title: iTF-Microglia Day 18, untreated, rep1

description: Library name: iTF-Microglia_rep1

data_processing:
PE150 raw reads were processed with the ENCODE ATAC-seq pipeline (v1.7.0)
Reads were trimmed with Cutadapt, aligned to the hg38 reference genome using Bowtie2, and filtered to remove duplicates and mitochondrial reads using Picard and Samtools. High-quality, properly paired reads were retained.
Peaks were called with MACS2 [--shift -75 --extsize 150], filtered for ENCODE blacklist regions, and reproducible peaks identified using IDR and naive overlap.
Assembly: hg38
Supplementary files format and content: Optimal set of reproducible ATAC-seq peaks identified by overlapping peak calls from two replicates. Peaks were called using MACS2 with narrowPeak output format.
Supplementary files format and content: Genome-wide -log10(p-value) signal track from pooled ATAC-seq replicates. It was generated using MACS2. The values indicate the statistical significance of enrichment for open chromatin.

extract_protocol:
Chromatin was extracted from 100,000 cells, which were harvested by mechanical dissociation, washed with 50 μL cold 1X PBS, and centrifuged at 300×g for 5 minutes. The pellet was resuspended in 50 μL cold lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin, and nuclease-free H2O) and incubated for 3 minutes on ice. After lysis, 1 mL of wash buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, and nuclease-free H₂O) was added, mixed by gentle inversion, and centrifuged at 500×g for 10 minutes at 4°C. The nuclei pellet was resuspended in 50 μL transposition mix (Tagment DNA TDE1 Enzyme and Buffer, Illumina) and incubated at 37°C for 30 minutes with shaking (1000 rpm). DNA was purified using the Qiagen MinElute Reaction Cleanup Kit and eluted in 11 μL.
Libraries were generated using Nextera XT Index primers (Illumina) and Q5 Hot Start Master Mix (NEB), followed by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 63°C, 1 min at 72°C] ×10 cycles).


title: E001

status: Public on Sep 09 2025

type: SRA

city: Shanghai

first: Tong

last: Fu

state: Shanghai

address: DongAn Road

country: China

institute: Fudan University

description: Library name: E001

library_source: transcriptomic single cell

data_processing:
The FASTQ files were processed and aligned to GRCh38 human reference genome using Cell Ranger software (version 8.0.1) from 10x Genomics, with unique molecular identifier (UMI) counts summarized for each barcode.
Assembly: GRCh38
Supplementary files format and content: Cell Ranger software output files (barcodes.tsv, features.tsv, matrix.mtx)

molecule: polyA RNA

organism: Homo sapiens

source_name: Breast cancer

extract_protocol:
GEM Generation & Barcoding: Prepare Master Mix, mix with cell suspension, and add Gel Beads. Dispense Partitioning Oil into wells and run the Chromium Controller to generate GEMs. GEM-RT Incubation: Store GEMs on ice for up to 1 hour or at 4°C for up to 72 hours, or at −20°C for longer storage.
Post GEM-RT Cleanup & cDNA Amplification: Clean up cDNA using Dynabeads and amplify using cDNA amplification mix. cDNA Cleanup: Clean up the amplified cDNA using SPRIselect reagent. Library Construction: Fragment cDNA, perform end repair and A-tailing, then ligate adaptors. Post Ligation Cleanup: Clean the library using SPRIselect reagent to remove excess adaptors. Sample Index PCR: Perform PCR to add sample indexes for multiplexing. Post PCR Cleanup: Clean the PCR product using SPRIselect reagent.

tag: tissue

value: Breast cancer

tag: Stage

value: early-stage TNBC


title: iTF-Microglia Day 18, untreated, rep2

city: Huntsville

first: Richard

middle: M.

last: Myers

state: AL

address: 601 Genome Way

country: USA

institute: HudsonAlpha Institute for Biotechnology

description: Library name: iTF-Microglia_rep2

library_source: genomic

molecule: genomic DNA

source_name: KOLF2.1J CLYBL_6-TF-iMG_KI1/KI2

growth_protocol: KOLF2.1J CLYBL_6-TF-iMG_KI1/KI2 iPSCs were maintained at 37°C with 5% CO2 in mTeSR Plus medium on dishes coated with Growth Factor Reduced (GFR), LDEV-Free Matrigel. Medium was changed every other day. Cells were passaged at 70–80% confluency using ReLeSR, resuspended in room-temperature mTeSR Plus, and transferred to freshly coated dishes.

treatment_protocol:
KOLF2.1J iTF-iPSCs were washed with D-PBS, dissociated with Accutase, and centrifuged at 300×g for 5 minutes. Cell pellets were resuspended in Essential 8 Basal Medium supplemented with 10 μM Y-27632 ROCK Inhibitor and 2 μg/mL doxycycline, then seeded onto double-coated plates with Poly-D-Lysine-precoated Bio plates and GFR Matrigel. On day 2, cells were switched to differentiation medium (Advanced DMEM/F12 supplemented with 1× GlutaMAX, 2 μg/mL doxycycline, 100 ng/mL human IL-34 and 10 ng/mL human GM-CSF). On day 4, the medium was replaced with iTF-Microglia medium (Advanced DMEM/F12 supplemented with 1× GlutaMAX, 2 μg/mL doxycycline, 100 ng/mL human IL-34, 10 ng/mL human GM-CSF, 50 ng/mL human M-CSF and 50 ng/mL human TGF-β1). On day 8, fresh iTF-Microglia medium was added. Cells were maintained for 9 more days with full medium changes every 2–3 days.
KOLF2.1J iTF-iPSCs were untreated and Day 18 iTF-Microglia were either untreated or stimulated with 100 ng/mL LPS (Invitrogen) and 20 ng/mL IFNG (Gibco) in fresh mTeSR Plus (for iTF-iPSCs) or iTF-Microglia medium (for iTF-Microglia) for 24 hours before harvesting.

tag: cell line

value: KOLF2.1J CLYBL_6-TF-iMG_KI1/KI2

tag: cell type

value: iTF-Microglia Day18

tag: genotype

tag: treatment

value: Untreated


title: iTF-iPSCs, untreated, rep1

description: Library name: iTF-iPSCs_rep1

value: iPSCs


title: iTF-iPSCs, untreated, rep2

description: Library name: iTF-iPSCs_rep2


title: blood, day 40, group 8 #3

city: shimogyo-ku

first: Tomohiro

last: Sensui

address: chudoji, awatacho

country: Japan

institute: Maruho

description: Library name: PR3287_163_b

library_source: transcriptomic

data_processing:
rRNA was filtered using RiboPicker, and mapped to the mouse genome GRCm39 using the DRAGEN Bio-IT Platform (Illumina).
Assembly: GRCm39
Supplementary files format and content: txt file including raw counts for each sample
Supplementary files format and content: txt file including TPM for each sample

molecule: total RNA

organism: Mus musculus

source_name: whole blood

extract_protocol:
Blood samples were collected from the tail vein (approximately 0.1 mL) using EDTA-2K-coated capillary tubes without anesthesia. Total RNA was isolated from blood using a NucleoSpin RNA Blood Kit (Macherey-Nagel, Easton, PA, USA), according to the manufacturer’s instructions.
Libraries were prepared using TruSeq Stranded Total RNA with RiboZero Globin (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.

value: whole blood

tag: strain

value: NC/Nga

tag: Sex

value: male

value: WT

tag: condition

value: conventional

tag: group

value: group 8

tag: dermatitis score

tag: time

value: day 40


title: Thap12h_RNAseq_rep2

first: Peipei

last: Jiang

address: No. 115 Jinzun Road, Pudong New Area, Shanghai

institute: Shanghai Jiao Tong University

description: Library name: Thap12h_RNAseq_rep2

data_processing:
Sequenced reads were trimmed for adaptor sequence using trim_galore with the default parameters
Trimmed sequence reads were mapped to mm10 genome using STAR (v2.5.3a ) with default parameters
All mapped bam files were converted to bigwig using bedtools (v2.31.0) for visualization in IGV
Diferentially expressed genes were analyzed by DEseq2
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

source_name: HeLa

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

extract_protocol:
RNA was harvested using MolPure Cell RNA Kit (YEASEN, 19231ES50)
RNA library for RNA-seq was prepared as fllows: mRNA was purified from total RNA using polyT and then fragmented into 300~350 bp fragments, the first strand cDNA was reverse-transcribed using fragmented RNA and dNTPs (dATP, dTTP, dCTP and dGTP) and second strand cDNA synthesis was subsequently performed. Remaining overhangs of double-strand cDNA were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, sequencing adaptors were ligated to the cDNA and the library fragments were purified. The template was enriched by PCR, and the PCR product was purified to obtain the final library.

