title: iPSC-EC, Han Chinese, normoxia, ind10

city: Chicago

first: Anna

last: Di Rienzo

state: IL

department: Human Genetics

institute: University of Chicago

data_processing:
The bulk RNAseq reads were aligned with the STAR aligner and read counts performed with RSEM using default settings.
Assembly: Gencode 2019-09-05 transcriptome (GRCh38)
Supplementary files format and content: Bulk RNAseq: tab separated values files.

growth_protocol: For the Tibetan samples, peripheral whole blood samples were collected by venipuncture, and LCLs were generated using a standard protocol (Coriell Institute) for establishment of lymphoblastoid cell cultures from peripheral blood mononuclear cells (PBMCs). All Han Chinese samples used in the study were obtained from Coriell in the form of LCLs. Reprogramming of LCLs to iPSCs was performed in accordance with Thomas et al., 2015. All generation and maintenance of iPSCs were performed in atmospheric oxygen concentration. Cells were grown in culture at 37°C, 5% CO2 in a standard incubator. iPSCs were then differentiated into vascular endothelium. Our panel of TAC-CHB iPSCs were grown to confluence in batches of 4-8 lines (balanced for population and sex). On day 0 cells were detached using Cell Release, an EDTA-based solution, counted, and replated at a density of ~50,000 cells/cm2 in Essential 8 media supplemented with Rho-associated kinase (ROCK) inhibitor Y27632 (BioVision, 1994-1). On day 1, medium was replaced with N2B27 medium – a 1:1 mixture of DMEM:F12 (Life technologies, 11320-033) and Neurobasal media (Life technologies, 21103049) with glutaMAX (Life technologies, 35050038), β-Mercaptoethanol (0.097%) ( Life technologies, 21985023), N2(Life technologies, 17502048), and B27 minus vitamin A (Life technologies, 12587010)] this was supplemented with 8uM CHIR (Tocris, 4953) and 25ng/ml hBMP4 (PeproTech, 120-05ET) and added to the cells at high volume (i.e. 15 mL in 22.1cm dish). The cells were left undisturbed for 3 days. On day 4, media was exchanged for EC Induction Medium - StemPro-34 SFM medium (Life technologies, 10639-011) supplemented with 200 ng/mL VEGF165 (PeproTech, 100-20) and 2μM forskolin (Abcam, ab120058), and penicillin-streptomycin]. EC Induction Medium was refreshed on day 5, and on day 6 CD144+ cells were either isolated via pulldown or replated for subsequent single-cell sequencing.

extract_protocol:
RNA extraction was performed in 5 balanced batched of 8 (balanced for population, sex, and treatment) in a Qiagen Qiacube (9001292) using the Rneasy Plus Mini Kit. RNA was assessed for concentration prior to library preparation
500ng of each sample was used for library prep using Illumina TruSeq2 RNA-seq library prep kit and set A indexes (RS-122-2001). All libraries were assessed via bioanalyzer to be of sufficient quality, concentration, and fragment size for further processing.

treatment_protocol: For the population-wide bulk RNA sequencing, bead-purified iPSC-derived endothelial cells were washed and lysed in-plate using RLT Plus buffer from the Qiagen Rneasy Plus Mini Kit (74134). Cell culture replicates in hypoxia were lysed and harvested in the hypoxia box with no exposure to normoxia prior to lysis. Lysates were flash frozen and stored at -80C degrees until all batches were complete.

value: LCL-iPS

value: iPS-derived vascular endothelium

value: wild type

value: normoxia


title: hd_ms_rep3

status: Public on Dec 01 2024

type: SRA

city: guangzhou

first: yizhi

last: chen

state: guangdong

address: No.601, West Huangpu Avenue

department: Guangdong-Hong Kong-Macau Institute of CNS Regeneration

country: China

institute: Jinan university

library_source: transcriptomic

data_processing:
Raw data (raw reads) of fastq format were firstly processed through Trimmomatic v0.39[2]. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing poly-A and low quality reads from raw data. FastQC v0.11.9[3] software was used to perform quality control on raw data and clean data respectively. Q20, Q30 and GC content were calculated through in-house python scripts. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from Ensembl website directly. Index of the reference genome was built using Hisat2 v2.2.1[4] and clean reads were aligned to the reference genome using Hisat2 v2.2.1 to generate a .sam or .bam files containing the alignments.
The gene expression level quantification was performed separately for each sample using the featureCounts v2.0.1[5] software in the subRead package, and the reads count of each gene was obtained with the parameter "-t exon -g gene_id -s 1". Then results of all samples were combined to obtain the expression matrix of all samples. The RPM (Reads per million mapped reads) of each gene was based on the reads count mapped to this gene and sequencing depth.
Assembly: Sus_scrofa.Sscrofa11.1
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

molecule: polyA RNA

organism: Mus musculus

source_name: striatum

extract_protocol:
Total RNAs from striatum of mice, and striatum of pigs were extracted using the Trizol method (Thermo Fisher Scientific, MA, USA).
Sequencing libraries were generated using the SEQUMED ® MustSeq ® 3'mRNA DEG kit（Sequmed，China）.The first step is to capture polyA mRNA with polyT primer and reverse transcript it to form DNA-RNA heterozygous chain. The primer consists of oligo-dT, a sample barcode, a unique and the Illumina-compatible 5ʹ adaptor. Then RNA is removed, the sample is purified with 1X magnetic beads.After the RNA removal,the random primer is used for two-chain synthesis,and the P5 and P7 adaptor that are suitable for the Illumina platform are used for PCR amplification.At last, PCR products were purified 250bp-1000bp fragments for sequencing library.

tag: tissue

value: striatum

tag: disease state

value: hd

tag: treatment

value: untreated


title: PA-1-BAY-850 treated replicate 2

city: Birmimgham

first: Romi

last: Gupta

state: Alabama

address: 720, 20th street south

country: USA

institute: University of Alabama at Birmingham

data_processing:
The reads were first mapped to the latest UCSC transcript set using Bowtie2 version 2.1.0 and the gene expression level was estimated using RSEM v1.2.15.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text file includes raw count values for each Sample

molecule: total RNA

organism: Homo sapiens

source_name: PA-1-BAY-850 treated

growth_protocol: PA-1 and SK-OV-3 cells were grown in DMEM medium and RPMI medium respectively with 10% Fetal bovine serum and Penicilline and Streptomycin at  37 C under 5% CO2 condition.

extract_protocol:
Total RNA was extracted using Trizol reagent (Invitrogen) as per the instruction from the manufacturer and then purified using RNAeasy mini columns (Qiagen) as per the manufacturer's instructions.
mRNA is purified from approximately 500ng of total RNA with oligo-dT beads and sheared by incubation at 94C.  Following first-strand synthesis with random primers, second strand synthesis is performed with dUTP for generating strand-specific sequencing libraries.  The cDNA library is then end-repaired, and A-tailed, adapters are ligated and second-strand digestion is performed by Uricil-DNA-Glycosylase. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul are used for sequencing.

treatment_protocol: PA-1 and SK-OV-3 cells were treated with  ATAD2 inhibitor (5 micomolar) BAY-850 for 48 hours,  were harvested and were used for preparing the total RNA and to perfrom RNA Seq.

tag: cell line

tag: cell type

value: ovarian cancer cell

value: BAY-850 treated, 5 micromolar, 48 hours


title: H_pH715_bio_rep_2

city: Memphis

first: Michael

middle: A

last: Dyer

state: Tennessee

address: 262 Danny Thomas

department: DNB

institute: St. Jude Children's Research Hospital

data_processing:
Read alignment to the reference genome was performed with STAR (version 2.7; PMID: 23104886) software. Gene level quantification was determined using RSEM (version 1.3.1 ; PMID: 21816040).
Assembly: mm10-mouse / GRCh38 – human
Supplementary files format and content: tab delimited text file with raw counts for each sample

source_name: retina

extract_protocol:
Retinal punches were dissolved in 800uL Trizol Reagent (Invitrogen #15596026). 200uL chloroform was added to each sample, inverted, incubated at room temperature for 3 minutes. Samples were then spun down at 12,000 3 g at 4 C for 15 min. The aqueous later was transferred to a new tube and 1uL glycogen and 500 uL isopropanol was added to each sample and mixed by vortex. Samples were incubated at room temperature for 10 minutes and spun down at 12,000 3 g at 4 C for 15 min. The isopropanol was removed and the pellet was washed twice with ice-cold 80% EtOH. The washed pellet was resuspened in water.
Libraries were constructed using Illumina library prep acording to manufacturer protocols

treatment_protocol: Retina were dissected from the eye cup in Retinal Explant Media (REM). 1.5mm punches were taken from each eye using a biopsy punchand were placed on a Nuclepore 1.0uM membrane floating on 2mL of REM in a 12-well dish. The retinal punches were incubated for 24 hours (37C, 5% CO2) for culture control retina. Punches were harvested after 24 hours.

value: retina

value: 24 hour culture in REM + 0.2ug/mL LPS


title: attB/P_mESC_K562_Lane1_FB_CS

city: Seattle

first: Sudarshan

last: Pinglay

state: WA

address: 3720 15th Ave NE

institute: University of Washington

description: Library name: SP000323

library_source: other

data_processing:
10x Genomics 3’ gene expression (transcriptome) libraries were processed using cellranger-6.0.1 count function (with reference refdata-cellranger-mm10-3.0.0 for mESC cells [loxPsym+ mESC experiment 1 and attB/P+ mESC/K562 experiment lane 1] and refdata-gex-GRCh38-2020-A for K562 cells [attB/P+ mESC/K562 experiment lane 1 & 2]). Resulting raw count matrices were converted to a Seurat (v4.3.1) object using functions Read10X and CreateSeuratObject (options: min.cells=3, min.features=50). The mitochondrial fraction was computed and cell barcodes with >1500 and >1000 transcriptome UMI/cell (K562 and mESC respectively) and with 2-8% and 1.3-6% mitochondrial fraction (K562 and mESC respectively) were retained. For loxPsym+ mESC experiment 1, cell barcodes with >1000 transcriptome UMI/cell and with 1-12% mitochondrial fraction were retained. Scrublet 0.2.3 was run on filtered cells, and cells with doublet score <0.4 were retained for further analysis. For the lane 1 mESC+K562 barnyard experiment, the following criteria were further used to unambiguously assign species identity to cells: cell barcodes passing each separate species singlet processing thresholds were retained, and any cell barcodes removed as a putative scrublet-called doublet in one species but not the other were further marked as doublets (e.g., cell called as singlet in K562 mapping but flagged as a doublet in mESC mapping). Total transcriptome UMIs mapping to both species from all these cell barcodes were then inspected, showing clear separation between singlet and doublet. Initial assignment from the single species threshold was finely refined to stringently identify cells as doublets if transcriptome UMI ratios were not sufficiently separate (threshold selected by inspection): mm10/hg38 <1.42 or hg38/mm10<3.  To obtain the BC-by-cell-count matrix, we first modified the fastq files to separate the cell barcode and shuffle barcodes in two distinct files (original read structure -- read 1: first 28 cycles [cell barcode]-umi, remaining cycles 29 to 75 to read [capture sequence]-BC-[att site]; read 2: cycles 1 to 47 cycles to [read capture sequence]-BC-[att site]). Modified fastq content -- read 1: 28 cyclces [cell barcode]-umi, read 2: cycles 1 to 47 cycles to [read capture sequence]-BC-[att site] followed by original cycles 29 to 75 from original read 1. The merge was generated using seqtk version 1.4 and the following script (appending line number to join and retaining only desired information in final fastq (seqtk trimfq -b 28 file_R1_001.fastq.gz | join <(zcat file_R2_001.fastq.gz | nl) <(cat - | nl) | awk -F ' ' '{if ($2 ~ /^@VH00979/ ) {print $4" "$5;} else {print $2$3;}}' | gzip > file_w_47bpR1R2transfer_R2_001.fastq.gz).  T7-BC sequencing data was then processed using cellranger-6.0.1 count to perform error correction on the cell barcodes. The unmapped reads with error-corrected cell barcodes were selected from the sorted BAM file output, and barcodes sequences were extracted for each read from the shuffle cassette by looking for matches for constant surrounding sequences. From the read 1 portion (searching in cyles 48 to 95 appended to read 2 as described above): attP CS2 if TGAGC(.{20})GTGG, attB CS2 if TGAGC(.{20})GGCC, attP CS1 if AAAGC(.{20})TGGG, attB CS1 if AAAGC(.{20})CCGG. From the original read 2 (searching in cycles 15 to 47): attB CS1 if AAAGC(.{20})CCGG, attP CS1 if AAAGC(.{20})TGGG, attP CS2 if AAAGC(.{20})TGGG, attB CS2 if TGAGC(.{20})GGCC. The resulting two (barcode)-(capture sequence)-(recombinase site) combinations were stored and joined to the error-corrected cell barcode and umi from the read. Reads counts and total set of UMIs for all cellBC/BC1/BC2/capture/recombinase site combinations were then tallied, discarding likely chimeric UMIs (taken to be UMIs for which the proportion of reads associated to a given BC1/BC2/capture-orientation all other BC1/BC2/capture/recombinase site in the specified cell barcode falls below 0.2). The number of error-corrected UMIs for a given BC1/BC2/capture/recombinase site was then taken as the number of connected components in a graph created by connecting all UMIs associated with that combination with a Hamming distance≤1. To filter out spurious molecules (sequencing errors or PCR chimeras, only cell barcode BC1/BC2 combinations with reads/UMI ≥15 and ≥9 for Lane 1 and Lane 2 respectively were retained commensurate with the level of sequencing saturation in both libraries).
Assembly: mm10 or hg38
Supplementary files format and content: For gene expression libraries, the processed file is a seurat object. For T7 libraries, processed file is a matrix containing count of reads, UMIs per shuffle cassette barcode combination, per cell barcode.

