TCR sequencing library was prepared according to the previous report (Tsunoda et al., 2021). In brief, to perform reverse transcription and template-switching, mRNA-trapped oligo-dT-immobilized Dynabeads M270-streptavidin (Thermo Fisher Scientific) were suspended in 10 µL of RT mix [1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP, 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO], and incubated for 60 min at 42ºC and immediately cooled on ice. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed following the purification of PCR product by an Agencort AM Pure XP kit (Beckman Coulter) at a 0.7:1 ratio of beads to sample and eluted with 20 µL of 10 mM Tris-HCl (pH 8.0). To amplify TCR libraries and add adaptor sequences for the next-generation sequencer, the third PCR was performed. The third-PCR products were purified as second PCR. The products were pooled and then purified and subjected to dual size selection using ProNex size-selective purification system (Promega) and eluted with 25 μL of 10 mM Tris-HCl (pH 8.5). Final TCR libraries, whose lengths were about 600 base pairs were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina). Only read2 contained the sequence regarding the definition of T cell clones.
Bulk TCR-seq

treatment_protocol: B16F10 cells (5 × 105 cells) were inoculated subcutaneously (s.c.) into the right flanks of the Fscn1-KI BMC mice. Anti-PD-L1 mAb (clone 10F.9G2, BioLegend) or Anti-CD4 mAb (clone GK1.5, BioLegend) was injected intraperitoneally (i.p.) at a dose of 200 μg per mouse on days 5 and 9 after tumor inoculation.

value: Bone marrow chimera of Fscn1 KI

value: anti-PD-L1 mAbs

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+ and CD44high T cells

value: 10 weeks post bone marrow transplantation

tag: Sex


title: B16_dLN_aPDL1_UT_CD8_5

value: female

value: B16F10 melanoma


title: LNA-SE351_rep2

value: SE351 knockdown


title: SAMP8 Female PBS R.104

tag: tissue

value: brain

value: Female


title: NC_GOM.replicate_3.hic

description: whole.samples.ABindex.results.txt.gz

value: greater omentum fat


title: LNA-SE351_rep1


title: NC_GOM.replicate_2.hic

description: whole.samples.ABindex.results.txt.gz


title: SAMP8 Female PBS R.73

value: PBS


title: B16_dLN_aPDL1_UT_CD8_3


title: B16_dLN_aPDL1_UT_CD8_2


title: NC_GOM.replicate_1.hic

description: whole.samples.ABindex.results.txt.gz


title: SAMP8 Female PBS R.84


title: LNA-NC_rep2

value: WT


title: LNA-NC_rep1

value: HeLa


title: B16_dLN_aPDL1_UT_CD8_1


title: 182: P18, colon, normal, anti-PD-1, scRNA-seq

city: Guangzhou

first: Cheng

last: Wu

address: No. 74, Zhongshan 2nd Road, Yuexiu District, Guangzhou

institute: Sun Yat-sen University

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software vcellranger-5.0.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: CRCh38
Supplementary files format and content: Tab-separated values files and matrix files