value: HeLa

value: Human Cervical Carcinoma Cell

value: CCCP 12h


title: F3, control [F3-rep2]

city: Debrecen

first: Laszlo Attila

last: Papp

state: Hajdú-Bihar Vármegye

address: Egyetem tér 1.

department: Department of Genetics and Applied Microbiology

country: Hungary

institute: University of Debrecen

description:
co-culture_vs_control-fusarium_Moderated T-Test.xlsx
fusarium_count.out.txt

data_processing:
Alignment with HISAT2 aligner
Generation of BAM files
FeatureCount values were generated using Liao Y, Smyth GK and Shi W (2014). featureCounts software.
Quantification and normalization using StrandNGS with DeSeq algorithm
Assembly: In case of co-culture experiment S. pombe ASM294v2 assembly was used.
Assembly: In case of F. verticillioides ASM14955v1 was used.
Supplementary files format and content: in the co-culture experiment: pombe_count.out.txt file contains the tab delimited raw count values of S. pombe control (14) vs S. pombe co-cultured (6-Fv3). co-culture_vs_control-pombe_Moderated T-Test.xlsx file contains the normalized data with statistics (moderated T-test, BH-FDR)
Supplementary files format and content: in the co-culture experiment: fusarium_count file contains the tab delimited raw count values of F. verticillioides control vs F. verticillioides co-cultured (6-Fv3). co-culture_vs_control-fusarium_Moderated T-Test.xlsx file contains the normalized data with statistics (moderated T-test, BH-FDR)
Supplementary files format and content: in the fumonisin B1 experiment: K1_vs_M1_featureCount.out.txt file contains tab delimited raw count values of S. pombe control (K1) vs S. pombe+0.5ppm fumonisin B1 (M1). M1_vs_K1_ModT-Test_with_BH-FDR.xlsx file contains the normalized data with statistics (moderated T-test, BH-FDR)

organism: Fusarium verticillioides

growth_protocol: Control S. pombe cells were grown on YEA (yeast-extract agar) media. Control F. verticillioides were grown on MXGB media. Both strains were cultured at 25°C.

extract_protocol:
RNA were extracted from cells using the method described by Lyne et al. (2003).RNA quality was checked with Agilent BioAnalyzer.
RNA libraries for RNA-seq were prepared using NEBNext Ultra II RNA Library Prep kit for Illumina

treatment_protocol:
The S. pombe cells treated with FB1 (0.5 ppm) for 1hour in MXGB media at 30°C.
In case of co-culturing conditions: 100 µl.of overnight S. pombe cells were inoculated into 150ml MXGB. We excised 5x5 mm of F. verticillioides hyphaes were transferred from another pre-cultured F. verticillioides flask. S. pombe and F. verticillioides were co-cultured for 2 days at 25°C without shaking. Then the cells were separated and submitted to RNA extraction respectively.


title: F3, control [F3-rep1]


title: Thap12h_RNAseq_rep1

description: Library name: Thap12h_RNAseq_rep1


title: blood, day 40, group 8 #2

description: Library name: PR3287_162_b


title: DMSO12h_RNAseq_rep3

description: Library name: DMSO12h_RNAseq_rep3

value: DMSO 12h


title: iTF-Microglia Day 18, LPS 100mg/ml + IFNG 20 ng/ml, 24h, rep2

description: Library name: iTF-Microglia_LPS_IFNG_rep2

value: LPS 100 ng/ml + IFNG 20 ng/ml

value: 24 hours


title: blood, day 40, group 8 #5

description: Library name: PR3287_165_b


title: DMSO12h_RNAseq_rep2

description: Library name: DMSO12h_RNAseq_rep2


title: iTF-Microglia Day 18, LPS 100mg/ml + IFNG 20 ng/ml, 24h, rep1

description: Library name: iTF-Microglia_LPS_IFNG_rep1

value: KOLF2.1J CLYBL_6-TF-iMG_KI1/KI2


title: blood, day 40, group 8 #4

description: Library name: PR3287_164_b


title: inCAGE scRNA-seq 1:1 mix of two cells lines

city: Boston

first: Ignas

last: Mazelis

state: MA

address: 210 Longwood Avenue

department: System Biology

institute: Harvard Medical School

description: Library name: K562 and L1210 run1

data_processing:
BCL sequencing files received from Illumina BaseSpace portal were converted to raw FASTQ files for each of the 4 (R1, R2, R3, R4) sequencing reads without read trimming or masking by using bcl2fastq software (Illumina, v2.20.0.422). Raw FASTQ files were processed using a custom pipeline adapted from inDrops.py (Zilionis et al., 2017). The new code, inCaps.py is available at github.com/AllonKleinLab/. In brief, sequencing reads were demultiplexed based on Read1 index sequence (stored as read3) (index used: CTAGTCGA,AGCTAGAA,ACTCTAGG,TCTTACGC,CTTAATAG,ATAGCCTT,TAAGGCTC,TCGCATAA), and were filtered based on expected structure (all barcodes matching a barcode whitelist with hamming distance of 1). The demultiplexed reads were then processed using Trimmomatic (1) (with parameters: LEADING 28; SLIDINGWINDOW 4:20; MINLEN 20), followed by Poly-A length trim to 4 nt and selecting reads that are longer than MINLEN. For surviving reads, the fraction of length composed out of same base repeats (only considering > 5) was determined, rejecting reads whose fraction is greater than 0.5.
Count matrices were generated using STARsolo pipeline (2) (STAR 2.7.9a) with the with parameters (--soloFeatures Gene, GeneFull, --genomeSAsparseD 3, --outSAMtype BAM SortedByCoordinate, --outSAMattributes CB CR UR UB UY GN GX, --outFilterScoreMinOverLread 0.3, --outFilterMatchNminOverLread 0.3, --soloCBmatchWLtype 1MM_multi_Nbase_pseudocounts, --soloUMIfiltering MultiGeneUMI_CR, --soloUMIdedup 1MM_CR, --soloMultiMappers EM, --soloCellFilter EmptyDrops_CR, --quantMode TranscriptomeSAM GeneCounts) Mixed Human GRCh38.101 and mouse GRCm.101 genome assembly was used.
Count matrices were filtered by selecting barcodes with >3000 UMIs and fraction of mitochondrial counts being below 0.4. To generate UMI saturation curves, random sampling was used to sample barcode-umi-gene combinations from output aligned and sorted bam files to generate a table of number of UMIs and reads for each barcode at different sequencing read depths.
Assembly: Mixed Human GRCh38.101 and mouse GRCm.101 genome assembly
Supplementary files format and content: h5ad cell x genes expression matrix, 2700 cells

organism:
Homo sapiens
Mus musculus

source_name: Blood

growth_protocol: K562 and L1210 cells were cultured in IMDM (Thermo Fisher, 10566016) + 10% FBS (ATCC, 30-2020) + 1x penicillin/streptomycin (Thermo Fisher, 10378016) in T25 flasks (Corning). Cell lines were cultured until they reached ~80-90% confluency. For passaging, suspension cells (K562, L1210) were resuspended gently and collected into a 15 mL tube. 5 mL of 37ºC pre-warmed complete media was used to wash the flask and was added into the same 15 mL tube. Cells were centrifuged at 300g for 4 min, followed by media aspiration and resuspension in 5 mL of fresh pre-warmed complete media. Cells were counted using an automatic cell counter and 500,000 cells were seeded into 5 mL of 37ºC pre-warmed complete media in a 25cm2 flask.