molecule: other

extract_protocol:
For loxPsym+ mESC scRNA-seq experiment, 300,000 parental cells were transfected in 6-well plates as described above with 1 μg of Cre and 200 ng of the reporter per transfection. 10 individual wells were transfected. At 24 hours post transfection, 5 wells were treated with 20 μM Pifithrin-α for 48 hours total. At 72 hours post transfection, both Pifithrin-α treated and untreated cells were harvested and approximately 500,000 RFP positive cells were FACS sorted into a combined tube based on the activity of the Cre reporter. Untransfected parental cells were harvested, and 1 million cells were used as input in parallel with Cre-sorted cells for the protocol.  For the attB/P+ K562 and attB/P+ mESC “Lane 1” experiment, 200,000 parental cells were nucleofected (2 reactions) or transfected (3 reactions) with 1 μg of Bxb1 plasmid as described above. All cells were harvested at day 3 post-transfection and used as input for the protocol. For the “Lane 2” experiment, attB/P+ K562 populations bottlenecked to 1000 cells after Bxb1 treatment were thawed and split once before being used as input in the protocol. For both Lanes, 500,000 cells from each population were mixed and were used for the subsequent steps.  After washing twice with cold 1X PBS (Gibco), 1 million cells per sample were resuspended in 400 μl of cold PBS. Cells were fixed with 1600 μl of cold 100% methanol, added dropwise with swirling. Cells were left on ice to fix with gentle swirling to mix every 5 minutes during the incubation. Cells were rehydrated with 4 mL of cold 1X PBS added slowly with gentle swirling of the tube. Cells were spun down and resuspended in 60 μl of PBS. For the loxPsym+ mESC experiment, approximately 60,000 cells and 380,000 cells in total were counted in the Cre and parental samples respectively using a Countess automated cell counter (Thermo) of which all and 100,000 cells in 18 μl of PBS were used for T7 IVT respectively. For the attB/P+ K562/mESC experiment, approximately 600,000 and 450,000 cells were counted of which, 100,000 cells per sample in 18 μl of PBS were used for T7 IVT. IVT reactions were set up in 30 μl total volume with the HiScribe T7 High Yield RNA Synthesis Kit (NEB E2040S) with 2 μl of each NTP, buffer and enzyme. Reactions were incubated at 37°C for 1 hour in a thermocycler. Cells were immediately moved to ice and 20 μl of cold PBS was added to each sample. Approximately 40,000 cells were processed per lane of a 10X Genomics Single Cell 3’ HT with Feature Barcoding kit.
Transcriptome libraries were prepared as per the manufacturer’s protocol. To prepare libraries from T7 transcripts captured using CS1 and CS2, we started with the supernatant from the clean-up after cDNA amplification of the standard 10X Genomics feature barcoding protocol for the loxPsym+ mESC experiment. For the attB/P+ K562/mESC experiment, we spiked in two construct specific primers (oSP1059, oSP1060) at 0.5 μM into the cDNA amplification step. Two rounds of PCR were performed with primers specific to the shuffle cassette. In PCR1, oSP997 (CS1-TruSeq2), oSP1022 (CS2-TruSeq2) and oSP1061 (feature-cDNA primer F) primers were used (1.25 μl of 10 μM each) in a 50 μl Q5 polymerase reaction with 5 μl of template. Cycling conditions: 98°C - 45 s; cycles of 98°C - 20s, 60°C - 5s, 72°C - 5s; 72°C - 60s, 4°C - hold. 15 cycles of PCR1 were performed for the loxPsym+ mESC experiment and 12 cycles for the attB/P+ K562/mESC experiment. loxPsym+ mESC  were cleaned up with AmpureXP beads (1X) and eluted in 15 μl of Qiagen buffer EB. attB/P+ K562/mESC reactions were cleaned up with AmpureXP beads (1X) and eluted in 30 ul of buffer EB. In PCR2, 5 μl of PCR1 eluate was used as input into either one or two (loxPsym+ mESC experiment)  50 μl Q5 Polymerase reactions per sample with sample index primers (1.25 μl of 10 μM each) that add Illumina adapters. Reaction progress was monitored using SYBR green and was stopped before saturation. Cycling conditions for the loxPsym+ mESC experiment: 98°C - 5 min; 9 cycles of 98°C - 10s, 65°C - 10s, 72°C - 20s; 72°C - 60s, 4°C - hold. Cycling conditions for the attB/P+ K562/mESC experiment: 98°C - 1 min; 8 cycles of 98°C - 20s, 63°C - 20s, 72°C - 1min; 72°C - 60s, 4°C - hold. After a clean up with AmpureXP beads (1X), quality of the libraries was confirmed (prominent single-peak) by running them on a TapeStation D1000 ScreenTape (Agilent).  Primer sequences:  oSP997_CS1_TruSeq2_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC TTGCTAGGACCGGCCTTAAAGC oSP1022_CS2_TruSeq_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT CCTTAGCCGCTAATAGGTGAGC oSP1061_FeaturecDNA_Primer1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG oSP1059_CS2_SpikeIn GGGGGGTTTTTTTTTTCCTTAGCC oSP1060_CS1_SpikeIn GGGGGGTTTTTTTTTTGCTAGGAC

tag: genotype

value: attB/P_mESC and attB/P_K562


title: H_LPS_bio_rep_2

value: 24 hour culture in REM at 33 degrees Celcius


title: iPSC-EC, Han Chinese, hypoxia, ind10

value: hypoxia


title: FSDE_FSDE_257_01

city: Morgantown

first: Pius

last: Joseph

address: 1000 Frederick Lane

institute: NIOSH

data_processing:
Basecalls were performed using bcl2fastq/2.19.
Sequence reads were trimmed using Trim Galore!, Cutadapt 3.4 and Trimgalore 0.6.7 using Nextflow 22.10.6 and the nf-core/rnaseq 3.12.0 pipeline.
All sequences were aligned to the Rattus norvegicus Rnor 6.0 genome from Ensembl downloaded from igenomes using Star 2.6.1d within  Nextflow 22.10.6 and the nf-core/rnaseq 3.12.0 pipeline.
Raw gene counts were assigned using Salmon 1.10.1 and Sammtools 1.17 within Nextflow 22.10.6 and the nf-core/rnaseq 3.12.0 pipeline.
Assembly: Rnor6.0
Supplementary files format and content: CSVt file including abundance measurements (raw counts) in a matrix containing all samples

organism: Rattus norvegicus

source_name: lung

growth_protocol: Healthy male Sprague-Dawley rats (Hilltop Lab Animals, Scottdale, PA), weighing 200-250 g were used in this study which was conducted in an Association for Assessment and Accreditation of Laboratory Animal Care International approved animal facility (NIOSH, Morgantown, WV) following a protocol approved by the Animal Care and Use Committee. The rats were housed in pairs and maintained on a 12-hour light-dark cycle in a temperature (68-72oF) and humidity (30-70%) controlled room with free access to filtered tap water and HaCXan Teklad Global 18% protein rodent diet (Harlan Teklad; Madison, WI).

extract_protocol:
Total RNA was isolated from a piece of the lung tissue using the RNeasy Fibrous Mini Kit (Qiagen, Inc, Valencia, CA) following the procedure provided by the manufacturer.
Next generation sequencing libraries prepared using TruSeq® Stranded mRNA Library Prep Kit (Illumina, Inc, San Diego, CA) were used in the sequencing.

treatment_protocol: The rats were exposed by whole-body inhalation to filtered air or DEP at concentrations 1.0 mg/m3 combined with FSD at concentrations of 30 mg/m3, 6 hours/day for 4 days.

value: lung

tag: strain

value: Sprague-Dawley

tag: time (days)

value: 30 mg/m3 fracking sand dust + 1.0 mg/m3 of diesel particles


title: attB/P_K562_parental_rep2

description: Library name: SP000290

library_source: genomic

data_processing:
Reads were demultiplexed using bcl2fastq (v2.20.0.422). Read1 contains the identity of the shuffle barcodes whereas read2 contains the associated genomic sequence. Reads were passed through a custom script (IVTextractBCs.py) to extract barcode sequences based on exact matches to the expected preceding and subsequent bases. The strand of each read (top-CS2 or bottom-CS1) was also assigned at this step. PiggyBac ITR sequences were trimmed from read2 using cutadapt (v2.5) with the following parameters: -cores=4 --discard-untrimmed -e 0.2 -m 10 -a CCCTAGAAAGATA. Trimmed reads were mapped to either the mm10 or hg38 reference genome using bwa mem (v0.7.17) with -Y option. SAM files were sorted using samtools (v1.9) and filtered out reads that do not align to known PiggyBac insertions sites (TTAA) or align to several locations (contain XA:Z flag) using a custom script (align_filter.py). Filtered SAM files were converted to BED format using the sam2bed tool in bedops (v2.4.35).  For mESC samples, bedtools (v2.29.2) intersect was used with the -loj -wa -wb -filenames -sorted options to extract those alignments overlapping with a known variant between the BL6 and CAST alleles from the Sanger Mouse Genome database. A custom python script (cleanup_sort_variantcall_update.py) was used to parse the CIGAR string of each alignment intersected BED file to assign each read to each of the following categories: BL6 (read overlaps with variant and sequence of alignment at that position matches the reference allele), CAST (read overlaps with variant and sequence of alignment at that position matches variant allele) and ‘no-Variant’ (read does not overlap with variant). Inconclusive alignments that contained some incongruence in allele assignment were discarded. The position of each alignment was determined by the genomic strand it mapped to. If strand is +, position is the end of the alignment and conversely, if strand is -, position is the start of alignment. For K562 samples, alignments were simply sorted and assigned to a position using a custom python script (cleanup_sort_variantcall_update_K562.py).  Next, another custom python script (ivt_clustered_groupcollapse_iterable.py) was used to collapse alignments into groups based on: 1) a shared chromosome and position and 2) a shared set of barcodes extracted from read1. Alignments at a given position were clustered together if their associated barcodes were within a Levenshtein distance of 6. Within each cluster, the most common values for barcode1, barcode2, strand of alignment to genome (+ or -) and shuffle cassette strand (top-CS2 or bottom-CS1) were assigned as the representative values for that cluster. For mESCs, each cluster was assigned to an allele based on the most common allele value within, without taking into account the number of no-Variant assignments within that cluster. Total read count (number of alignments per cluster), total read1 UMIs (coming from forward primer of second strand synthesis), total read2 UMIs (first 8 bp of genome coming from the degenerate 3’ end of the RT primer) and total number of unique alignment lengths per cluster were also determined.
Assembly: mm10 or hg38
Supplementary files format and content: Count of reads, UMIs per shuffle cassette barcode combination clustered by sequence similarity at each chromosomal position with strand of alignment and shuffle cassette orientation. *_freq columns indicate frequency of that value in the cluster.