source_name: colon

extract_protocol:
Tumor biopsy samples were first stored in liquid nitrogen with the SenotechTM Tissue Storage Buffer(#JZ-SC-5802，Senotech Genomics, Shanghai, China)or proceed directly to tissue disassociation. Heat the frozen tissue in a 37 ° C water bath,then transfer the tissue samples together with the freezed-storage solution to a new 15mL centrifuge tube, add 5mL preheated RPMI-1640 medium (Gibco).Centrifuge at room temperature at 300×g for 5min to remove the supernatant without disturbing the tissue sample at the bottom of the centrifuge tube.The tissue samples enzymatically digested with Neuronal Isolation Enzyme(Thermo Scientific) for 20-30 min at 37 C according to the manufacturer’s protocol.The dissociated cells were next passed through a 40-mm cell-strainer (BD) in the RPMI-1640 medium (Invitrogen) with 10% FBS until uniform cell suspensions were obtained. Subsequently, the suspended cells were passed through cell strainers and centrifuged at 400 g for 10 min. Red blood cells were removed via the same procedure described above. The recommended cell washing and resuspension solution is 1X PBS (calcium and magnesium free) containing 0.04% weight/volume BSA (400 µg/ml).
Single-cell RNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3’ Kit v2 or v3 from 10x Genomics, following the manufacturer’s instructions. In brief, single cells were  resuspended in PBS containing 0.04% BSA to a final concentration of 500~1,200 cells/ml as determined by Countess 3 Automated Cell Counters(Invitrogen). ~10,000 cells were captured in droplets to generate nanoliter-scale Gel bead in EMulsion (GEMs). GEMs were then reverse transcribed in a C1000 Touch Thermal Cycler (Bio-Rad) programmed at 53C for 45 min, 85C for 5 min, and held at 4C. After reverse transcription and cell barcoding, emulsions were broken and cDNA was isolated and purified with Cleanup Mix containing DynaBeads and SPRIselect reagent (Thermo Fisher Scientific), followed by PCR amplification. Amplified cDNA was fragmented and end-repaired, double-sided size-selected, PCR-amplified with sample indexing primers, and double-sided size-selected or RNA-seq library construction. Libraries prepared according to the manufacturer’s user guide were then purified and profiled for quality assessment. Single-cell RNA libraries were sequenced by an Illumina NovaSeq 6000 or BGISEQ DNBSEQ-T7 sequencer with 150 bp paired-end reads.

tag: subject

value: Goblet cells

value: colon

value: normal


title: LNA-unrelated_rep2

value: Cervical cancer cells

tag: genotype

value: MISP knockdown

value: LNA transfection

value: 24 hours


title: SAMP8 Female PBS R.105


title: SAMP8 Female PBS R.85


title: SAMP8 Female PBS R.103


title: ME25, replicate 2, scRNAseq

city: kunming

first: Ci

middle: bai

last: quan

address: bodalu

institute: lpbr

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v4.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Subsequently, unique molecular identifiers (UMI) matrices from each sample were merged in R and imported to Seurat(v4.1.1).
Assembly: Mmul_10/rheMac10
Supplementary files format and content: Tab-separated values files and matrix files

organism: Macaca fascicularis

source_name: in vitro

extract_protocol:
Freshly collected DC and Si8 from RedMUC298trTg mice were flushed with ice-cold HBSSwo (Mg2+/Ca2+ free supplemented with 10mM HEPES, pH 7.2) to remove luminal content. The tissue was inverted and inflated by injection of HBSSwo. Epithelial isolation was performed by incubation of tissue with pre-digestion buffer (HBSSwo, 5 mM EDTA, 5% v/v FCS, 1 mM DTT) in a shaking incubator. The isolated epithelial cells were resuspended in digestion buffer (HBSS with Mg2+/Ca2+ supplemented with 2 mg/ml collagenase type I and 40 U/ml DNase I) and incubated at 37 °C for 30 min. Cells were washed and re-suspended in PBS and stained with a Viability Dye, washed and resuspended in FACS buffer (HBSSwo, 2% v/v FCS, 5mM EDTA). Alive GCs were sorted using a FACS Jazz (Becton Dickinson) according to the presence or absence of mCherry signal using a C57Bl6 mouse as negative control. Goblet cells were washed in HBSS-/- and resuspended in HBSS-/- supplemented with 0.04% w/v BSA.
Library was performed according to the manufacter’s instructions (Chromium Single Cell 3’ Reagent Kit v3.1 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip G to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. On average, approximately 13,200 single cells were loaded on each channel and approximately 8,000 cells were recovered. Libraries were sequenced on the Illumina NovaSeq 6000 platform

value: Embryo

value: in vitro


title: 181: P18, colon, tumor, anti-PD-1, scRNA-seq

description: 10x Genomics

value: tumor


title: B16_dLN_aPDL1_DT_CD8_4

value: Bone marrow chimera of Fscn1 KI

value: anti-PD-L1 mAbs with DT

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+, CD4- CD8+ and CD44high T cells