extract_protocol:
Cells were washed twice with ice cold DPBS with 0.1% BSA with 0.1% BSA and were encapsulated into CAGEs at a final concentration of 1.0e6 cells/mL in core mix. Next, CAGE core dextran was removed using dextranase enzyme. Following this, all steps were performed on ice. First, capsules were washed three times with an ice-cold 3X SSCI buffer with 800g spins for 1 min. Cells were lysed by washing with ice-cold 7.5pH TEIS with 5 min incubations on ice. After lysis, capsules were imaged (check above), washed three times with 3X SSCI and three times with HI10, packed at 800g for 1 min and stored on ice. Reverse-transcription reaction was set on ice and performed in PCR strip tubes. A standard 100μL reaction was composed out of 60.5μL of packed capsules, 2.5μL of 10μM RT poly(T) primer housing a 10nt UMI and 6nt barcode (Table S3, RT), 3μL of 10% Igepal CA-630 (Millipore Sigma, I8896-50ML), 20μL of 5X SMARTseq3 RT buffer (100mM Tris pH 8.3, 150mM NaCl, 12.5mM MgCl2, 5mM GTP, 40mM DTT), 0.5μL of 10mM dNTPs (NEB, N0447L), 2.5μL of 40U/μL Recombinant RNase Inhibitor (Takara Bio, 2313A), 1.5μL of 50μM TSO, 5μL of Maxima H- reverse transcriptase (ThermoFisher, EP0753). Reaction volumes were scaled according to the amount of capsules used and number of reactions performed. To set up the reaction, capsules, RT primer and Igepal CA-630 (Millipore Sigma, I8896-50ML) were mixed on ice in a PCR tube, with each housing a different RT barcode. Capsules were incubated on ice for 10 min to allow the RT primer to diffuse in, followed by heating at 70ºC for 5 min and cooling down on ice. Next, RT master mix, composed out of the remaining reagents was added to each tube, ensuring a thought mixing of capsules with pipetting. Tubes were incubated on ice for 5 min and the reaction was carried out at 42ºC for 90 min, followed by cycling between 50ºC and 42ºC incubation of 2 mins for 15 cycles. Reactions were left at room temperature overnight and processed the next day. After the RT reaction, capsules were washed three times with TI100 (pH 8.0), twice with TIbS (pH 8.0), three times with ice-cold TI100 (pH 8.0) with 2 min incubations on ice. Finally capsules were washed twice with TI10 (pH 8.0). Next, Capsule dU PCR was performed to carry out cDNA amplification. After the cDNA amplification, capsules were washed three times with TI100 (pH 8.0), twice with TIbS (pH 8.0), three times with TI100 (pH 8.0), twice with TI10 (pH 8.0). Washed capsules were subjected to tree rounds of split-and-pool barcoding. Following split-and-pool barcoding, capsules were split into 8 tubes and were dissolved  using 10M NaOH. DNA was purified using MinElute PCR purification kit and barcoded cDNA was eluted into 20μL of the elution buffer. Barcoded and purified DNA was amplified using PCR. After the reaction, amplified barcoded cDNA was purified using 0.8X AMPure XP beads (Beckman Coulter, A63881) and was eluted into a 15μL Elution Buffer.
To construct Illumina sequencing libraries, 50 ng of barcoded and amplified cDNA was fragmented, blunted and dA-tailed by mixing it with 3.5 μL of NEBNext Ultra II FS Reaction buffer (NEB), 1 μL of fully mixed NEBNext Ultra II FS Enzyme mix (NEB) and nuclease free water to a final volume of 17.5 μL. The reaction mix was vortexed for 10 s, spanned down and placed into a preheated PCR machine for incubation at 37°C for 10 min; 65°C for 30 min; hold 4°C (lid at 75ºC). Fragmented DNA was purified using 0.8X AMPure XP beads (Beckman Coulter, A63881) and eluted into 16μL of water. Single-stranded DNA adapter, containing Illumina’s TruSeq read 1 (Adp. TruSeq R1 FW and RV) sequences were annealed together at the final concentration of 1.5 μM by mixing 1.5 μL of forward ligation primer and reverse ligation primer (each at 100 μM) with 97 μL of nuclease-free water and heating the solution to 95°C for 2 min and cooling it at room temperature for 5 min. 16 μL of the purified fragmented DNA was mixed with 1.25 μL of 1.5 μM annealed adapters, 1.5μL of 10X T4 DNA ligase buffer, 15 μL of NEBNext Ultra II Ligation Master Mix (NEB, E7805S kit) and 0.5 μL of NEBNext Ultra II Ligation Master Enhancer (NEB, E7805S kit). The ligation reaction was carried out by incubating in the thermocycler for 15 min at 20°C (lid at 30ºC). After ligation, the reaction mix was diluted to 100 μL and was purified using AMPure XP beads (Beckman Coulter, A63881) with 0.8X volume ratio and eluted into 40 μL nuclease-free water. Indexing PCR was carried out by mixing purified DNA with 25 μL of 2x NEBNext® High-Fidelity 2X PCR Master Mix (NEB, M0543L) and 2.5 μL of 20μM P7 binding primer, 8 sets of 2.5μL of 20μM indexing primer that houses an 8bp library index and water to 50μL. PCR cycling program: 98 °C, 2 min; 3 cycles of 98 °C, 20 s; 55 °C, 30 s; 72 °C, 20 s and 9 cycles 98 °C, 20 s; 65 °C, 30 s; 72 °C, 20 s; with a 1 min final extension at 72 °C, and 4 °C, hold at the end. Following final library indexing, DNA was double-side purified using 0.6x-0.8x SPRIselect (Beckman Coulter, B23318) magnetic beads and eluted into 15μL

value: Blood

value: Lymphoblast


title: KO Tumor_2

city: Guangzhou

first: Liu

middle: chu

last: wei

state: Guangdong province

address: 58 ZhongshanSecond Road, Yuexiu District,

institute: The First Affiliated Hospital of Sun Yat-sen University

description:
Library name: SAMPLE4
10×Genomics

data_processing:
The raw expression matrix was processed using the Seurat 5.1.0 R package, applying the following quality control criteria: 1) Cells with fewer than 500 unique molecular identifiers (UMIs) were excluded. 2) Cells expressing fewer than 300 or more than 7,000 genes were removed. 3) Cells with mitochondrial gene counts exceeding 10% of total counts were excluded. 4) Genes detected in fewer than three cells were omitted. After filtering, a dataset comprising 40,586 cells and 29,019 genes was retained for analysis.
Assembly: Mouse mm10
Supplementary files format and content: tab-delimited text files include count values for each Sample

source_name: Tumor tissue

extract_protocol:
For sc-RNA sequence, tumors were excised and minced into 1–2 mm fragments in a sterile petri dish.  The tissue fragments were digested in a buffer containing 10 mM HEPES, 1.25 mg/mL collagenase D, 0.85 mg/mL collagenase V, 30 U/mL DNase I, 1 mg/mL dispase II, and 10% fetal bovine serum (FBS) in Hank’s Balanced Salt Solution (HBSS). Digestion was carried out on a rotator at 37°C for 45 minutes. Post-digestion, the cell suspension was filtered sequentially through 70 µm and 40 µm cell strainers (BD) to remove debris and large aggregates. The filtered cells were centrifuged at 600×g for 5 minutes at 4°C to pellet the single-cell suspension. The resulting suspension was used for single-cell RNA-sequencing.
Gene expression libraries were aligned to the mm10 genome using Cell Ranger 5.0.1 (10X Genomics) with default parameters to generate gene-cell count matrices.

treatment_protocol: Eight-week-old C57BL/6J mice were inoculated with Hnrnpa2b1 KO and WT MC38 cells (2× 106 cells per mouse) via subcutaneous injection into the left flank. For sc-RNA sequence, tumors were excised and minced into 1–2 mm fragments in a sterile petri dish.  The tissue fragments were digested in a buffer containing 10 mM HEPES, 1.25 mg/mL collagenase D, 0.85 mg/mL collagenase V, 30 U/mL DNase I, 1 mg/mL dispase II, and 10% fetal bovine serum (FBS) in Hank’s Balanced Salt Solution (HBSS). Digestion was carried out on a rotator at 37°C for 45 minutes. Post-digestion, the cell suspension was filtered sequentially through 70 µm and 40 µm cell strainers (BD) to remove debris and large aggregates. The filtered cells were centrifuged at 600×g for 5 minutes at 4°C to pellet the single-cell suspension. The resulting suspension was used for single-cell RNA-sequencing.

value: Tumor tissue


title: blood, day 26, group 8 #8

description: Library name: PR3287_158_b

value: whole blood

value: day 26


title: inCAGE scRNA-seq of NIK/3t3 cells encapsulated in IMDM media with dextranase trated CAGEs

description: Library name: 3t3_Media_dextranase

data_processing:
BCL sequencing files received from Illumina BaseSpace portal were converted to raw FASTQ files for each of the 4 (R1, R2, R3, R4) sequencing reads without read trimming or masking by using bcl2fastq software (Illumina, v2.20.0.422). Raw FASTQ files were processed using a custom pipeline (available at https://github.com/AllonKleinLab/paper-data/tree/master/Mazelis2025_CAGEs). In brief, sequencing reads were demultiplexed based on Read1 index sequence (stored as read3) (index used: GAGAATTG, GTGGATGG), and were filtered based on expected structure (all barcodes matching a barcode whitelist with hamming distance of 1). The demultiplexed reads were then processed using Trimmomatic (1) (with parameters: LEADING 28; SLIDINGWINDOW 4:20; MINLEN 20), followed by Poly-A length trim to 4 nt and selecting reads that are longer than MINLEN. For surviving reads, the fraction of length composed out of same base repeats (only considering > 5) was determined, rejecting reads whose fraction is greater than 0.5.
Count matrices were generated using STARsolo pipeline (2) (STAR 2.7.9a) with the with parameters (--soloFeatures Gene, GeneFull, --genomeSAsparseD 3, --outSAMtype BAM SortedByCoordinate, --outSAMattributes CB CR UR UB UY GN GX, --outFilterScoreMinOverLread 0.3, --outFilterMatchNminOverLread 0.3, --soloCBmatchWLtype 1MM_multi_Nbase_pseudocounts, --soloUMIfiltering MultiGeneUMI_CR, --soloUMIdedup 1MM_CR, --soloMultiMappers EM, --soloCellFilter EmptyDrops_CR, --quantMode TranscriptomeSAM GeneCounts) Mixed Human GRCh38.101 and mouse GRCm.101 genome assembly was used.
Count matrices were filtered by selecting barcodes with >1000 UMIs and fraction of mitochondrial counts being below 0.4. Mouse and human cells were filered based on gene expression and different conditions were demultiplexed based on RT barcode
Assembly: Mixed Human GRCh38.101 and mouse GRCm.101 genome assembly
Supplementary files format and content: h5ad cell x genes expression matrix for different conditions