molecule: genomic DNA

extract_protocol:
Qiagen DNA mini kit
The same protocol was followed both for shuffle cassette insertion site mapping from the parental population and for rearrangement call validation post Cre transfection. Genomic DNA purified using the Qiagen DNeasy kit was used as template for all IVT reactions. For loxPsym+ mESC samples, 300 ng of template, for loxPsym+ K562, attB/P+ mESC and attB/P+ K562, 500ng of template was used per reaction. T7 IVT was performed using the HiScribe T7 High Yield RNA Synthesis Kit (NEB E2040S), in a 60 μl total reaction for loxPsym+ mESCs and in a 30 μl total reaction for all other samples. Reactions were incubated at 37°C for 16 hours in a thermocycler with the lid set to 50°C. Reactions were treated with Turbo DNase (Thermo AM2238) to remove template DNA according to manufacturer’s instructions and RNA was extracted using Trizol LS reagent (Thermo 10296010). Briefly, each sample volume was normalized to 250 μl with water, 750 μl of Trizol LS reagent was added. Samples were mixed by pipetting and incubated at room temperature for 4 min. 200 μl of chloroform was added and samples were incubated at room temperature for 3 min. Samples were then spun at 12,000 x g for 15 min and the aqueous phase was transferred to a new tube. 1 μl of 5 mg/mL Glycogen was added (Invitrogen) per sample. Next, RNA was precipitated by adding 1 volume of isopropanol. Samples were mixed by inverting and incubated at -80°C for 1 hour and subsequently spun at 21,000 x g for 1 hour at 4°C. RNA pellet was washed with ice cold 80% ethanol and resuspended in 11.5 μl of H2O. Reverse transcription was performed using the SuperScript IV Reverse Transcriptase (Thermo 18090200) with 0.5 uL 100 μM RT primer that contains an 8 base-pair degenerate 3’ end (oSP1012). RNA was initially incubated with 0.5 μL 100 μM RT primer (oSP1012) and 1 μL 10 mM dNTP at 65°C for 5 min and cooled on ice. Enzyme, buffer, RNAse inhibitor and DTT were added and reactions were incubated in a thermocycler at 23°C for 10 min, 50°C for 15 min and 80°C for 10 min, followed by a hold at 10°C.  For loxPsym+ mESC replicate 1, 2nd strand synthesis for both top and bottom strands was performed in the same reaction with primers oSP1008, oSP1021 and oSP1013. For loxPsym+ mESC replicate 2, 2nd strand synthesis for top and bottom strands were performed separately, one with oSP1008 and oSP1013 and another with oSP1021 and oSP1013. Four 50 μl PCR reactions were performed per sample with Q5 Polymerase (NEB M0492S) with the following cycling parameters: 98°C - 3 min; 4 cycles of 98°C - 20s, 65°C - 20s, 72°C - 30s; 72°C - 60s, hold at 4°C. Reactions corresponding to a particular sample and primer pair were pooled. For replicate 1, double-sided size selection (0.5X, 1.1X) was performed with Ampure XP beads (Beckman A63882) on 200 μl of sample and eluted in 50 μl of H2O. For replicate 2, double-sided size selection (0.5X, 1.1X) was performed on 100 μl of sample and eluted in 25 μl of H2O. From each sample, a second PCR was set up with indexing primers using 12 μl of the previous eluate as input. Two 50 μl PCR reactions were performed per sample with Q5 Polymerase (NEB M0492S) with real-time tracking using SYBR green dye with the following cycling parameters 98°C - 3 min; 4 cycles of 98°C - 15s, 65°C - 15s, 72°C - 30s until the curves reached saturation (14-16 cycles). Running these libraries on a D1000 ScreenTape (Agilent) revealed a smear of expected size but also some lower molecular weight products that may dominate the sequencing reaction. To address this, we created 3 equimolar pools: samples from replicate 1, top strand samples from replicate 2 and bottom strand samples from replicate 2. Pools were run on a 6% TBE PAGE gel and DNA between 400 bp and 1000 bp was excised and purified.  For both replicates of loxPsym+ K562, attB/P+ mESC and attB/P+ K562 samples, 2nd strand synthesis was performed according to the replicate 2 protocol described above with minor modifications. Two 50 μl PCR 1 reactions were performed per sample, one corresponding to the top strand and one for the bottom strand. After bead clean-up, one 50 μl PCR 2 reaction was performed per sample, per strand. PCR2 was performed with the previous cycling parameters for a total of 16-18 cycles. Libraries were run on agarose gel and DNA between 400 bp and 1000 bp was excised and purified.  Library sequencing was performed on an Illumina NextSeq2000 P2 300 cycle kit. For loxPsym+ mESCs, the read lengths were: 106 read1, 10 index1, 10 index2 and 212 on read2. For all other samples, read lengths were: 124 read1, 6 index1, 10 index2 and 198 on read2.  Primer sequences:  oSP1012_IST_RT_TruSeqR2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNN oSP1013_TruSeq_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT oSP1008_996_CS2_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNCCTTAGCCGCTAATAGGTGAGC oSP1021_1014_CS1_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNTTGCTAGGACCGGCCTTAAAGC


title: hd_ms_rep1


title: casrx_ms_rep3

value: casrx


title: loxPsym_K562_parental_rep2

description: Library name: SP000289


title: iPSC-EC, Han Chinese, hypoxia, ind9

value: LCL-iPS

value: iPS-derived vascular endothelium


title: H_bio_rep_2.3%O2_bio_rep_2

value: 24 hour culture in REM + 70uM Ouabain


title: H_bio_rep_33C_bio_rep_2

value: 24 hour culture in REM at 2.3% Oxygen


title: attB/P_K562_parental_rep1

description: Library name: SP000284


title: casrx_ms_rep2

value: striatum

value: hd


title: attB/P_mESC_K562_Lane1_GEX

description: Library name: SP000327

library_source: transcriptomic single cell


title: H_Kcl_bio_rep_2

value: 24 hour culture in REM + 80mM Kcl


title: attB/P_K562_Lane2_FB_CS

description: Library name: SP000324


title: casrx_ms_rep1


title: iPSC-EC, Han Chinese, hypoxia, ind8

value: wild type


title: loxPsym_K562_parental_rep1

description: Library name: SP000283


title: H_Ouabain_bio_rep_2

value: retina

value: 24 hour culture in REM at 40 degrees Celcius


title: H_40C_bio_rep_2

value: 24 hour culture in REM at 40% Oxygen


title: attB/P_K562_Bxb1_d6_rep2

description: Library name: SP000314

data_processing:
Reads were demultiplexed using bcl2fastq (v2.20.0.422). Reads were passed through a custom script (read_extract_iterable.py or read_extract_iterable_4primers.py or amplicon_BCExtract_20240906.py) to extract barcode sequences and UMIs based on matches to the expected preceding and subsequent bases. In the case of 4-primer amplicon-seq, the strand of each read (top-CS2 or bottom-CS1) was also assigned at this step. The identity of the recombination site (loxP, attB, attP, attL or attR) was determined from the combination of bases specific to each site expected to be detected in read 1 and read 2.Total number of reads per barcode pair was taken as the readcount and the total number of unique UMIs detected was taken as the UMI count (df_group.py or df_group_4primers.py). In 4-primer amplicon-seq, the same shuffle cassette can result in two distinct amplicons. We collapsed reads coming from the same shuffle cassette based on the shared set of barcodes and summed up the read counts and UMI counts. Those barcode pairs where we did not detect both types of amplicons were discarded. In all cases, the barcode closest to CS2 was named barcode1 and the barcode closest to CS1 in the shuffle cassette was named barcode2.
Supplementary files format and content: Count of reads, UMIs per shuffle cassette barcode combination without normalization for sequencing depth.

extract_protocol:
Qiagen DNA mini kit
We employed two different strategies for amplicon-seq library construction: 2-primer  and 4-primer. The major difference between these two strategies is that either 2 or 4 primers corresponding to the capture sequences were used to generate the PCR product. In the case of the 2-primer strategy, the P5 Illumina sequencing adapter can only come from the primer that binds CS2 and the P7 adapter can only come from the primer that binds CS1. This precludes identification of recombined shuffle cassettes that contain the same capture sequence on both sides of the loxPsym site. In the 4 primer strategy, primers with P5 and P7 adapters that bind to both CS2 and CS1 are included in the PCR. Theoretically, this would enable us to detect recombined shuffle cassettes with the same capture-sequence on both sides. Two rounds of PCR were performed on genomic DNA extracted from indicated samples to construct sequencing libraries. For the 2-primer experiments, we used 250 ng of genomic DNA prepared using the Qiagen DNeasy kit as input. PCR1 (UMI addition) was performed in a 50 μl reaction with Q5 Polymerase (NEB M0492S), with 2.5 μL of each 10 μM primer (oSP1008 and oSP997) with the following cycling parameters: 98°C - 5 min; 4 cycles of 98°C - 20s, 62°C - 20s, 72°C - 30s; 72°C - 60s. PCRs were cleaned up using AmpureXP beads (1X) and eluted in 11 μl of H2O. PCR2 (sample indexing and sequencing adapter addition) was again performed with Q5 Polymerase in a 50 μl reaction using 10 μl of eluate from the previous step as template and 2.5 μL of each 10 μM sample indexing primer. Progression of PCR2 was monitored in real-time using SYBR green dye and reactions were stopped before saturation (usually 15-18 cycles). Cycling conditions for PCR2:  98°C - 5 min; 15 cycles of 98°C - 10s, 65°C - 10s, 72°C - 20s. Reactions were cleaned up with AmpureXP beads (1X) and eluted in 12 μl of H2O. Sample quality was confirmed on a TapeStation D1000 ScreenTape (Agilent), after which reactions were pooled. Sequencing was performed on an Illumina NextSeq2000 100 cycle kit with the following read lengths: 69 read1, 6 index1, 10 index2 and 53 on read2. For the 4-primer experiments involving loxPsym+ mESCs, 100 ng of genomic DNA prepared using the Qiagen DNeasy kit as used as input. For the 4-primer experiments involving loxPsym+ K562, attB/P+ mESC and attB/P+ K562, 250ng of genomic DNA prepared using the Qiagen DNeasy kit was used as input. PCR1 was performed with a mix of 4 primers (oSP1008, oSP997, oSP1021 and oSP1022) with 0.125 μl of each primer at 100 μM per 50 μl reaction. The rest of the protocol was identical to the 2-primer workflow described above. Primer sequences: oSP1008_996_CS2_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNN CCTTAGCCGCTAATAGGTGAGC oSP1021_1014_CS1_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNN TTGCTAGGACCGGCCTTAAAGC oSP997_CS1_TruSeq2_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC TTGCTAGGACCGGCCTTAAAGC oSP1022_CS2_TruSeq_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT CCTTAGCCGCTAATAGGTGAGC


title: Retina, chronic ocular hypertension with eyes, Cut&Tag-seq

city: shanghai

first: Hong

last: Zhou

state: shanghai

address: dongan road 130

department: Institutes of Brain Science

institute: Fudan University

description: COH 1w-1

data_processing:
*library strategy: CUT&TAG
Raw sequence reads passed quality control by Cutadapt (Version 1.9.1).
Clean data was aligned to reference genome via Bowtie2 (version).
Peaks calling and annotation were proceeded by MACS (V2).
Assembly: mm9
Supplementary files format and content: bigWig, mapping
Supplementary files format and content: txt, annotated peaks

extract_protocol:
The retinas were collected from control and COH mice at G1w, and cell nuclei were prepared using a Nucleus Isolation Kit (#52009-10, SHBIO, Shanghai, China).
CUT&Tag seq libraries were prepared using Hyperactive® Universal CUT&Tag Assay Kit for Illumina (Vazyme#TD903) complemented with TruePrep Index Kit V2 for Illumina (Vazyme#TD202). Libraries were amplified using 15 cycles on the thermocycler and then purified using VAHTS DNA Clean Beads (Vazyme#N411).