value: 10 weeks post bone marrow transplantation

value: female

value: B16F10 melanoma

tag: date


title: 172: P17, colon, normal, anti-PD-1, scRNA-seq

description: 10x Genomics


title: ME25, replicate 1, scRNAseq

description: 10x Genomics


title: ME24, replicate 2, scRNAseq

description: 10x Genomics


title: HeLa_rep2

description: replicates were merged for processing

data_processing:
Illumina bcl2fastq (v2.16) conversion software used for basecalling.
After sequencing, RIC-seq reads were aligned to the human genome using the RICpipe program with minor revisions. Briefly, RIC-seq reads were first mapped to the reference genome (assembly version: hg19 for human and mm9 for mouse) using the STAR software (v2.5.2b). The parameters were set as: --runMode alignReads --genomeDir index --readFilesIn read.fq --outFileNamePrefix prefix --outReadsUnmapped Fastx --outFilterMultimapNmax 100 --outSAMattributes All --alignIntronMin 1 --scoreGapNoncan -4 --scoreGapATAC -4 --chimSegmentMin 15 --chimJunctionOverhangMin 15 --limitOutSJcollapsed 10000000 --limitIObufferSize 1500000000 --runThreadN 16 --alignSJoverhangMin 15 --alignSJDBoverhangMin 10 --alignSJstitchMismatchNmax 5 -1 5 5 --outFilterMatchNminOverLread 0.5 --outFilterScoreMinOverLread 0.5. Next, unmapped reads reported by STAR were aligned again to the reference genome by the bwa program (v0.7.17) with the following parameters: bwa mem -k 12 -T 15. Alignments from STAR and bwa were combined and sent to RICpipe for the subsequent analysis to extract chimeric reads.
Enhancer and promoter intervals for each cell are defined as previously described (Cai et al. Nature, 2020, 582(7812): 432-437.), using ChIP-seq data of H3K4me3, H3K4me1 and H3K27ac downloaded from the ENCODE or GEO database.
Using these defined promoter and enhancer regions, we adopted a Monte Carlo simulation strategy to detect significant enhancer-promoter interactions (P < 0.05, unique chimeric reads ≥ 2) as previously described.
Assembly: hg19, mm9
Supplementary files format and content: tabular files that contain enhancer-promoter RNA interacion chimeric reads, columns 1-4 denote the enhancer RNA fragment, column 5-8 denote the promoter RNA fragment
Library strategy: RIC-seq

source_name: human HeLa cells

growth_protocol: GM12878 and K562 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Life Technologies; 15140). IMR-90 cells were grown in DMEM/F12 + Glutamax (Life Technologies; 10565) supplemented with 5 ng/ml fibroblast growth factor-basic (R&D), 1 μg/ml hydrocortisone hemisuccinate, 50 μg/ml ascorbic acid, 5 μg/ml rhInsulin, 2% fetal bovine serum and 100 U/ml penicillin/streptomycin. Human embryonic stem cells (WA01, Line H1) were obtained from Dr. Hongjie Yao laboratory and cultured according to the protocol from WiCell Research Institute. The hESCs were maintained in mTeSR1 medium (Stem Cell Technologies) in feeder-free conditions on hESC qualified Matrigel (BD Biosciences). mTeSR1 media was changed daily and cells were passaged every 6-8 days with 1 ml of ReLeSR (Stem Cell Technologies). hNPC cells were grown in DMEM/F12 + Glutamax (Life Technologies; 10565) supplemented with 20 ng/ml fibroblast growth factor-basic (R&D), 0.5× N-2, 0.5× B-27,  and 100 U/ml penicillin/streptomycin. MEF and HepG2 cells were grown in DMEM containing 10% fetal bovine serum plus 100 U/ml penicillin/streptomycin.

extract_protocol:
Cells were treated with micrococcal nuclease and proteinase K. Total RNA was extracted with TRIzol LS.
Total RNA was extracted, and rRNA was depleted with specific probes. After fragmentation, the biotin-marked RNA fragments were enriched with streptavidin magnetic beads, and converted into Illumina HiSeq X10 compatible libraries.

treatment_protocol: Cells were fixed with  PBS containing 1% formaldehyde, and then treated with 1/20 volume of 2.5 M glycine.

tag: cell line

value: HeLa