source_name: mouse embryonic fibroblast

growth_protocol: HEK293t and NIH/3t3 cells were cultured in RPMI1640 (Thermo Fisher, 61870036) + 10% FBS (ATCC, 30-2020) + 1x penicillin/streptomycin (Thermo Fisher, 10378016) in T25 flasks (Corning). Cell lines were cultured until they reached ~80-90% confluency. For passaging cells were passaged by aspirating the used media and washing 2 times with 2 mL of 37ºC pre-warmed 1x DPBS buffer. Cells were detached by adding 1 mL of TrypLE reagent (Thermofisher Scientific, 12605010), waiting 10s and quickly aspirating all of the TrypLE. Cells were incubated in 37ºC for 2 minutes and were resuspended in 5 mL of 37ºC pre-warmed complete media. Cells were centrifuged at 300g for 4 min, followed by media aspiration and resuspension in 5 mL of fresh pre-warmed complete media. Cells were counted using an automatic cell counter and 500,000 cells were seeded into 5 mL of 37ºC pre-warmed complete media in a 25cm2 flask.

extract_protocol:
Cells were washed twice with IMDM or DPBS with 0.1% BSA, mixed at 1:1 ration and were encapsulated into CAGEs at a final concentration of 1.4e6 cells/mL in core mix. Next, for some samples the CAGE core dextran was removed using dextranase enzyme. Following this, all steps were performed on ice. First, capsules were washed three times with an ice-cold 3X SSCI buffer with 800g spins for 1 min. Cells were lysed by washing with ice-cold 7.5pH TEIS with 5 min incubations on ice. After lysis, capsules were imaged, washed three times with 3X SSCI and three times with HI10, packed at 800g for 1 min and stored on ice. Part of the sample was frozen at -80ºC in 10% DMSO. Reverse-transcription reaction was set on ice and performed in PCR strip tubes. A standard 100μL reaction was composed out of 60.5μL of packed capsules, 2.5μL of 10μM RT poly(T) primer housing a 10nt UMI and 6nt barcode (Table S3, RT), 3μL of 10% Igepal CA-630 (Millipore Sigma, I8896-50ML), 20μL of 5X SMARTseq3 RT buffer (100mM Tris pH 8.3, 150mM NaCl, 12.5mM MgCl2, 5mM GTP, 40mM DTT), 0.5μL of 10mM dNTPs (NEB, N0447L), 2.5μL of 40U/μL Recombinant RNase Inhibitor (Takara Bio, 2313A), 1.5μL of 50μM TSO, 5μL of Maxima H- reverse transcriptase (ThermoFisher, EP0753). Reaction volumes were scaled according to the amount of capsules used and number of reactions performed. To set up the reaction, capsules, RT primer and Igepal CA-630 (Millipore Sigma, I8896-50ML) were mixed on ice in a PCR tube, with each housing a different RT barcode. Capsules were incubated on ice for 10 min to allow the RT primer to diffuse in, followed by heating at 70ºC for 5 min and cooling down on ice. Next, RT master mix, composed out of the remaining reagents was added to each tube, ensuring a thought mixing of capsules with pipetting. Tubes were incubated on ice for 5 min and the reaction was carried out at 42ºC for 90 min, followed by cycling between 50ºC and 42ºC incubation of 2 mins for 15 cycles. Reactions were left at room temperature overnight and processed the next day. After the RT reaction, capsules were washed three times with TI100 (pH 8.0), twice with TIbS (pH 8.0), three times with ice-cold TI100 (pH 8.0) with 2 min incubations on ice. Finally capsules were washed twice with TI10 (pH 8.0). Next, Capsule dU PCR was performed to carry out cDNA amplification. After the cDNA amplification, capsules were washed three times with TI100 (pH 8.0), twice with TIbS (pH 8.0), three times with TI100 (pH 8.0), twice with TI10 (pH 8.0). Washed capsules were subjected to tree rounds of split-and-pool barcoding. Following split-and-pool barcoding, capsules were split into 8 tubes and were dissolved  using 10M NaOH. DNA was purified using MinElute PCR purification kit and barcoded cDNA was eluted into 20μL of the elution buffer. Barcoded and purified DNA was amplified using PCR. After the reaction, amplified barcoded cDNA was purified using 0.8X AMPure XP beads (Beckman Coulter, A63881) and was eluted into a 15μL Elution Buffer.
To construct Illumina sequencing libraries, 50 ng of barcoded and amplified cDNA was fragmented, blunted and dA-tailed by mixing it with 3.5 μL of NEBNext Ultra II FS Reaction buffer (NEB), 1 μL of fully mixed NEBNext Ultra II FS Enzyme mix (NEB) and nuclease free water to a final volume of 17.5 μL. The reaction mix was vortexed for 10 s, spanned down and placed into a preheated PCR machine for incubation at 37°C for 10 min; 65°C for 30 min; hold 4°C (lid at 75ºC). Fragmented DNA was purified using 0.8X AMPure XP beads (Beckman Coulter, A63881) and eluted into 16μL of water. Single-stranded DNA adapter, containing Illumina’s TruSeq read 1 (Adp. TruSeq R1 FW and RV) sequences were annealed together at the final concentration of 1.5 μM by mixing 1.5 μL of forward ligation primer and reverse ligation primer (each at 100 μM) with 97 μL of nuclease-free water and heating the solution to 95°C for 2 min and cooling it at room temperature for 5 min. 16 μL of the purified fragmented DNA was mixed with 1.25 μL of 1.5 μM annealed adapters, 1.5μL of 10X T4 DNA ligase buffer, 15 μL of NEBNext Ultra II Ligation Master Mix (NEB, E7805S kit) and 0.5 μL of NEBNext Ultra II Ligation Master Enhancer (NEB, E7805S kit). The ligation reaction was carried out by incubating in the thermocycler for 15 min at 20°C (lid at 30ºC). After ligation, the reaction mix was diluted to 100 μL and was purified using AMPure XP beads (Beckman Coulter, A63881) with 0.8X volume ratio and eluted into 40 μL nuclease-free water. Indexing PCR was carried out by mixing purified DNA with 25 μL of 2x NEBNext® High-Fidelity 2X PCR Master Mix (NEB, M0543L) and 2.5 μL of 20μM P7 binding primer, 8 sets of 2.5μL of 20μM indexing primer that houses an 8bp library index and water to 50μL. PCR cycling program: 98 °C, 2 min; 3 cycles of 98 °C, 20 s; 55 °C, 30 s; 72 °C, 20 s and 9 cycles 98 °C, 20 s; 65 °C, 30 s; 72 °C, 20 s; with a 1 min final extension at 72 °C, and 4 °C, hold at the end. Following final library indexing, DNA was double-side purified using 0.6x-0.8x SPRIselect (Beckman Coulter, B23318) magnetic beads and eluted into 15μL.