tag: antibody

value: Rac1 antibody:ab33186

value: chronic ocular hypertension with the mouse eye

tag: age

value: 7 weeks old

tag: strain background


title: Retina, no treatment, Cut&Tag-seq


title: iPSC-EC, Han Chinese, normoxia, ind7

value: normoxia


title: attB/P_K562_Bxb1_d3_rep2

description: Library name: SP000312


title: iPSC-EC, Han Chinese, hypoxia, ind7

value: hypoxia


title: BMSCs, Gel@MDI, 8 weeks, rep3

city: 武汉

first: Gilbert

last: Han

state: 湖北

address: 双林南路

institute: Wuhan University

data_processing:
Bcl2fastq software was used to perform Base Calling on the raw image data of sequencing results, and the Pass Filter Data was obtained after preliminary quality analysis. The results were stored in FASTQ file format.
Raw reads were quality-checked with FastQC and trimmed of adapter sequences using Trimmomatic. The short reads were then aligned using the HISAT2 aligner. Gene expression was calculated using Htseq software. RPKMs atthe current sequencing depth were evaluated by jackknifing using RSeQC, and the accuracy of the evaluated RPKM was measured by percent relative error.
Differential expression analysis was carried out utilizing the DESeq2 package in R, with a significance threshold set at an adjusted p-value of <0.05 and an absolute log2 fold change greater than 1. Gene enrichment was conducted via EnrichR (https://maayanlab.cloud/Enrichr/) and Gene Set Enrichment Analysis (GSEA). Data visualization, including dot plots andheatmaps, were rendered using ggplot2 and the ComplexHeatmap package, respectively.
Assembly: mm10
Supplementary files format and content: tab-limited text file included raw count and RPKM count for each sample

source_name: bone

extract_protocol:
RNA  from  the  sample  was  extracted  using  the  RNAeasyMini  Kit  according  to  the manufacturer's  instructions.  The  quality  and  quantity  of  the  extracted  RNA  were  measured using  a  NanoDrop  spectrophotometer  (Thermo  Fisher  Scientific)  and  an  Agilent  2100 Bioanalyzer (Agilent Technologies), respectively.
Library preparation and sequencing were performed at the XYZ Genomics Facility. Libraries were prepared using the TruSeq RNA Library Prep Kit (Illumina), following the manufacturer's protocols. Briefly, mRNA was purified from total RNA and fragmented. First-strandcDNA was synthesized using random hexamers and reverse transcriptase. Following second strand synthesis, the cDNA was end-repaired, A-tailed, and ligated to Illumina sequencing adapters. The resulting libraries were size-selected, PCR amplified and sequenced on an Illumina NovaSeq 6000, producing paired-end reads of 150 base pairs

value: bone

value: BMSCs


title: loxPsym_K562_Cre_d6_rep2

description: Library name: SP000313


title: iPSC-EC, Han Chinese, normoxia, ind6

value: LCL-iPS

value: iPS-derived vascular endothelium


title: loxPsym_K562_Cre_d3_rep2

description: Library name: SP000311


title: H_40pct_bio_rep_2

value: 24 hour culture in REM


title: H_T0_bio_rep_2

value: Normal human retina


title: BMSCs, Gel@MDI, 8 weeks, rep2


title: attB/P_K562_parental_rep2

description: Library name: SP000310


title: H_IFN_bio_rep_3

value: 24 hour culture in REM + 100U/mL IFN g


title: hd_ms_rep2

value: striatum

value: hd

value: untreated


title: iPSC-EC, Han Chinese, normoxia, ind9

value: wild type


title: iPSC-EC, Han Chinese, normoxia, ind5


title: iPSC-EC, Han Chinese, hypoxia, ind6


title: BMSCs, Gel@MDI, 8 weeks, rep1


title: H_Control_bio_rep_2

value: retina


title: iPSC-EC, Han Chinese, normoxia, ind8


title: iPSC-EC, Han Chinese, hypoxia, ind5


title: H_MeOH_bio_rep_3

value: 24 hour culture in REM + 8uM Formic Acid


title: attB/P_K562_Bxb1_DNA_1k_bottleneck_B

description: Library name: SP000305


title: BMSCs, GEL, 8 weeks, rep2

value: GEL


title: H_Gluc_bio_rep_3

value: 24 hour culture in REM + 20mM Glucose


title: BMSCs, GEL, 8 weeks, rep1


title: loxPsym_K562_parental_rep2

description: Library name: SP000309


title: iPSC-EC, Han Chinese, normoxia, ind4

value: normoxia


title: attB/P_K562_Bxb1_DNA_1k_bottleneck_A

description: Library name: SP000304


title: H_pH770_bio_rep_3

value: 24 hour culture in REM at pH 7.70


title: loxPsym_K562_Cre_DNA_1k_bottleneck

description: Library name: SP000303


title: H_pH715_bio_rep_3

value: 24 hour culture in REM at pH 7.15


title: iPSC-EC, Han Chinese, hypoxia, ind4

value: hypoxia


title: attB/P_K562_Bxb1_DNA_5k_bottleneck_B

description: Library name: SP000302


title: BMSCs, control, 8 weeks, rep2

value: bone

value: BMSCs

value: control


title: iPSC-EC, Han Chinese, hypoxia, ind3

value: LCL-iPS

value: iPS-derived vascular endothelium


title: H_33C_bio_rep_3

value: 24 hour culture in REM at 33 degrees Celcius


title: loxPsym_K562_Cre_DNA_5k_bottleneck

description: Library name: SP000300


title: iPSC-EC, Han Chinese, normoxia, ind2


title: BMSCs, control, 8 weeks, rep1


title: iPSC-EC, Han Chinese, hypoxia, ind2


title: attB/P_K562_Bxb1_DNA_5k_bottleneck_A

description: Library name: SP000301


title: iPSC-EC, Han Chinese, normoxia, ind3


title: D13_Ngn3_c

city: Guangzhou

first: SHUYI

last: CHEN

state: Guangdong

address: 54 S. Xianlie Rd

department: State Key Laboratory of Ophthalmology

institute: Zhongshan Ophthalmic Center, SYSU

data_processing:
Basecalls performed using CASAVA
clean reads were aligned to the mouse reference genome (mm10) using Hisat2
featureCounts was used to count the reads numbers mapped to each genes
Assembly: mm10
Supplementary files format and content: read counts for each genes were given in the processed data files

source_name: MEF

growth_protocol: MEFs were cultured in DMEM+10% FBS, at 37℃ in incubator.

extract_protocol:
RNAs were extracted using TRIzol reagent
NEBNext UltraTM RNA library Prep Kit for Illumina following manufacturer's protocol

treatment_protocol: MEFs were infected with Ngn1/Ngn3 overexpressing lentiviruses, doxycycline was added on day 0 to induce Ngn1/Ngn3 expression, cells were switched to neuronal medium (DMEM/F12+GlutaMAX+N2+B27) on day 2 of reprogramming; medium was half-changed every other day.

value: MEF


title: attB/P_K562_Bxb1_DNA_10k_bottleneck_B

description: Library name: SP000299


title: BMSCs, GEL, 8 weeks, rep3


title: D13_Ngn3_b


title: H_LPS_bio_rep_3

value: 24 hour culture in REM + 0.2ug/mL LPS


title: H_2.3%O2_bio_rep_3

value: 24 hour culture in REM at 2.3% Oxygen


title: H_Kcl_bio_rep_3

value: retina

value: 24 hour culture in REM + 70uM Ouabain


title: iPSC-EC, Han Chinese, normoxia, ind1

value: wild type


title: D13_Ngn3_a


title: BMSCs, control, 8 weeks, rep3


title: H_Ouabain_bio_rep_3

value: 24 hour culture in REM + 80mM Kcl


title: iPSC-EC, Han Chinese, hypoxia, ind1


title: loxPsym_K562_Cre_DNA_10k_bottleneck

description: Library name: SP000297


title: D13_Ngn1_c


title: MOLM13 cells, treated with combination of SD70 and MI-503 for 12h

city: Tianjin

first: Wenqi

last: zhu

state: Tianjin

address: 288 Nanjing Road

institute: State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence,then use BWA (Li, H. and Durbin, R., 2009) to map clean reads to genome reference and use Bowtie (Langmead B, et al., 2009) to gene reference.
Assembly: hg38
Supplementary files format and content: tab-deliminated text file include fpkm values for each sample

source_name: MOLM13 cells

extract_protocol:
Treated MOLM13 cells were collected,and RNA was harvested using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: MOLM13 cells

value: AML cells


title: H_40C_bio_rep_3

value: 24 hour culture in REM at 40 degrees Celcius


title: MOLM13 cells, treated with MI-503 for 12h

value: treated with MI-503


title: D13_Ngn1_b


title: attB/P_K562_Bxb1_DNA_50k_bottleneck_B

description: Library name: SP000296


title: attB/P_K562_Bxb1_DNA_50k_bottleneck_A

description: Library name: SP000295


title: D13_Ngn1_a


title: H_40pct_bio_rep_3

value: 24 hour culture in REM at 40% Oxygen


title: MOLM13 cells, treated with SD70 for 12h

value: treated with SD70


title: attB/P_K562_parental_rep1

description: Library name: SP000264


title: D1_Ngn3_c


title: H_T0_bio_rep_3

value: Normal human retina


title: D1_Ngn3_b


title: H_Control_bio_rep_3

value: 24 hour culture in REM


title: MOLM13 cells, treated with DMSO for 12h

value: treated with DMSO


title: loxPsym_K562_parental_rep1

description: Library name: SP000263


title: loxPsym_K562_Cre_DNA_50k_bottleneck

description: Library name: SP000294


title: day7 replicate1

city: Chavannes-près-Renens

first: Can

last: Aztekin

address: Swiss Federal Institute of Technology Lausanne (EPFL)

country: Switzerland

institute: EPFL

description: Library name: rep1_day7

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v7.1.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: h5 format
Supplementary files format and content: Contains the per molecule read information from Cell Ranger. The instructions to read the matrix in hdf5 format can be found here: https://support.10xgenomics.com/single-cell/software/pipelines/latest/advanced/h5_matrices

source_name: 129/SvEv mouse embryonic stem cells (mESCs)

extract_protocol:
Samples (day0 replicate1, day3 replicate1, day5 replicate1, day7 replicate1, day5 replicate2, day7 replicate2 and day12 replicate2), were collected, dissociated with 0.25% trypsin-EDTA, deactivated with mESC media without inhibitors (2i/Lif). Cells were collected, dissociated, centrifuged, resuspended in 1X PBS supplemented with 1% BSA, and filtered through a strainer. Samples were multiplexed using CellPlex, according to the manufacturer’s recommendations. Samples (Day2budoids and Day5budoids) were collected, washed with 1X PBS, dissociated with 0.25% trypsin-EDTA and deactivated with mESCs media without inhibitors (2i/Lif). Cells were collected, centrifuged, resuspended in 1X PBS supplemented with 0.04 % BSA and filtered. Please refer to the main text for more details.
Library was performed according to the manufacturer's instructions (3'GE v3.1).


title: attB/P_K562_Bxb1_DNA_10k_bottleneck_A

description: Library name: SP000298


title: U2OS cells, MED13 IDR-3

city: Houston

first: Mo-Fan

last: Huang

state: TX

address: 6431 Fannin Street

department: Integrative Biology and Pharmacology

institute: University of Texas Health science center

data_processing:
Obtained reads were mapped to the human GRCh38 genome using or Hisat2 (version 2.0.5).
Reads on annotated genes were counted using featureCounts (version 1.5.0-p3).
FPKM and TPM values were calculated from mapped reads by normalizing to total counts.
Assembly: GRCh38

extract_protocol:
Total RNA was extracted using Trizol reagent
mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.

value: osteosarcoma cells

value: MED13 IDR overexpression


title: U2OS cells, MED13 IDR-2


title: attB/P_K562_Bxb1_d6_rep1

description: Library name: SP000250


title: day3 replicate1

description: Library name: rep1_day3

value: 129/SvEv mouse embryonic stem cells (mESCs)


title: D1_Ngn3_a

value: MEF


title: attB/P_K562_Cre_d6_rep1

description: Library name: SP000249


title: U2OS cells, MED13 IDR-1


title: D1_Ngn1_b


title: day0 replicate1

description: Library name: rep1_day0


title: U2OS cells, control-3

value: WT


title: loxPsym_K562_Bxb1_d6_rep1

description: Library name: SP000248


title: D1_Ngn1_a


title: U2OS cells, control-2


title: U2OS cells, control-1


title: attB/P_K562_Bxb1_d3_rep1

description: Library name: SP000246


title: Ctrl_b


title: Ctrl_a


title: day5 replicate1

description: Library name: rep1_day5


title: attB/P_K562_Cre_d3_rep1

description: Library name: SP000245


title: M0-FPCO

city: Minatoku

first: Shunsuke

last: Tabe

address: 4-6-1 shirokanedai

department: Regenetive medicine

country: Japan

institute: Institute of medical and science,the university of Tokyo

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v7.1.0(https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: hg19
Supplementary files format and content: Tab-separated values file and matrix files

source_name: FPCO including macrophages

extract_protocol:
M0-FPCO at day14 was digested into single cell suspension with collagenase (Sigma-Aldrich) and Pronase (Roche) in reaction buffer. The obtained cell suspension were filtered 70-μm cell straner (SPL), and re-suspended in PBS(-) to avoid cell-cell attachment.
Chromium NEXT GEM Single Cell 3' Kit v3.1 (PN-1000130, 10X Genomics), Chromium NEXT GEM Chip G Single Cell Kit (PN- 1000127) and Chromium NEXT GEM Single Cell 3' v3.1 Gel Beads (PN-1000129, 10X Genomics) were used to create the library according to Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) USER GUIDE. 5000 cells in FPCO-derived single cell suspension were loaded into the Chromium Controller to create single-cell Gel-Beads emulsions (GEMs). GEMs were subjected to reverse transcription in a T100 TM Thermal Cycler (Biolad). The resulting cDNA products were recovered from the GEMs using Recovery Agent and cleaned up using Dynabeads MyONE Saline (Beckman Coulter). The cDNA products were then subjected to amplification in a T100TM Thermal Cycler according to the manufacturer's protocol. The amplified cDNA products were cleaned up by SPRIselect (Beckman Coulter) and then their concentrations were measured using a high sensitivity D5000 ScreenTape (Agilent technology). The cDNA products were then fragmented by enzyme, end-repaired and poly A-tailed, and cleaned up again by SPRIselect. Next, adaptor ligation and subsequent cleanup by SPRIselect were performed, Only appropriately labeled samples were obtained by double-size selection using SPRIslect. Samples were quality-checked using a high sensitivity D5000 ScreenTape (Agilent technology) and sequenced on a NovaSeq6000 (Illumina) at pair-end 150 bp.