value: mouse embryonic fibroblast

value: embryonic fibroblast


title: inCAGE scRNA-seq of NIK/3t3 cells encapsulated in DPBS buffers with dextranase trated CAGEs

description: Library name: 3t3_DBPS_dextranase


title: blood, day 26, group 8 #9

description: Library name: PR3287_159_b

value: NC/Nga

value: male

value: WT

value: conventional


title: KO Tumor_1

description:
Library name: SAMPLE3
10×Genomics


title: Thap6h_RNAseq_rep3

description: Library name: Thap6h_RNAseq_rep3

value: HeLa

value: Human Cervical Carcinoma Cell

value: CCCP 6h


title: WT Tumor_2

description:
Library name: SAMPLE2
10×Genomics


title: inCAGE scRNA-seq of NIK/3t3 cells encapsulated in DPBS buffers

description: Library name: 3t3_DBPS


title: inCAGE scRNA-seq of NIK/3t3 cells encapsulated in DPBS buffers and cryopreserved for a week

description: Library name: 3t3_frozen


title: WT Tumor_1

description:
Library name: SAMPLE1
10×Genomics


title: Thap6h_RNAseq_rep2

description: Library name: Thap6h_RNAseq_rep2


title: Old, replicate 4, scRNAseq

city: Vienna

first: Sergey

last: Isaev

address: Spitalgasse, 4

department: Department of Neuroimmunology

country: Austria

institute: Medical University of Vienna

description: 10x 3’ (v3)

data_processing:
Single-cell RNA sequencing data was processed using the CellRanger suite (v. 6.1.2). For human samples, the GRCh38 reference (GENCODE v32/Ensembl 98) was used. Mouse samples were aligned to mm10 (GENCODE vM23/Ensembl 98) reference. Standard CellRanger filtration strategy was used for empty droplet identification. Additionally, cells with more than 10% mitochondrial UMI counts and 15,000 total UMIs (25,000 for samples that were obtained using 10x v3 chemistry) were also removed (fig. S1C). Doublet detection was performed with Scrublet (v. 0.2.2) [PMID: 30954476].
Assembly: Mouse: mm10 / Human: GRCh38
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Thymus

extract_protocol:
All human tissue specimens were collected with institutional review board (IRB) approval. The tissue was processed immediately upon isolation to ensure highest possible cell quality. Murine samples were cut into fine pieces and digested in Medium 199 (M199, Gibco) with 2% (v/v) fetal bovine serum (FBS, Gibco), Liberase (0.5WU/ml, Roche) and DNAse I (0.1 KU, ThermoFisher) 3 x 15 minutes at 37°C under constant agitation. Human samples were processed by digestion with M199 with 2% FBS, DNAse I (0.1 KU) and 2 mg/ml Stemxyme 1 (Worthington) for 2 x 30 minutes at 37°C under constant agitation. For the last 30 minutes the samples were digested with the Stemxyme/DNAse I cocktail in combination with 0.125% Trypsin (Gibco). All samples were digested in the presence of RNase inhibitors (RNasin (Promega) and RNase OUT (Invitrogen). Sorted thymus stromal cells were encapsulated into emulsion droplets using the Chromium Controller (10X Genomics). scRNA sequencing libraries were subsequently prepared using Chromium Single Cell 3 v2 and v3 Reagent kits (10X Genomics).
Sorted thymus stromal cells were encapsulated into emulsion droplets using the Chromium Controller (10X Genomics). scRNA sequencing libraries were subsequently prepared using Chromium Single Cell 3 v2 and v3 Reagent kits (10X Genomics).

value: Thymus

tag: age

value: 96 weeks old

value: Female


title: blood, day 26, group 8 #6

description: Library name: PR3287_156_b

value: group 8


title: blood, day 40, group 8 #1

description: Library name: PR3287_161_b

value: day 40


title: Thap6h_RNAseq_rep1

description: Library name: Thap6h_RNAseq_rep1


title: blood, day 26, group 8 #10

description: Library name: PR3287_160_b


title: 72_V_3

city: Taipei City

first: Ting-Fen

last: Tsai

address: No. 155, Sec. 2, Linong St. Beitou Dist.,

department: Department of Life Sciences and Institute of Genome Sciences

country: Taiwan

institute: National Yang Ming Chiao Tung University

description:
Library name: V_72h_rep3
Vehicle heart, 72h rep3

data_processing:
Reads were aligned to the Mus musculus reference genome (GRCm38), and expected counts were generated by the core facility. Differential expression analysis was performed using DESeq2.
CLC Genomics Workbench. CLC Genomics Workbench, for analyzing and visualizing next generation sequencing data, incorporates cutting-edge technology and algorithms, while also supporting and integrating with the rest of your typical NGS workflow. (https://www.qiagenbioinformatics.com/products/clc-genomics-workbench/)
Raw sequencing and quality control. Raw sequencing data were generated by the sequencer in FASTQ format, containing nucleotide sequences and corresponding quality scores.
Read trimming. Reads were trimmed by removing the first 12 bases at the 5′ end (to eliminate low-quality bases and potential random hexamer sequences introduced during reverse transcription), bases with quality scores < Q20, reads shorter than 15 bp, and adapter sequences.
RNA-seq analysis. Filtered sequencing reads were used for gene expression analysis. rRNA mapping. Reads were also mapped to ribosomal RNA to evaluate rRNA content. The rRNA reference database was derived from the SILVA high-quality ribosomal RNA databases, release 123.1, specifically using the SILVA_123.1_LSURef_tax_silva_trunc and SILVA_123.1_SSURef_tax_silva_trunc bacterial subsets (http://www.arb-silva.de).
Assembly: GRCm38 (mm10)
Supplementary files format and content: Tab-delimited text file. Rows represent Ensembl gene IDs, columns represent individual samples, values are raw expected counts per gene generated by RSEM.

source_name: heart

growth_protocol: Mice were maintained under standard conditions with free access to food and water

extract_protocol:
Total RNA was isolated from cardiac tissues using TRI Reagent (Sigma) and phenol/chloroform extraction. RNA integrity was assessed using an Agilent 2100 Bioanalyzer, and only samples with RIN > 8 were used.
Libraries were prepared from total RNA using poly(A)-selected mRNA. After fragmentation, cDNA synthesis and adapter ligation were performed, followed by PCR amplification, according to standard Illumina protocols

treatment_protocol: Mice were treated with doxorubicin (25 mg/kg) or hesperitin (10 mg/kg/day), and cardiac tissues were harvested at 16 and 72 hours after treatment.

value: heart

tag: time point

value: vehicle

tag: batch


title: blood, day 26, group 8 #5

description: Library name: PR3287_155_b

value: whole blood

value: day 26


title: inCAGE scRNA-seq of NIK/3t3 cells encapsulated in IMDM media with dextranase trated CAGEs, replicate

description: Library name: 3t3_Media_dextranase_rep

value: mouse embryonic fibroblast

value: embryonic fibroblast


title: 72_V_2

description:
Library name: V_72h_rep2
Vehicle heart, 72h rep2


title: Old, replicate 3, scRNAseq

description: 10x 3’ (v3)


title: blood, day 26, group 8 #4

description: Library name: PR3287_154_b

value: NC/Nga

value: male


title: 72_V_1

description:
Library name: V_72h_rep1
Vehicle heart, 72h rep1

value: WT


title: Old, replicate 2, scRNAseq

description: 10x 3’ (v3)

value: Male


title: DMSO12h_RNAseq_rep1

description: Library name: DMSO12h_RNAseq_rep1

value: HeLa

value: Human Cervical Carcinoma Cell

value: DMSO 12h


title: Old, replicate 1, scRNAseq

description: 10x 3’ (v3)

value: Thymus

value: 96 weeks old


title: DMSO6h_RNAseq_rep2

description: Library name: DMSO6h_RNAseq_rep2

value: DMSO 6h


title: blood, day 26, group 8 #3

description: Library name: PR3287_153_b

value: conventional

value: group 8


title: DMSO6h_RNAseq_rep3

description: Library name: DMSO6h_RNAseq_rep3


title: blood, day 26, group 8 #2

description: Library name: PR3287_152_b


title: 72_H_3

description:
Library name: HES_72h_rep3
Hesperitin-treated heart, 72h rep3

value: heart

value: hsperitin


title: HeLa_CCCP6h_CEBPG_2

description: Library name: HeLa_CCCP6h_CEBPG_2

data_processing:
The raw reads were trimmed by Trim Galore (v0.6.10)
Trimmed reads were mapped to the mouse transcriptome (mm10) by Bowtie2
CUT&Tag peaks were identified using MACS2 (v2.2.7.1, -f BAMPE) with p<0.05, and using IgG as control.  Pronounced ATF4 peaks were then filter with the summit>100
Assembly: hg38
Supplementary files format and content: bigWig
Library strategy: CUT&Tag

growth_protocol: HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% C02 at 37℃

extract_protocol:
The CUT&Tag assay was performed using Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme #TD903)
The CUT&Tag assay was performed using Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme #TD903)

treatment_protocol: HeLa cells were treated with 10μM DMSO and 10μM CCCP respectively for 6h.