value: FPCO including macrophages


title: QR27 - case

city: New York

first: Bin

last: Xu

state: NY

address: 1051 Riverside Dr

department: Psychiatry

institute: Columbia University

data_processing:
We used RTA (Illumina) for base calling and bcl2fastq2 (version 2.19) for converting BCL to fastq format, coupled with adaptor trimming.
STAR alignment with gene level count
DESeq2 analysis
Assembly: Hg38, Ensembl version 98
Supplementary files format and content: tab delimited file of raw counts

source_name: iPSC-derived cerebral organoid

growth_protocol: Cerebral Organoids were generated as described in Rao et. al

extract_protocol:
total RNA was extracted using QIAgen RNA extraction kit
Poly-A pull-down to enrich mRNAs from total RNA samples, followed by library construction using Illumina TruSeq chemistry.  We multiplexed samples in each lane, which yielded 20 million paired-end 100bp reads for each sample.

value: iPSC-derived cerebral organoid

value: mixed, iPSC-derived cerebral organoid

value: AAV-SYN-GFP


title: QR20 - control

value: Wild type


title: VHIR-MS21-40

city: Barcelona

first: María

middle: Dema

last: Etxezarreta

state: Barcelona

address: Passeig de la Vall d'Hebron, 119-129 Edifici Collserola - Laboratorio 150Passeig de la Vall d'Hebron, 119-129

department: Clinic neuroimmunology

country: Spain

institute: VHIR

data_processing:
nf-core/rnaseq pipeline version 3.5
Data filtering and pre-processing by FastQC (read quality control), UMI-tools (UMI extraction), TrimGalore (adapter and quality trimming), BBSplit (removing of genome contaminants), SortMeRNA (removal of ribosomal RNA).
Read alignment against mm39 genome using STAR
Read quantification and tmm normalization using SALMON (parameter –useEM)
Assembly: mm39
Supplementary files format and content: XLSX file, containing gene count matrix
Supplementary files format and content: XLSX file, containing TMM normalized gene count matrix
Supplementary files format and content: XLSX file, containing transcript count matrix
Supplementary files format and content: XLSX file, containing TMM normalized transcript count matrix

source_name: Splenocytes, 8weeks, day28

extract_protocol:
Total RNA was isolated using RNeasy Mini Kit (74104, Qiagen) with on-column DNase digestion (79254, Qiagen). RNA quality and concentration was analysed by capillary electrophoresis using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and  1 µg per sample was used to start the libraries.
PolyA tail selection was performed with Poly(A) RNA Selection Kit V1.5 (157.96, Lexogen). Retrotranscription into DNA libraries ready for sequencing was performed with CORALL Total RNA-Seq Library Prep Kit (095, Lexogen).

value: Spleen

value: Splenocytes

value: 8 weeks

tag: time point

value: Day 28

tag: Sex

value: female


title: loxPsym_K562_Bxb1_d3_rep1

description: Library name: SP000244


title: VHIR-MS21-39


title: D1_Ngn1_c


title: QR23 - case


title: H9NGN2 hPSC cells iSNs replicate 3

city: Wollongong

first: Dominic

last: Kaul

state: NSW

address: Northfields Ave

country: Australia

institute: University of Wollongong

description: Library name: Replicate 3

data_processing:
Transcripts per million (TPM) were calculated using the cpm function in edgeR (version 3.17, R).
Assembly: GRCh38.109
Supplementary files format and content: .csv

growth_protocol: In preparation for iSN differentiation, cell culture plasticware was first coated with 10 μg/ml Poly-D-Lysine (Sigma–Aldrich) for 30 min at room temperature, then coated with Matrigel overnight at 37 °C. CD271+ NCCs were then seeded at a density of approximately 8.0 ´ 103 cells/cm2 on 12 mm circular glass coverslips for patch-clamp studies, 2.6 ´ 104 cells/cm2 on 13 mm circular glass coverslips for immunocytochemistry, or 2.6 ´ 104 cells/cm2 directly on plasticware for transcriptomic analyses. iSN differentiation was completed following previously published methods with some modifications (Hulme et al., 2020) St Clair-Glover et al, 2024). Briefly, cells were cultured in neuronal medium containing Neurobasal™ medium containing 1% N2, 1% B-27 minus vitamin A, 1% Insulin-transferrin-Selenium-A, and 1% GlutaMAX™ (all from Gibco, Waltham, MA, USA). Throughout iSN differentiation, the media was supplemented with 10 μM Y-27632, 10 ng/ml BDNF, 10 ng/ml GDNF, 10 ng/ml NT-3, and 10 ng/ml b-NGF (all from STEMCELL Technologies), with media changes  conducted every 2-3 days. For the initial 96 h after plating (day 6-10), NGN2 expression was induced by the application of doxycycline at 2 µg/mL (Sigma-Aldrich, D9891). From day 14, cells were phased into BrainPhys™ neuronal medium (BrainPhys™ Neuronal Medium, 0.02% NeuroCult™ SM1 without Vitamin A, 0.01% N2 Supplement-A [all from STEMCELL Technologies]) for maturation, with a ratio of 25:75, 50:50, 75:25, 100:0 (BrainPhys™: neuronal media) at each media change. Where required, proliferating cells were removed by adding anti-mitotic agent, cytosine β-D-arabinofuranoside at 2.5 μM (AraC, Sigma-Aldrich), for 48 h between days 16-18

extract_protocol:
Total RNA extraction was performed on iSNs using the PureLink™ RNA Mini Kit (Invitrogen) following the manufacturer’s directions (n = 3 independent cell culture preparations)
Resulting FASTQ files were aligned with the human genome (genome build GRCh38.109) using the nf-core/rnaseq pipeline (Ewels et al., 2020). Briefly, FASTQ files were pre-processed to infer strandedness (Salmon), trim reads (Trim Galore!), and remove genome/ribosomal contamination (BBSplit and SortMeRNA, respectively). Reads were then aligned to reference genome using STAR and Salmon (Dobin et al., 2013; Patro et al., 2017). Quality control of read alignment was conducted using MulitQC, qualimap, and FastQC.

value: hPSC

value: Sensory neuron differentiation


title: VHIR-MS21-38


title: QR19 - control


title: VHIR-MS21-37


title: Q6 - case


title: VHIR-MS21-36


title: H9NGN2 hPSC cells iSNs replicate 2

description: Library name: Replicate 2


title: loxPsym_K562_Cre_d6_rep1

description: Library name: SP000247


title: H9NGN2 hPSC cells iSNs replicate 1

description: Library name: Replicate 1


title: Ctrl_c


title: Q1 - case


title: VHIR-MS21-35


title: JJN3 response, PDS+Lena condition

city: Montpellier

first: Jerome

last: MOREAUX

address: 141, rue de la Cardonille 34396 Montpellier Cedex 5 FRANCE

country: France

institute: IGH

data_processing:
RNA-seq read pairs were mapped to the reference human GRCh37 genome using the STAR aligner.
All of the statistical analyses were performed with the R statistics software (version 4.3.0; available from https://www.r-project.org) and with R packages developed by the BioConductor project (available from https://www. bioconductor.org/).
The expression level of each gene was summarized and normalized using the DESeq2 R/Bioconductor package.
Assembly: GRCh37
Supplementary files format and content: Text file with values separated with tabs

source_name: cell line

growth_protocol: Cells were cultured at 10e5 cells/ml (JJN3, XG7) or 20e5cells/ml (XG20) in 10ml culture flask in RPMI 1640 medium, 10% FCS. For XG cell lines in addition to this, we added 2ng/ml of IL-6

extract_protocol:
A Total RNA was extreated using Qiagen AllPrep DNA/RNA/miRNA Universal Kit (ref 80224)
The RNA-seq library preparation was completed with 300 ng of input RNA using the Illumina TrueSeq Stranded mRNA Library Prep Kit. Paired end RNA-seq was performed with an Illumina NEBNext Ultra II Directional RNA library Prep kit sequencing instrument (Integragen, Evry, France).

treatment_protocol: XG20, XG7, JJN3 cell lines were treated with compound lenalidomide  (XG7, JJN3: 25µM, XG20: 50µM), or pyridostatin (2.5µM), or the combination for 48h.

value: cell line

value: PDS+Lena


title: JJN3 response, Lena condition

value: Lena


title: Q5 - control


title: VHIR-MS21-34


title: Q2 - control


title: JJN3 response, PDS condition

value: PDS


title: VHIR-MS21-33


title: XG20 response, PDS+Lena condition


title: loxPsym_K562_Cre_d3_rep1

description: Library name: SP000243


title: VHIR-MS21-46

source_name: Splenocytes, 40 weeks, day28

value: 40 weeks


title: QR27, case, 150DIV

data_processing:
Processing, gene counting and aggregation was performed using Cell Ranger software v2.1.1
Assembly: Hg38, Ensembl version 98
Supplementary files format and content: hda5 generated by scanpy

extract_protocol:
High Viability single cell suspension was generated using papain dissociation of DIV70 and DIV 150 cerebral organoids
Library was generated using manufaturer's protocol (10x Genomics)

value: 150DIV cerebral organoid


title: Q6, case, 150DIV

value: iPSC-derived cerebral organoid


title: QR20, control, 150DIV

value: mixed, iPSC-derived cerebral organoid


title: XG20 response, Lena condition


title: VHIR-MS21-43


title: VHIR-MS21-44


title: XG20 response, PDS condition


title: Q5, control, 150DIV


title: Q1, case


title: VHIR-MS21-42


title: XG7 response, PDS+Lena condition


title: VHIR-MS21-41


title: Q2, control


title: QR27, case, 70DIV

value: 70DIV cerebral organoid


title: XG7 response, Lena condition


title: VHIR-MS21-8

source_name: Splenocytes, 8weeks, day0

value: Day 0


title: QR20, control, 70DIV

value: Wild type


title: ATAC_T8_WT_IFNb_01h30min_MMAK015

city: Vienna

first: Christian

last: Schoefer

address: Schwarzspanierstraße 17

department: Center for Anatomy and Cell Biology

country: Austria

institute: Medical University of Vienna

description: open chromatin (ATAC-seq) ATAC-seq sample of spleen CD8+ T cells and IFNb treatment

data_processing:
Raw reads were trimmed with trimmomatic (version 0.32) aligned to the mouse reference genome (mm10) using bowtie2 (version 2.2.4) Primary alignments with mapping quality greater than 30 were retained. ATAC-seq peaks were called using MACS (version 2.7.6 (Zhang et al., 2008)) on each individual sample. Peaks were aggregated into a list of consensus peaks using the function reduce of the package GenomicRanges (version 1.38.0) in R (version 3.6.1). Consensus peaks that overlapped with blacklisted genomic regions (downloaded from https://github.com/Boyle-Lab/Blacklist/tree/master/lists) were discarded. Quantitative measurements were obtained by counting reads within consensus peaks using the function summarizeOverlaps from the GenomicAlignments (version 1.22.1) package in R (version 3.6.1).
Assembly: mm10/GRCm38
Supplementary files format and content: Peaks in narrowPeak format

source_name: T CD8+ cells

growth_protocol: Ex vivo harvesting of cells was conducted following this protocol: We established and validated a robust isolation and staining protocol, which was used consistently across all experiments. Spleens were resected and immediately placed into tubes containing cold PBS (Gibco). Tissue was smashed with a 100µm strainer (SPL Life Sciences) using a syringe plunger and a 50ml tube. A new strainer was used for each spleen and rinsed with 10 to 20ml DMEM (Gibco) containing 10% FCS (Sigma) and 5ml penicillin streptomycin with 10,000 U/ml (Gibco). We pooled three littermates to obtain sufficient cell numbers. Samples were centrifuged at 500g for 5 min at 4 °C. Pellets were resuspended in 1ml Red Blood Cell Lysis Solution (Promega, Z3141) and incubated for 5min on ice. The lysis was stopped by adding 50ml 1x PBS. Samples were centrifuged at 500g for 5 min at 4 °C. Supernatant was discarded and pellets were resuspended in 1ml PBS supplemented with 2% BSA (Sigma). Samples were filtered through a 70µm strainer (SPL Life Sciences). The strainer was washed with 1ml PBS supplemented with 2% BSA. Samples were centrifuged at 500g for 5 min at 4 °C and supernatant was discarded. Cell pellets were resuspended in 100µl PBS (2% BSA) and anti-CD16/CD32 (clone 93, Biolegend) was added at a concentration of 1:500 for 15 min to prevent nonspecific binding. Single-cell suspensions were then stained with combinations of antibodies against TER-119 (APC-Cy7, clone TER-119), CD8 (APC, clone 53-6.7), F4/80 (FITC, clone BM8), CD19 (PerCPCy5.5, clone 6D5), CD3 (PE, clone 17A2), Ly-6C (PECy7, clone HK1.4), Ly-6G (PECy7, clone 1A8), NK1.1 (PECy7, clone PK136), CD45 (AF700, clone 30-F11, Biolegend) and Fixable Viability Dye eFluor 780 (APC-eFluor 780, eBio-science). In case of dendritic cell purification CD11c (PECy7, clone N418, eBioscience), MHCII (PE, MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), eBioscience) was used. Cells were stained for 30 min at 4 °C in the dark. 1ml PBS supplemented with 2% BSA was added and suspensions were centrifuged at 500g for 5 min at 4 °C. Pellets were resuspended in 300µl PBS supplemented with 2% BSA and filtered over 40µm strainer (SPL Life Sciences), filters were rinsed with 1ml PBS supplemented with 2% BSA. Cells were sorted with a BD FACS-Aria III Fusion instrument into PBS supplemented with 20% BSA.