value: CCCP 6h


title: IL7R KO, replicate 4, scRNAseq

description: 10x 3’ (v2)

value: IL7R knockout

value: 8 weeks old


title: blood, day 26, group 8 #1

description: Library name: PR3287_151_b

value: whole blood

value: NC/Nga

value: day 26


title: IL7R KO, replicate 3, scRNAseq

description: 10x 3’ (v2)

value: Female


title: blood, day 26, group 8 #7

description: Library name: PR3287_157_b

value: male

value: WT


title: HeLa_CCCP6h_CEBPG_1

description: Library name: HeLa_CCCP6h_CEBPG_1

value: HeLa

value: Human Cervical Carcinoma Cell


title: DMSO6h_RNAseq_rep1

description: Library name: DMSO6h_RNAseq_rep1


title: 72_H_2

description:
Library name: HES_72h_rep2
Hesperitin-treated heart, 72h rep2


title: 72_H_1

description:
Library name: HES_72h_rep1
Hesperitin-treated heart, 72h rep1


title: blood, day 44, group 7 #10

description: Library name: PR3287_150_b

value: group 7

value: day 44


title: 72_DOXH_3

description:
Library name: DOXH_72h_rep3
DOX plus hes-treated heart, 72h replicate 3

value: doxorubicin plus hsperitin


title: HeLa_DMSO6h_CEBPG_2

description: Library name: HeLa_DMSO6h_CEBPG_2

value: DMSO 6h


title: IL7R KO, replicate 1, scRNAseq

description: 10x 3’ (v2)

value: Thymus


title: blood, day 44, group 7 #9

description: Library name: PR3287_149_b

value: conventional


title: HeLa_DMSO6h_CEBPG_1

description: Library name: HeLa_DMSO6h_CEBPG_1


title: HeLa_CCCP6h_H3K27Ac_2

description: Library name: HeLa_CCCP6h_H3K27Ac_2

value: CCCP 6h


title: blood, day 44, group 7 #8

description: Library name: PR3287_148_b


title: 72_DOXH_1

description:
Library name: DOXH_72h_rep1
DOX plus hes-treated heart, 72h replicate 1

value: heart


title: Transplantation, replicate 2, scRNAseq

description: 10x 3’ (v2)

value: 8 weeks old

value: Male


title: Transplantation, replicate 3, scRNAseq

description: 10x 3’ (v2)


title: 72_DOX_3

description:
Library name: DOX_72h_rep3
DOX-treated heart, 72h replicate 3

value: doxorubicin


title: 72_DOX_2

description:
Library name: DOX_72h_rep2
DOX-treated heart, 72h replicate 2


title: blood, day 44, group 7 #6

description: Library name: PR3287_146_b

value: whole blood

value: NC/Nga


title: HeLa_CCCP6h_H3K27Ac_1

description: Library name: HeLa_CCCP6h_H3K27Ac_1


title: blood, day 44, group 7 #7

description: Library name: PR3287_147_b

value: male


title: Transplantation, replicate 1, scRNAseq

description: 10x 3’ (v2)

value: WT

value: Female


title: 72_DOX_1

description:
Library name: DOX_72h_rep1
DOX-treated heart, 72h replicate 1


title: blood, day 44, group 7 #5

description: Library name: PR3287_145_b


title: HeLa_DMSO6h_H3K27Ac_1

description: Library name: HeLa_DMSO6h_H3K27Ac_1

value: HeLa

value: Human Cervical Carcinoma Cell


title: HeLa_IgG_2

description: Library name: HeLa_IgG_2


title: blood, day 44, group 7 #4

description: Library name: PR3287_144_b

value: group 7

value: day 44


title: blood, day 44, group 7 #3

description: Library name: PR3287_143_b


title: Control, replicate 2, scRNAseq

description: 10x 3’ (v2)


title: inC-ATAC-seq of a 2:3:3 mixutre of K562, L1210 and L929 cells to benchmark inC-seq split-and-pool barcoding

description: Library name: K562_L1210_L929_inC-ATACseq

library_source: genomic single cell

data_processing:
BCL sequencing files received from Illumina BaseSpace portal were converted to raw FASTQ files for each of the 4 (R1, R2, R3, R4) sequencing reads without read trimming or masking by using bcl2fastq software (Illumina, v2.20.0.422). Raw FASTQ files were processed using a custom pipeline (available at https://github.com/AllonKleinLab/paper-data/tree/master/Mazelis2025_CAGEs). In brief, sequencing reads were demultiplexed based on Read1 index sequence (stored as read3) (index used: TACTCCTT, AGGCTTAG, ATTAGACG, CGGAGAGA), and were filtered based on expected structure (all barcodes matching a barcode whitelist with hamming distance of 1). The demultiplexed reads were then processed using Trimmomatic (1) (with parameters: LEADING 28; SLIDINGWINDOW 4:20; MINLEN 20), followed by Poly-A length trim to 4 nt and selecting reads that are longer than MINLEN. For surviving reads, the fraction of length composed out of same base repeats (only considering > 5) was determined, rejecting reads whose fraction is greater than 0.5. CAGE ATAC-seq approach used pair-end sequencing to map out gDNA fragment length. While Read1 sequencing reads contain only the gDNA fragment, Read4 contains CAGE barcodes followed by a gDNA fragment. First, parts of Read4 sequencing reads corresponding to gDNA fragments were extracted using seqtk trimfq command with a parameter -b 50. Sequencing reads were then processed as described above to generate demultiplexed and filtered  FASTQ files twice, once for each gDNA read (Read1 and Read4). Next, fastq-pair was used to pair the two demultiplexed and filtered gDNA fragment FASTQ files, which were then merged using NGmerge. Merged FASTQ files were aligned to merged human GRCh38.101 and mouse GRCm.101 index using bowtie2 (with parameter --very-sensitive, -p 4 –local -X 2000) and bam files were sorted using samtools sort function. Mitochondrial reads were counted and removed, followed by BAM file deduplication, sorting and indexing using samtools. Next a pysam script was used to move barcode sequences from BAM read headers to bam CB tag and a string tag was appended to each barcode to label runs corresponding to different indices. Following this, bam files were merged for all the runs.
BAM files were analyzed using the SnapATAC2 python package. First, BAM files were converted into fragments files using snapatac2.pp.make_fragment_file which were filtered (TSSE >5; fragment number >400; FRiP>0.4) after metric calculation (snapatac2.metrics.tsse, snapatac2.tl.macs3, snapatac2.metrics.frip). UMAPs were generated by selecting 50000 features, removing doublet using scrublet. Gene activity matrices were generated using snapatac2.pp.make_gene_matrix.
Assembly: Mixed Human GRCh38.101 and mouse GRCm.101 genome assembly
Supplementary files format and content: h5ad cell x fragments matrix
Supplementary files format and content: h5ad cell x gene activity matrix

source_name: Human and mouse Blood, connective mouse tissue

growth_protocol: K562 and L1210 cells were cultured in IMDM (Thermo Fisher, 10566016) + 10% FBS (ATCC, 30-2020) + 1x penicillin/streptomycin (Thermo Fisher, 10378016) in T25 flasks (Corning). L929 cells were cultured in EMEM (ATCC, 30-2003) + 10% horse serum (Thermo Fisher, A5669502) + 1x penicillin/streptomycin (Thermo Fisher, 10378016) in T25 flasks (Corning). Cell lines were cultured until they reached ~80-90% confluency. For passaging, suspension cells (K562, L1210) were resuspended gently and collected into a 15 mL tube. 5 mL of 37ºC pre-warmed complete media was used to wash the flask and was added into the same 15 mL tube. Adherent cells (L929) cells were passaged by aspirating the used media and washing 2 times with 2 mL of 37ºC pre-warmed 1x DPBS buffer. Cells were detached by adding 1 mL of TrypLE reagent (Thermofisher Scientific, 12605010), waiting 10s and quickly aspirating all of the TrypLE. Cells were incubated in 37ºC for 2 minutes and were resuspended in 5 mL of 37ºC pre-warmed complete media. Cells were centrifuged at 300g for 4 min, followed by media aspiration and resuspension in 5 mL of fresh pre-warmed complete media. Cells were counted using an automatic cell counter and 500,000 cells were seeded into 5 mL of 37ºC pre-warmed complete media in a 25cm2 flask. For encapsulation experiments, cells were washed twice with ice cold DPBS with 0.1% BSA

extract_protocol:
Cells were washed twice with DPBS with 0.1% BSA, mixed at 2:3:3 (K562:L1210:L929) ratio and were encapsulated into CAGEs at a final concentration of 1.2e6 cells/mL in core mix. Next, for some samples the CAGE core dextran was removed using dextranase enzyme. Following this, all steps were performed on ice. To perform gDNA tagmentation, CAGEs were washed with DPBS with 0.1% (w/v) F127 and 0.1% L31 (w/v). For a 25μL reaction, 9μL of package CAGEs were mixed with 12.5μL of 2x TD buffer (20 mM Tris 10 mM MgCl2; 20% (vol/vol) DMF, pH 7.6), 1μL of loaded Tn5, 2.5μL of 10X Lysis additive (5% Tween20, 0.05% Digitonin in DPBS). The solution was incubated on ice for 5 min, followed by incubation at 37ºC for 30 min. The CAGEs were distributed into six wells for gDNA tagmentation in 40μL reactions. After the reaction, CAGEs were washed three times with TEIS (pH 7.5) and then Proteinase K treated in 200μL of TEIS (pH 7.5) with 10μL of Proteinase K (NEB, P8107S) for 15 min at 55ºC. After the reaction, CAGEs were washed three times with TI100 (pH 7.5) and twice with TI (pH 7.5), six times with an ice-cold 3X SSCI buffer with 800g spins for 1 min and three times with HI10, packed at 800g for 1 min and stored on ice. CAGEs were also then subjected to a reverse-transcription reaction. To this end, the reaction was prepared on ice and performed in 6 PCR strip tubes as described above in CAGE reverse-transcription reaction followed by post reverse-transcription wash steps. Next, the CAGEs were split into four wells for a CAGE dU PCR reaction, using using 2.5μL of 20μM Read1_Biotin FW, 2.5μL of 20μM TSO FW, 5μL of 10μM dU universal RV primer with 25μL of packed CAGEs in 50μL reactions. The reaction was carried out by first incubating the tubes on ice for 5 mins, followed by a PCR cycling program: 98ºC for 30s; 6 cycles of 98ºC for 15s, 68ºC for 20s, 72ºC for 4 min; a final extension of 72ºC for 2min and hold at 25ºC. Following this, CAGEs were subjected to 3 round of 48x48x48 split-and-pool barcoding. After barocding CAGEs were dissolved into 4 1.5mL tubes using NaOH. DNA was purified using MinElute PCR purification kit and barcoded DNA was eluted into 20μL of the elution buffer. Barcoded and purified DNA was amplified using PCR using 1.25μL of 20μM Read1_Biotin FW FW and 1.25μL of 20μM TSO FW primer. DNA was purified using AMPure XP beads (Beckman Coulter, A63881) with 1.8X ratio and eluted into 20μL. Next, biotin pulldown was performed to purify amplified and barcoded biotinylated gDNA fragments from non-biotinylated cDNA libraries.
Next, 4 indexing and CAGE ATAC-seq library amplification and normalization reactions were performed using 4 distinct NexteraR1_x_p5 FW primers. 7, 9, 9 and 11 additional PCR cycles were added for each library respectively. Final inC-ATAC-seq libraries were pooled and quantified using Qubit and qPCR before sequencing.

value: Lymphoblast, fibroblast


title: Control, replicate 3, scRNAseq

description: 10x 3’ (v2)

value: Thymus


title: HeLa_IgG_1

description: Library name: HeLa_IgG_1

value: DMSO 6h


title: blood, day 44, group 7 #2

description: Library name: PR3287_142_b

value: conventional


title: IL7R KO, replicate 2, scRNAseq

description: 10x 3’ (v2)

value: IL7R knockout


title: 72_DOXH_2

description:
Library name: DOXH_72h_rep2
DOX plus hes-treated heart, 72h replicate 2

value: heart

value: doxorubicin plus hsperitin


title: Control, replicate 1, scRNAseq

description: 10x 3’ (v2)

value: 8 weeks old


title: inC-ATAC-seq of a 1:1 mixutre of K562 and L1210 cells to benchmark inC-seq split-and-pool barcoding

description: Library name: K562_L1210_inC-ATACseq

data_processing:
BCL sequencing files received from Illumina BaseSpace portal were converted to raw FASTQ files for each of the 4 (R1, R2, R3, R4) sequencing reads without read trimming or masking by using bcl2fastq software (Illumina, v2.20.0.422). Raw FASTQ files were processed using a custom pipeline (available at https://github.com/AllonKleinLab/paper-data/tree/master/Mazelis2025_CAGEs). In brief, sequencing reads were demultiplexed based on Read1 index sequence (stored as read3) (index used: ATAGAGAG, AGAGGATA), and were filtered based on expected structure (all barcodes matching a barcode whitelist with hamming distance of 1). The demultiplexed reads were then processed using Trimmomatic (1) (with parameters: LEADING 28; SLIDINGWINDOW 4:20; MINLEN 20), followed by Poly-A length trim to 4 nt and selecting reads that are longer than MINLEN. For surviving reads, the fraction of length composed out of same base repeats (only considering > 5) was determined, rejecting reads whose fraction is greater than 0.5. CAGE ATAC-seq approach used pair-end sequencing to map out gDNA fragment length. While Read1 sequencing reads contain only the gDNA fragment, Read4 contains CAGE barcodes followed by a gDNA fragment. First, parts of Read4 sequencing reads corresponding to gDNA fragments were extracted using seqtk trimfq command with a parameter -b 50. Sequencing reads were then processed as described above to generate demultiplexed and filtered  FASTQ files twice, once for each gDNA read (Read1 and Read4). Next, fastq-pair was used to pair the two demultiplexed and filtered gDNA fragment FASTQ files, which were then merged using NGmerge. Merged FASTQ files were aligned to merged human GRCh38.101 and mouse GRCm.101 index using bowtie2 (with parameter --very-sensitive, -p 4 –local -X 2000) and bam files were sorted using samtools sort function. Mitochondrial reads were counted and removed, followed by BAM file deduplication, sorting and indexing using samtools. Next a pysam script was used to move barcode sequences from BAM read headers to bam CB tag and a string tag was appended to each barcode to label runs corresponding to different indices. Following this, bam files were merged for all the runs.
BAM files were analyzed using the SnapATAC2 python package. First, BAM files were converted into fragments files using snapatac2.pp.make_fragment_file which were filtered (TSSE >5; fragment number >400; FRiP>0.4) after metric calculation (snapatac2.metrics.tsse, snapatac2.tl.macs3, snapatac2.metrics.frip).
Assembly: Mixed Human GRCh38.101 and mouse GRCm.101 genome assembly
Supplementary files format and content: h5ad cell x fragments matrix

source_name: Human and mouse Blood

growth_protocol: K562 and L1210 cells were cultured in IMDM (Thermo Fisher, 10566016) + 10% FBS (ATCC, 30-2020) + 1x penicillin/streptomycin (Thermo Fisher, 10378016) in T25 flasks (Corning). Cell lines were cultured until they reached ~80-90% confluency. For passaging, suspension cells (K562, L1210) were resuspended gently and collected into a 15 mL tube. 5 mL of 37ºC pre-warmed complete media was used to wash the flask and was added into the same 15 mL tube. Cells were centrifuged at 300g for 4 min, followed by media aspiration and resuspension in 5 mL of fresh pre-warmed complete media. Cells were counted using an automatic cell counter and 500,000 cells were seeded into 5 mL of 37ºC pre-warmed complete media in a 25cm2 flask. For encapsulation experiments, cells were washed twice with ice cold DPBS with 0.1% BSA

extract_protocol:
Cells were washed twice with DPBS with 0.1% BSA, mixed at 1:1 (K562:L1210) ratio and were encapsulated into CAGEs at a final concentration of 1.2e6 cells/mL in core mix. Next, for some samples the CAGE core dextran was removed using dextranase enzyme. Following this, all steps were performed on ice. To perform gDNA tagmentation, CAGEs were washed with DPBS with 0.1% (w/v) F127 and 0.1% L31 (w/v). For a 25μL reaction, 9μL of package CAGEs were mixed with 12.5μL of 2x TD buffer (20 mM Tris 10 mM MgCl2; 20% (vol/vol) DMF, pH 7.6), 1μL of loaded Tn5, 2.5μL of 10X Lysis additive (5% Tween20, 0.05% Digitonin in DPBS). The solution was incubated on ice for 5 min, followed by incubation at 37ºC for 30 min. The CAGEs were distributed into 5 wells for gDNA tagmentation in 80μL reactions. After the reaction, CAGEs were washed three times with TEIS (pH 7.5) and then Proteinase K treated in 200μL of TEIS (pH 7.5) with 10μL of Proteinase K (NEB, P8107S) for 15 min at 55ºC. After the reaction, CAGEs were washed three times with TI100 (pH 7.5) and twice with TI (pH 7.5. Next, the CAGEs were split into four wells for a CAGE dU PCR reaction, using using 2.5μL of 20μM Nextera R1 FW, 2.5μL of 20μM dU universal RV primer with 25μL of packed CAGEs in 50μL reactions. The reaction was carried out by first incubating the tubes on ice for 5 mins, followed by a PCR cycling program: 98ºC for 30s; 6 cycles of 98ºC for 15s, 68ºC for 20s, 72ºC for 4 min; a final extension of 72ºC for 2min and hold at 25ºC. Following this, CAGEs were subjected to 3 round of 48x48x48 split-and-pool barcoding. After barocding CAGEs were dissolved into 2 1.5mL tubes using NaOH. DNA was purified using MinElute PCR purification kit and barcoded DNA was eluted into 20μL of the elution buffer. Barcoded and purified DNA was amplified using PCR using 1.25μL of 20μM Read1_Biotin FW FW and 1.25μL of 20μM TSO FW primer. DNA was purified using AMPure XP beads (Beckman Coulter, A63881) with 1.8X ratio and eluted into 20μL
4 indexing and CAGE ATAC-seq library amplification and normalization reactions were performed using 4 distinct NexteraR1_x_p5 FW primers. 9 additional PCR cycles were added for each library respectively. Final inC-ATAC-seq libraries were pooled and quantified using Qubit and qPCR before sequencing.