extract_protocol:
Chromatin accessibility mapping by ATAC-seq was performed as previously described (Buenrostro et al., 2013; Corces et al., 2016), with minor adaptations. After centrifugation, the pellet was carefully resuspended in the transposase reaction mix (12.5µl 2xTD buffer, 2µl TDE1 (Illumina), 10.25µl nuclease-free water, and  0.25µl 1% digitonin (Promega) for all other cell types) for 30 min at 37 °C. Following DNA purification with the MinElute kit eluting in 11µl, 1µl of the eluted DNA was used in a quantitative PCR reaction to estimate the optimum number of amplification cycles. The remaining 10μl of each library were amplified for the number of cycles corresponding to the Cq value from the qPCR (i.e., the cycle number at which fluorescence has increased above background levels, rounded down). Library amplification was followed by SPRI beads (Beckman Coulter) size selection to exclude fragments larger than 1,200bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). Library amplification was performed using custom Nextera primers (Buenrostro et al., 2013). The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50bp single-end configuration.
After centrifugation, the pellet was carefully resuspended in the transposase reaction mix (12.5 µl of 2 × TD buffer, 2 µl of TDE1 (Illumina), 10.25 µl of nuclease-free water and 0.125 µl of 10% NP-40 (Sigma) for macrophages and dendritic cells or 0.25 µl of 1% digitonin (Promega) for all other cell types) and incubated for 30 min at 37 °C. Following DNA purification using the MinElute kit, DNA was eluted in 11 µl. We used 1 µl of the eluted DNA in a quantitative PCR reaction to estimate the optimum number of amplification cycles. The remaining 10 μl of each library was amplified for the number of cycles corresponding to the Cq value from the quantitative PCR (that is, the cycle number at which fluorescence has increased above background levels, rounded down). Library amplification was followed by SPRI bead (Beckman Coulter) size selection to exclude fragments larger than 1,200 bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). Library amplification was performed using custom Nextera primers85. The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-end configuration.

treatment_protocol: In vitro culture and treatment of ex vivo sort purified cells was done following this protocol: Aliquots of the sort-purified cell populations were stored for RNA/DNA isolation in RLT-buffer (Qi-agen) or directly processed using the ATAC-seq assay (as described below). Splenic CD8+ T cells were cultured in 48 well tissue culture treated plates for in vitro treatment with different stimuli. To that end, the cells were centrifuged at 500g for 5 min at 4 °C.  3*105 T cells were seeded per well in 300µl media. T cells were resuspended in RPMI (10% FCS, 5 mL penicillin streptomycin with 10000 U/ml). The following conditions were applied: (i) 20 hours in culture untreated, (ii) 20 hours in culture with treatment, and (iii) 18.5 hours in culture followed by treatment for the last 1.5 hours. Treatments included murine recombinant IFN-β carrier-free (PBL as-say science, Cat. No. 12401-1) at a final concentration of 1,000 U/ml or recombinant murine IL-2 (PeproTech, Cat. No. 212-12) at a final concentration of 1,000 ng/ml or murine M-CSF (PeproTech, Cat. No. 315-02) at a final concentration of 100 ng/ml. T cells were harvested by transferring them into a reaction tube, additional cold PBS (0.2% BSA) was added, centrifuged at 500g for 5 min at 4 °C and supernatant was removed. T cells were resuspended in 1ml PBS (0.2% BSA) and split equally between the two tubes. After vortexing the sample for one minute, it was stored at -80°C until further processing. RNA and DNA were isolated with the AllPrep RNA/DNA Micro Kit (Qiagen) following the manufacturer’s instructions and stored as recommended.

tag: organ

value: Spleen

value: T CD8+ cells

value: 8-12 weeks

value: WT

value: IFNb 1h30min


title: JJN3 response, control condition

value: DMSO


title: RNA_T8_WT_H_H_MMAK004

description: RNA-seq sample of splenic T CD8+ cells

data_processing:
Raw reads were trimmed using trimmomatic (version 0.32) aligned to the mouse reference genome (mm10) using STAR (version 2.7.1) Gene expression was quantified by counting uniquely aligned reads in exons using the function summarizeOverlaps from the GenomicAlignments package (version 1.6.3) in R (version 3.2.3) Gene annotations were based on the Ensembl GENCODE Basic set (genome build GRCm38 release 93)
Assembly: mm10/GRCm38
Supplementary files format and content: csv file of read counts

source_name: CD8+ T cells

growth_protocol: Ex vivo harvesting of cells was conducted following this protocol: We established and validated a robust isolation and staining protocol, which was used consistently across all experiments. Spleens were resected and immediately placed into tubes containing cold PBS (Gibco). Tissue was smashed with a 100µm strainer (SPL Life Sciences) using a syringe plunger and a 50ml tube. A new strainer was used for each spleen and rinsed with 10 to 20ml DMEM (Gibco) con-taining 10% FCS (Sigma) and 5ml penicillin streptomycin with 10,000 U/ml (Gibco). For the isolation of dendritic cells (DCs) spleens were injected with and placed in a digestion mixture (RPMI (Sigma), 2% FBS, 1mg/ml Collagenase D, 20µg/ml DNase I) and then incubated at 37°C for 30 minutes in a 24-well cell culture dish, before proceeding with the same mashing through a 100µm strainer. We pooled three littermates to obtain sufficient cell numbers. Samples were centrifuged at 500g for 5 min at 4 °C. Pellets were resuspended in 1ml Red Blood Cell Lysis Solution (Promega, Z3141) and incubated for 5min on ice. The lysis was stopped by adding 50ml 1x PBS. Samples were centrifuged at 500g for 5 min at 4 °C. Supernatant was discarded and pellets were resuspended in 1ml PBS supplemented with 2% BSA (Sigma). Samples were filtered through a 70µm strainer (SPL Life Sciences). The strainer was washed with 1ml PBS supplemented with 2% BSA.

extract_protocol:
RNA was isolated with the AllPrep RNA/DNA Micro Kit (Qiagen) following the manufacturer’s instructions and stored as recommended. 500pg of RNA was used as input. Reverse transcription and PCR were performed as described (Picelli et al., 2014).
Library preparation was conducted on 1ng of cDNA using the Nextera XT DNA Sample Preparation Kit (Illumina) followed by SPRI (Beckman Coulter) size selection. Sequencing was performed by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50bp single-end configuration.

treatment_protocol: In vitro culture and treatment of ex vivo sort purified cells was done following this protocol: Aliquots of the sort-purified cell populations were stored for RNA/DNA isolation in RLT-buffer (Qi-agen) or directly processed using the ATAC-seq assay (as described below). Splenic CD8+ T cells were cultured in 48 well tissue culture treated plates for in vitro treatment with different stimuli. To that end, the cells were centrifuged at 500g for 5 min at 4 °C. 1*105 macrophages and 3*105 T cells were seeded per well in 300µl media. T cells were resuspended in RPMI (10% FCS, 5 mL penicillin streptomycin with 10000 U/ml). The following conditions were applied: (i) 20 hours in culture untreated, (ii) 20 hours in culture with treatment, and (iii) 18.5 hours in culture followed by treatment for the last 1.5 hours. Treatments included murine recombinant IFN-β carrier-free (PBL as-say science, Cat. No. 12401-1) at a final concentration of 1,000 U/ml or recombinant murine IL-2 (PeproTech, Cat. No. 212-12) at a final concentration of 1,000 ng/ml or murine M-CSF (PeproTech, Cat. No. 315-02) at a final concentration of 100 ng/ml. T cells were harvested by transferring them into a reaction tube, additional cold PBS (0.2% BSA) was added, centrifuged at 500g for 5 min at 4 °C and supernatant was removed. T cells were resuspended in 1ml PBS (0.2% BSA) and split equally between the two tubes. Tubes were then centrifuged at 500g for 5 min at 4 °C and either taken for RNA/DNA isolation or ATAC-seq (as de-scribed below). After centrifugation, the supernatant was carefully removed and the pellet was resus-pended in 350μl RLT buffer (Qiagen) with 3.5µl ß-mercaptoethanol (Sigma). After vortexing the sam-ple for one minute, it was stored at -80°C until further processing. RNA and DNA were isolated with the AllPrep RNA/DNA Micro Kit (Qiagen) following the manufacturer’s instructions and stored as recommended.

value: spleen

value: CD8+ T cells

value: Homeostatic


title: Q6, case, 70DIV


title: VHIR-MS21-7

value: Splenocytes

value: 8 weeks

value: female


title: XG7 response, control condition

value: cell line


title: VHIR-MS21-45

value: 40 weeks

value: Day 28


title: VHIR-MS21-5

value: Day 0


title: XG7 response, PDS+PANO condition

growth_protocol: Cells were cultured at 10e5 cells/ml (JJN3, XG7) or 15e5cells/ml (XG2) in 10ml culture flask in RPMI 1640 medium, 10% FCS. For XG cell lines in addition to this, we added 2ng/ml of IL-6

treatment_protocol: XG2, XG7, JJN3 cell lines were treated with compound panobinostat  at 5nM, or pyridostatin (XG2: 0.63µM, XG7: 1.25µM, JJN3: 1.25µM), or the combination for 48h.

value: PDS+PANO


title: Q5, control, 70DIV

value: iPSC-derived cerebral organoid

value: mixed, iPSC-derived cerebral organoid

value: Wild type

value: 70DIV cerebral organoid


title: RNA_T8_TYK2KO_H_H_MMAK004

description: RNA-seq sample of splenic T CD8+ cells

value: 8-12 weeks

value: Tyk2 knockout


title: XG7 response, PANO condition

value: PANO


title: RNA_T8_TYK2KO_H_H_MMAK003

description: RNA-seq sample of splenic T CD8+ cells

value: spleen

value: CD8+ T cells


title: RNA_T8_WT_H_H_MMAK002

description: RNA-seq sample of splenic T CD8+ cells

value: WT

value: Homeostatic


title: XG20 response, control condition

value: DMSO


title: Q2, control, 70DIV


title: VHIR-MS21-6

value: Spleen

value: Splenocytes

value: 8 weeks

value: female


title: XG7 response, PDS condition

value: cell line

value: PDS


title: VHIR-MS21-4

value: Day 0


title: VHIR-MS21-3


title: RNA_T8_TYK2KO_H_H_MMAK002

description: RNA-seq sample of splenic T CD8+ cells


title: VHIR-MS21-2


title: XG7 response, PDS condition


title: CAOV3_P7B_3D

city: Birmingham

first: Mythreye

last: Karthikeyan

address: 1824 6th avenue south

department: pathology

institute: university of alabama

description:
P72
2x150bp

data_processing:
Quality done with FASTQC, trimming with Trim Galore (v 0.6.4), reassessed with FASTQC.
Aligned with STAR (v2.6.0) with  GENCODE GRCh38 primary assembly and annotation GTF (release 37)
BAM index files generated with SAMtools, count generation with featurecounts. Tertiary Analysis with DESeq2.
Sequence alignment, quality control, and variant calling was done following GATK best practices using the germline and somatic variant calling algorithms haplotype caller and mutect2, respectively
MAFtools version 2.10.05 was used to compare the sequenced time points.
Assembly: GRCh38 release 37
Supplementary files format and content: raw counts from feature counts
Supplementary files format and content: MAF files

source_name: OVARY

growth_protocol: cultured in RPMI-1640 ((ATCC® 30-2001ATCCTM) containingl-glutamine, 10% FBS, without Pen/Strep

extract_protocol:
Total DNA was isolated using Qiagen DNeasy Blood and Tissue Kit (ref 69504). Total RNA was isolated using Trizol/Chloroform extraction method and RNA quality was validated on RNA-1000 chip using Bioanalyzer (Agilent). 1.0 ug of total RNA was used for the construction of sequencing libraries.
For DNA, Novogene utilized Agilent SureSelect Human All Exon V6 Kit. For RNA, Novogene utilized the NEBNext UltraTM RNA Library Prep Kit for Illumina.

treatment_protocol: cells were grown in regular growth media for the cell line without any additional treatments to the media.

value: OVARY

value: serous epithelial

value: 24hr suspension


title: XG7 response, control condition

value: Ctrl


title: CAOV3_P7C_3D

description:
P73
2x150bp


title: RNA_NK_TYK2KO_H_H_MMAK006

description: RNA-seq sample of splenic NK cells

source_name: NK cells

value: NK cells

value: 8-12 weeks

value: Tyk2 knockout


title: CAOV3_P7A_3D

description:
P71
2x150bp


title: VHIR-MS21-24

source_name: Splenocytes, 8weeks, day14

value: Day 14


title: VHIR-MS21-1


title: Q1, case, 70DIV

value: iPSC-derived cerebral organoid

value: mixed, iPSC-derived cerebral organoid

value: 70DIV cerebral organoid


title: RNA_NK_TYK2KO_H_H_MMAK005

description: RNA-seq sample of splenic NK cells

value: spleen

value: Homeostatic


title: JJN3 response, PDS condition


title: JJN3 response, PANO condition

value: PANO


title: CAOV3_P6C_2D

description:
P63
2x150bp

value: Attached


title: VHIR-MS21-23

value: Spleen

value: Splenocytes

value: 8 weeks

value: female


title: CAOV3_P6B_2D

description:
P62
2x150bp

value: OVARY

value: serous epithelial


title: JJN3 response, control condition

value: cell line

value: Ctrl


title: Tumor-derived NK cells, WT, Rep2

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using Trimmomatic
Trimmed sequence reads were mapped to GrCM38/mm10 using STAR
Read counts to exons (EnsEMBL annotations release 93) were performed using R package Genomic-Alignments (function summarizeOverlaps)
Assembly: mm10
Supplementary files format and content: Tab-delimited text files include raw counts per gene (exons)

source_name: MC38 adenocarcinoma

extract_protocol:
Oligo-dT primer hybridization to poly(A) tail of all RNA was performed by incubating the RNA samples with oligo-dT primer (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′) and dNTP mix at 72 C for 3 min. 500 pg of RNA was used as input. Reverse transcription and preamplification were performed as previously described in Picelli 2014 (Nature protocols DOI: 10.1038/nprot.2014.006).
Stranded sequencing libraries were generated using Smart-seq2 protocol with Nextera DNA Library Preparation Kit (Illumina, #FC-121-1030).

treatment_protocol:
Tumor transplants were performed with the MC38 adenocarcinoma cell line. 10^6 MC38 cells were injected subcutaneously into each flank and tumor growth was monitored every other day. After 8-10 days tumors were isolated, weighed and cut tumors were digested in DMEM complete medium containing 1 mg/ml of collagenase D from Clostridium histolyticum (COLLD-RO, Sigma-Aldrich, cat. no. 11088858001) and 0.05 mg/ml of DNase I (Sigma-Aldrich, cat. no. 11284932001) for 1h in 37°C. The digested tissue was squeezed through 100 µm cell strainers twice to obtain single cell suspensions. CD45+ cells were enriched/isolated by magnetic separation using the MagniSortTM CD45 positive selection kit (Thermo Fisher Scientific cat. no. 8802-6865).
Cells were stained for CD45 (30-F11), CD3ε (145-2C11), NK1.1 (PK136) and CD8a (53-6.7) and 300 NK cells (CD45+, CD3ε-, NK1.1+) or 300 CD8+ T cells (CD45+, CD3ε+, CD8a+) were sorted and lysed in buffer containing 0.2% Triton X-100 solution (Sigma-Aldrich, cat. no. T9284) supplemented with RNAse inhibitor (1:20, Takara Clontech, 2313A). Samples were stored at -80°C until RNA extraction.

value: MC38 adenocarcinoma

value: Tumor-derived NK cells

value: WT

tag: time


title: VHIR-MS21-18

value: Day 14


title: CAOV3_P6A_2D

description:
P61
2x150bp


title: CAOV3_P1C_3D

description:
P13
2x150bp

value: 24hr suspension


title: Tumor-derived NK cells, WT, Rep1


title: VHIR-MS21-19


title: XG2 response, PDS+PANO condition

value: PDS+PANO


title: VHIR-MS21-17


title: JJN3 response, PDS+PANO condition


title: Tumor-derived NK cells, TYK2KO, Rep1


title: XG2 response, PDS condition

value: PDS


title: CAOV3_P1B_3D

description:
P12
2x150bp


title: CAOV3_P1A_3D

description:
P11
2x150bp


title: RNA_NK_TYK2KO_H_H_MMAK007

description: RNA-seq sample of splenic NK cells

value: spleen

value: NK cells

value: 8-12 weeks

value: Tyk2 knockout

value: Homeostatic


title: VHIR-MS21-16

source_name: Splenocytes, 40 weeks, day0

value: Spleen

value: Splenocytes

value: 40 weeks

value: Day 0

value: female


title: Tumor-derived NK cells, TYK2KE, Rep2

value: MC38 adenocarcinoma


title: XG2 response, control condition

value: cell line

value: Ctrl


title: CAOV3_P0C_2D

description:
P03
2x150bp

value: OVARY

value: serous epithelial

value: Attached


title: VHIR-MS21-15


title: VHIR-MS21-14


title: Tumor-derived CD8 T cells, WT, Rep2

value: Tumor-derived CD8 T Lymphocytes

value: WT


title: VHIR-MS21-13


title: D456, GLUT3oe, S485A,T492A, repB

city: Huntsville

first: Sara

last: Cooper

state: AL

address: 601 Genome Way

institute: HudsonAlpha Institute for Biotechnology

data_processing:
Lexogen BlueBee QuantSeq FWD Analyses pipeline
Cutadapt to remove artifacts and adapter sequences
FASTQC report for trimmed reads
STAR aligner
Assembly: hg38
Supplementary files format and content: raw cont table at the gene level for all samples

growth_protocol: THP1 cells were purchased from ATCC. THP1 cells were grown in RPMI 1640 media with 2000mg/L glucose, supplemented with 10% FBS, 1% penicillin/streptomycin (Gibco), and 1% GlutaMax (Gibco). 2.5ug/mL phytohemagglutinin (PHA) to induce proliferation after thawing. Cells were split with Accutase (Gibco) and counted prior to plating. THP1 cells were lifted, as needed, and pipetted vigorously before counting and plating.

extract_protocol:
RNA was isolated using Norgen Total RNA kit plus DNase treatment according to the manufacturer's protocol (Norgen # 37500, 25720). RNA quality was verified with the Agilent BioAnalyzer RNA Nano 600 kit (cat# 5067-1512) with the RIN range between (9.7–10 RIN). RNA was quantified using Qubit 2.0 Fluorometer (Thermofisher).
RNA-sequencing libraries were made using Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina kit (cat# 015.24) with 250 ng of RNA input.We pooled all samples for sequencing on a NextSeq 500 with single-end 75 base pair reads. Read counts averaged 3.9 million reads per sample with an average Q30>92%.

treatment_protocol: CSC293T cells were transiently co-transfected with psPAX2, pCMV-VSVG, and pLX304 lentiviral plasmid containing SLC2A3 or the empty vector control plasmid (Genecopia). Transfection was performed using Fugene HD Transfection Reagent (Promega), as previously reported48. Viral titer was determined using the Lenti-X RT-qPCR Titration Kit (Takara Bio). Addition of Blasticidin S to culture media was used to select cells with stable overexpression. 8 million D456 EV and GLUT3OE cells were plated on geltrex (Gibco) and allowed to recover overnight. Media was changed and cells were incubated for 48 hours, before spent media was collected. Exosomes were isolated from spent media using differential ultracentrifugation. Briefly, spent media will be centrifuged at 300g to remove cells, then at 2,000g to remove dead cells. The supernatant is again spun at 10,000g to remove cell debris, before being filtered through a 0.22um filter and spun at 100,000g to pellet extracellular vesicles. Extracellular vesicles were purified using the ExoQuick-TC ULTRA (System Biosciences). Quantification and characterization of exosomes. Extracellular vesicles were characterized based on size using nanoparticle tracking analysis via the NanoSight NS300. Additionally, the relative concentration of the exosomes was determined, and used later to calculate treatment volumes. Nanoparticle tracking analysis was performed in the NanoSight NS300 software. Extracellular vesicles were checked for common exosome markers, such as CD9, CD81, CD63, using the Amnis Imagestream XMark II. The ImageStream machine combines imaging with flow cytometry allowing for simultaneous imaging of the fluorescently labeled extracellular vesicles, while also using flow cytometry to determine the percentage of cells positive for each marker. Amnis Imagestream data was visualized and analyzed in the IDEAs software in collaboration with the UAB comprehensive flow cytometry core. Antibodies used are as follows: CD9 Mouse anti-human, PE, clone: M-L13 (BD Bioscience) or CD9 Human, Alexa Fluor 594 (Biotechne); CD63 Mouse anti-human, eFlour 450, Clone: H5C6 (eBioscience); CD81 mouse anti-human, PE/Cyanine7 , TAPA-1 (Biolegend). One million THP1 cells were plated and allowed to recover overnight. Exosomes were added at a concentration of 100 exosomes/cell (D456 EV or GLUT3OE) or an equivalent amount of PBS, for 24 hours and cell pellets were harvested and snap frozen.


title: D456, GLUT3oe, S485A,T492A, repD


title: XG2 response, PANO condition

value: PANO


title: Tumor-derived CD8 T cells, TYK2KO, Rep2


title: CAOV3_P0A_2D

description:
P01
2x150bp


title: D456, GLUT3oe, S485A,T492A, repC


title: Tumor-derived CD8 T cells, WT, Rep1


title: Tumor-derived NK cells, TYK2KO, Rep2

value: Tumor-derived NK cells


title: OV90_P7B_WES

description:
P72_CKDN210022308-1A_H7MKMDSX3
2x150bp

data_processing:
Quality done with FASTQC, trimming with Trim Galore (v 0.6.4), reassessed with FASTQC.
Aligned with STAR (v2.6.0) with  GENCODE GRCh38 primary assembly and annotation GTF (release 37)
BAM index files generated with SAMtools, count generation with featurecounts. Tertiary Analysis with DESeq2.
Sequence alignment, quality control, and variant calling was done following GATK best practices using the germline and somatic variant calling algorithms haplotype caller and mutect2, respectively
MAFtools version 2.10.05 was used to compare the sequenced time points.
Assembly: GRCh38 release 37
Supplementary files format and content: raw counts from feature counts
Supplementary files format and content: MAF files
Library strategy: whole exome-seq

value: 24hr suspension


title: VHIR-MS21-12


title: D456, GLUT3oe, S485A,T492A, repA


title: Tumor-derived CD8 T cells, TYK2KO, Rep1


title: THP1, GLUT3oe, S485A,T492A, EXOSOME, 24hr, repC

description: THP1_GLUT3_S485A_T492A_OE_EXOSOME_24hr_C


title: OV90_P7A_WES

description:
P71_CKDN210022307-1A_H7MKMDSX3
2x150bp


title: Tumor-derived CD8 T cells, TYK2KE, Rep2


title: VHIR-MS21-9


title: Tumor-derived NK cells, TYK2KE, Rep1


title: Tumor-derived CD8 T cells, TYK2KE, Rep1


title: VHIR-MS21-10


title: OV90_P0A_WES

description:
P01_CKDN210022301-1A_H7MKMDSX3
2x150bp


title: THP1, GLUT3oe, S485A,T492A, EXOSOME, 24hr, repB

description: THP1_GLUT3_S485A_T492A_OE_EXOSOME_24hr_B


title: VHIR-MS21-32

source_name: Splenocytes, 40weeks, day14

value: Day 14


title: OV90_P0B_WES

description:
P02_CKDN210022302-1A_H7MKMDSX3
2x150bp


title: M_MeOH_1

city: Memphis

first: Michael

last: Dyer

address: 262 Danny Thomas

institute: St. Jude Children's Research Hospital

data_processing:
Data was processed using Cellranger v6.0.0
Assembly: mm10-mouse / GRCh38-human
Supplementary files format and content: barcode, feautres, and matrix files for each sample

source_name: retina

extract_protocol:
Papain (40U) was added to 400uL of papain buffer and incubate at 37°C for ~10 min. Retinal punches were added to papain and incubated at 37°C for 5-10 minutes while flicking. Dnase (40uL) was added as the retina began dissociating. The suspension was transferred into a 50-ml conical tube through a 40-μm mesh cell strainer and washed with PBS−/− and layered onto a 5 ml BSA cushion medium (4% BSA in Retinal Explant media). The sample were centrifuged at 500gfor 10 min at 4 °C and the cell pellets were resuspended in REM. Cells were counted and viability was confirmed on a hemocytometer.
Libraries were constructed using 10x Genomics Chromium Single Cell 3' Reagent Kit

value: retina

value: Retina, 24 hour culture in REM + 8uM Formic Acid


title: OV90_P7C_3D

description:
A63
2x150bp


title: THP1, GLUT3oe, EXOSOME, 24hr, repC


title: VHIR-MS21-31

value: Spleen

value: Splenocytes

value: 40 weeks


title: CAOV3_P0B_2D

description:
P02
2x150bp


title: VHIR-MS21-11

value: Day 0

value: female


title: THP1, GLUT3oe, EXOSOME, 24hr, repB

value: cell line


title: OV90_P7A_3D

description:
A61
2x150bp

value: OVARY


title: VHIR-MS21-30


title: M_Control_2

value: Retina, 24 hour culture in REM + 1mM Glutamate


title: M_Control_1

value: Retina, 24 hour culture in REM


title: OV90_P6C_2D

description:
A53
2x150bp

value: serous epithelial

value: Attached


title: THP1, GLUT3oe, EXOSOME, 24hr, repA


title: OV90_P7B_3D

description:
A62
2x150bp


title: VHIR-MS21-29


title: THP1, EXOSOME, 24hr, repC


title: VHIR-MS21-27


title: M_C.Albic_bio_rep_2

data_processing:
Read alignment to the reference genome was performed with STAR (version 2.7; PMID: 23104886) software. Gene level quantification was determined using RSEM (version 1.3.1 ; PMID: 21816040).
Assembly: mm10-mouse / GRCh38 – human
Supplementary files format and content: tab delimited text file with raw counts for each sample

extract_protocol:
Retinal punches were dissolved in 800uL Trizol Reagent (Invitrogen #15596026). 200uL chloroform was added to each sample, inverted, incubated at room temperature for 3 minutes. Samples were then spun down at 12,000 3 g at 4 C for 15 min. The aqueous later was transferred to a new tube and 1uL glycogen and 500 uL isopropanol was added to each sample and mixed by vortex. Samples were incubated at room temperature for 10 minutes and spun down at 12,000 3 g at 4 C for 15 min. The isopropanol was removed and the pellet was washed twice with ice-cold 80% EtOH. The washed pellet was resuspened in water.
Libraries were constructed using Illumina library prep acording to manufacturer protocols

value: 24 hour culture in REM + 100CFU/mL C. Albicans


title: OV90_P6B_2D

description:
A52
2x150bp


title: M_C.Albic_bio_rep_1


title: THP1, EXOSOME, 24hr, repB


title: THP1, GLUT3oe, S485A,T492A, EXOSOME, 24hr, repA

description: THP1_GLUT3_S485A_T492A_OE_EXOSOME_24hr_A


title: Sorted_vHipp_D2_replicateD

first: Molly

middle: S

last: Estill

address: 1 Gustave L. Levy Place

department: Neuroscience

institute: Mount Sinai School of Medicine

description: 10x Genomics

data_processing:
Demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v3.1.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
The filtered feature-barcode matrices were used as input to downstram processing by the Seurat R package (v4.3.0).
Assembly: mm10 pre-mRNA mouse genome
Supplementary files format and content: Filtered HDF5 Feature-Barcode Matrices

source_name: FAN-sorted nuclei

extract_protocol:
Mouse brains were collected after cervical dislocation and followed by rapid bilateral vHipp dissections from 1 mm-thick coronal brain sections using a 14G needle and frozen on dry ice. To obtain a nuclei suspension, frozen vHipp samples were homogenized in 4 mL of low-sucrose lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM EDTA, 10 mM Tris-HCl) using a large clearance then a small clearance pestle of a glass dounce tissue grinder (Kimble Kontes). Homogenates were filtered through a 40 µm cell strainer (Pluriselect) into ultracentrifuge tubes (Beckman Coulter), underlaid with 5 mL of high-sucrose solution (1.8 M sucrose, 3 mM Mg(Ace)2, 1 mM DTT, 10 mM Tris-HCl) and centrifugated at 107,000 g for 1 hour at 4°C in a SW41Ti Swinging-Bucket Rotor (Beckman Coulter). Supernatant was discarded and nuclei pellets were re-suspended in 800 µL of PBS supplemented with 0.5% bovine serum albumin (BSA). DAPI was added at a 1 µg/mL final concentration. Nuclei were sorted on a BD FACS Aria II three-laser device with a 70 µm nozzle and using BD FACSDiva Software v8.0.2. Gating strategy from a representative sort are visualized in fig. S1. Briefly, debris and doublets were excluded using FSC and SSC filters, nuclei were then selected as DAPI-positive (Violet1-A laser) events, and finally GFP-positive nuclei (Blue1-A laser) were sorted directly into BSA-coated low-binding tubes. 25,000 nuclei were recovered for each sample.
Following FANS, nuclei were quantified (Countess II, Life Technologies) and 8,000 per sample were loaded on a single 10X lane using Chromium Single Cell 3’ Library Construction Kit (10X Genomics). cDNA libraries were prepared according to the manufacturer’s protocol (10X Genomics).

value: FAN-sorted nuclei

value: vHipp dissection

value: D2-Cre x fl/fleGFP::L10a

value: Male

value: 8-12 weeks old


title: OV90_P6A_2D

description:
A51
2x150bp


title: OV90_P4C_3D

description:
A43
2x150bp

value: 24hr suspension


title: Sorted_vHipp_D2_replicateC

description: 10x Genomics


title: THP1, PBS, EXOSOME, 24hr, repC


title: Sorted_vHipp_D2_replicateB

description: 10x Genomics


title: OV90_P4B_3D

description:
A42
2x150bp


title: M_Control_bio_rep_5

value: retina

value: 24 hour culture in REM


title: M_Gluc_bio_rep_2

value: 24 hour culture in REM + 20mM Glucose


title: THP1, PBS, EXOSOME, 24hr, repB


title: OV90_P4A_3D

description:
A41
2x150bp


title: M_C33_bio_rep_2

value: 24 hour culture in REM at 33 degrees Celcius


title: Sorted_vHipp_D2_replicateA

description: 10x Genomics


title: THP1, PBS, EXOSOME, 24hr, repA

value: cell line


title: Sorted_vHipp_D1_replicateD

description: 10x Genomics

value: D1-Cre x fl/fleGFP::L10a


title: M_MeOH_bio_rep_2

value: 24 hour culture in REM + 8uM Formic Acid


title: D456, GLUT, S485A_T492Aoe, repE


title: Sorted_vHipp_D1_replicateC

description: 10x Genomics


title: OV90_P3C_2D

description:
A33
2x150bp

value: OVARY


title: D456, GLUT, S485A_T492Aoe, repD


title: Sorted_vHipp_D1_replicateB

description: 10x Genomics


title: M_C33_bio_rep_1


title: OV90_P3B_2D

description:
A32
2x150bp

value: serous epithelial


title: OV90_P3A_2D

description:
A31
2x150bp

value: Attached


title: D456, GLUT, S485A_T492Aoe, repC


title: M_Control_bio_rep_2


title: M_MeOH_bio_rep_1


title: THP1, EXOSOME, 24hr, repA


title: Sorted_vHipp_D1_replicateA

description: 10x Genomics


title: OV90_P1C_3D

description:
A23
2x150bp


title: D456, GLUT, S485A_T492Aoe, repA


title: M_PH7.69_bio_rep_2

value: 24 hour culture in REM at pH 7.69


title: OV90_P1B_3D

description:
A22
2x150bp


title: OV90_P1A_3D

description:
A21
2x150bp


title: D456, GLUT3oe, repC


title: M_PH7.69_bio_rep_1


title: WT_polyp

city: Noda

first: HAIYANG

last: SUN

state: Chiba

address: Yamazaki

institute: Research Institute for Biomedical Sciences,  Tokyo University of Science

data_processing:
Illumina Novaseq software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to GRCm38 (mm10) whole genome.
BAM files resulting from the alignments were summarized to genes using the Stringtie algorithm.
Transcript per million (TPM) were calculated.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each sample.

source_name: colon

extract_protocol:
colon and polyp form colon were removed, flash frozen on liquid nitrogene, and RNA was harvested using Mammalian Total RNA Miniprep kit (MilliporeSigma, USA).
RNA libraries were prepared for sequencing using NEBNext® Ultra RNA Library Prep Kit for Illumina® (E7530).

value: colon

value: BALB/cA

value: wild type


title: M_Gluc_bio_rep_1


title: D456, GLUT3oe, repB


title: M_PH7.15_bio_rep_1

value: 24 hour culture in REM at pH 7.15


title: DCIR_KO_nonpolyp

value: DCIR-/-


title: WT_nonpolyp


title: D456, GLUT3oe, repA


title: DCIR_KO_DSS


title: OV90_P0B_2D

description:
A12
2x150bp


title: D456, empty, repC


title: OV90_P0C_2D

description:
A13
2x150bp


title: WT_DSS


title: M_ 40pct _bio_rep_2

value: 24 hour culture in REM at 40% Oxygen


title: OV90_P0A_2D

description:
A11
2x150bp


title: D456, empty, repB


title: DCIR_KO


title: M_ Control _bio_rep_1

value: retina


title: D456, empty, repA


title: DCIR_KO_polyp


title: Myometrial Arteries_6

city: Newcastle Upon Tyne

last: Taggart

state: Tyne And Wear

address: International Centre for Life,

department: Biosciences Institute,

country: United Kingdom

institute: Newcastle University

description:
sample_20_S20
MA_6

data_processing:
QC checks with FastQC v0.11.9 and FastQ Screen v0.14.1, adapter contamination <0.1% across all samples.
Sequence reads aligned to Homo_sapiens.GRCh38.104 genome using hisat2 v2.2.1
Quantified raw read counts per gene using HTSeq-count version 1.99.2 and Homo_sapiens.GRCh38.104.gtf (genome feature file)
Pre-filter row with at least read count of 21.
Regularised logarithm (rlog) normalisation using DESeq2.
Assembly: Homo_sapiens.GRCh38.104
Supplementary files format and content: normalised_counts.csv-  Comma-seperated text file consists of normalised counts of each samples
Supplementary files format and content: rawCountsTable.csv- Comma-sperated text file consists of un-normalised raw counts of each samples

source_name: myometrial arteries

extract_protocol:
Tissue pieces weighed about 3.5mg to 5mg was used for RNA extraction. RNA was extracted using Rneasy Micro Kit (QIAGEN 74004) according to the manufacturer's instructions.
RNA library was prepared  using standard illumina protocols.

value: myometrial arteries


title: Myometrial Arteries_7

description:
sample_21_S21
MA_7


title: hWJ EV 3

city: Tsukuba shi

first: Neni

last: Anggraeni

state: Ibaraki

address: 1-1-1 Tennodai

institute: University of Tsukuba

data_processing:
FASTQ files were imported to CLC Genomics Workbench (Ver.10.1.1, Qiagen).
Reads were grouped by sequence then matched to miR-base annotated microRNAs (miRbase v21)
Total counts were normalized by adjusting total counts to 1x10^6.
Assembly: mirbase 21
Supplementary files format and content: Microsoft Excel file format, containing gene name, raw read counts and normalized read counts

source_name: Wharton's Jelly

growth_protocol: The cells were culture using EV-depleted medium at 37°C under 5% CO2  for 24 hr and under a hypoxic condition (1%O2)

extract_protocol:
The cell samples were collected, and RNA was purified using Trizol reagent according to the standard protocol. 500 ng of total RNA was applied for sequencing. The NEBNext Small RNA Library Prep Kit was used to build a sequencing small RNA library, which was selected according to size using AMPure beads and verified using a Bioanalyzer DNA High-sensitivity Kit.
NEBNext Small RNA Library Prep Kit, small RNA library

treatment_protocol: Hypoxic condition (1%O2)

value: Wharton's Jelly

value: Mesenchmal Stem cell

value: Wild Type

value: treated


title: WT


title: M_ T0 _bio_rep_2

value: WT Retina


title: M_ T0 _bio_rep_1