value: Human and mouse Blood

value: Lymphoblast


title: HeLa_CCCP6h_ATF4_2

description: Library name: HeLa_CCCP6h_ATF4_2

value: CCCP 6h


title: gDNA amplicon sequencing derived from single-cell gDNA in CAGEs (gDNA_Amplicons)

description: Library name: GFP_RFP_gDNAseq

data_processing:
BCL sequencing files received from Illumina BaseSpace portal were converted to raw FASTQ files for each of the 4 (R1, R2, R3, R4) sequencing reads without read trimming or masking by using bcl2fastq software (Illumina, v2.20.0.422). . Raw FASTQ files were processed using a custom pipeline (available at https://github.com/AllonKleinLab/paper-data/tree/master/Mazelis2025_CAGEs). In brief, sequencing reads were demultiplexed based on Read1 index sequence (stored as read3) (index used: AGGCTTAG, ATTAGACG), and were filtered based on expected structure (all barcodes matching a barcode whitelist with hamming distance of 1). The demultiplexed reads were then processed using Trimmomatic (1) (with parameters: LEADING 28; SLIDINGWINDOW 4:20; MINLEN 20), followed by Poly-A length trim to 4 nt and selecting reads that are longer than MINLEN. For surviving reads, the fraction of length composed out of same base repeats (only considering > 5) was determined, rejecting reads whose fraction is greater than 0.5.
Read count were generated by aligning the gDNA amplicon sequences to GFP and RFP references using Bowtie aligner
Read matrices were filtered by selecting barcodes with >2000 reads.
Assembly: GFP and RFP reference sequence
Supplementary files format and content: h5ad cell x GFP/RFP reads matrix

extract_protocol:
Cells were washed twice with ice cold DPBS with 0.1% BSA with 0.1% BSA and were encapsulated into CAGEs at a final concentration of 1.2e6 cells/mL in core mix. Next, CAGE core dextran was removed using dextranase enzyme. Following this, all steps were performed on ice. First, capsules were washed three times with an ice-cold 3X SSCI buffer with 800g spins for 1 min. Cells were lysed by washing with ice-cold 7.5pH TEIS with 5 min incubations on ice. After lysis, capsules were imaged (check above), washed three times with 3X SSCI and three times with HI10, packed at 800g for 1 min and stored on ice. Reverse-transcription reaction was set on ice and performed in PCR strip tubes. A standard 100μL reaction was composed out of 60.5μL of packed capsules, 2.5μL of 10μM RT poly(T) primer housing a 10nt UMI and 6nt barcode (Table S3, RT), 3μL of 10% Igepal CA-630 (Millipore Sigma, I8896-50ML), 20μL of 5X SMARTseq3 RT buffer (100mM Tris pH 8.3, 150mM NaCl, 12.5mM MgCl2, 5mM GTP, 40mM DTT), 0.5μL of 10mM dNTPs (NEB, N0447L), 2.5μL of 40U/μL Recombinant RNase Inhibitor (Takara Bio, 2313A), 1.5μL of 50μM TSO, 5μL of Maxima H- reverse transcriptase (ThermoFisher, EP0753). Reaction volumes were scaled according to the amount of capsules used and number of reactions performed. To set up the reaction, capsules, RT primer and Igepal CA-630 (Millipore Sigma, I8896-50ML) were mixed on ice in a PCR tube, with each housing a different RT barcode. Capsules were incubated on ice for 10 min to allow the RT primer to diffuse in, followed by heating at 70ºC for 5 min and cooling down on ice. Next, RT master mix, composed out of the remaining reagents was added to each tube, ensuring a thought mixing of capsules with pipetting. Tubes were incubated on ice for 5 min and the reaction was carried out at 42ºC for 90 min, followed by cycling between 50ºC and 42ºC incubation of 2 mins for 15 cycles. Reactions were left at room temperature overnight and processed the next day. After the RT reaction, capsules were washed three times with TI100 (pH 8.0), twice with TIbS (pH 8.0), three times with ice-cold TI100 (pH 8.0) with 2 min incubations on ice. Finally capsules were washed twice with TI10 (pH 8.0). The CAGEs containing gDNA and cDNA were then processed to amplify target gDNA regions by carrying out 16 cycles of CAGE gDNA amplicon PCR. The PCR reactions were performed in 50μL volume using 5μL of 10μM RFP_R1 FW primer, 5μL of 10μM GFP_R1 FW primer, 5μL of 10μM universal dU CMV RV primer, and 20μL of packed CAGEs. The reaction was carried out by first incubating the tubes on ice for 5 mins, followed by a PCR cycling program: 98ºC for 30s; 16 cycles of 98ºC for 15s, 70ºC for 20s, 72ºC for 2 min; a final extension of 72ºC for 2min and hold at 25ºC. After PCR, CAGEs were washed once with TIS (pH 8.0), three times with TI100 (pH 8.0) and three times with TI10 (pH 8.0). A second PCR reaction was then performed in a 50μL volume using 2.5μL of 10μM Read1_Biotin FW primer, 2.5μL of 10μM universal dU CMV RV primer, 2.5μL of 10μM universal dU RV primer, 2.5μL of 10μM of TSO FW primer with 25μL of packed CAGEs. Following split-and-pool barcoding, CAGEs were split into multiple 1.5mL tubes and were dissolved as described above. DNA was purified using MinElute PCR purification kit and barcoded DNA was eluted into 20μL of the elution buffer. Barcoded and purified DNA was amplified using PCR using 2.5μL of TSO_fw and Read1_Biotin FW primers. After the reaction, amplified barcoded DNA was purified using 0.8X AMPure XP beads (Beckman Coulter, A63881) and was eluted into a 20μL Elution Buffer. Next biotinylated DNA corresponding to gDNA amplicons was purified using Biotinylated DNA pulldown beads.
Indexing PCR was carried out by mixing 20µL of purified DNA with 25 μL of 2x NEBNext® High-Fidelity 2X PCR Master Mix (NEB, M0543L) and 2.5 μL of 20μM P7_rv, 2.5μL of 20μM indexing primer that houses an 8bp library index. PCR cycling program: 98 °C, 2 min; 3 cycles of 98 °C, 20 s; 55 °C, 30 s; 72 °C, 20 s and 9 cycles 98 °C, 20 s; 65 °C, 30 s; 72 °C, 20 s; with a 1 min final extension at 72 °C, and 4 °C, hold at the end. Following final library indexing, DNA was purified using 0.8x SPRIselect magnetic beads (Beckman Coulter, B23318) and eluted into 15μL.

value: Blood


title: blood, day 44, group 7 #1

description: Library name: PR3287_141_b

value: whole blood

value: NC/Nga

value: male

value: WT


title: inC-seq barcoding of plasmid DNA encapsulated in CAGEs (Plasmid_sequencing)

description:
Library name: Plasmid_Barcoding
bacteria plasmid DNA

library_source: other

data_processing:
BCL sequencing files received from Illumina BaseSpace portal were converted to raw FASTQ files for each of the 4 (R1, R2, R3, R4) sequencing reads without read trimming or masking by using bcl2fastq software (Illumina, v2.20.0.422). . Raw FASTQ files were processed using a custom pipeline (available at https://github.com/AllonKleinLab/paper-data/tree/master/Mazelis2025_CAGEs). In brief, sequencing reads were demultiplexed based on Read1 index sequence (stored as read3) (index used: AGGCTTAG, ATTAGACG), and were filtered based on expected structure (all barcodes matching a barcode whitelist with hamming distance of 1). The demultiplexed reads were then processed using Trimmomatic (1) (with parameters: LEADING 28; SLIDINGWINDOW 4:20; MINLEN 20). For surviving reads, the fraction of length composed out of same base repeats (only considering > 5) was determined, rejecting reads whose fraction is greater than 0.5.
Read count were generated by aligning the gDNA amplicon sequences to GFP and RFP references using Bowtie aligner
Read matrices were filtered by selecting barcodes with >2000 reads.
Assembly: GFP and RFP reference sequence
Supplementary files format and content: h5ad cell x GFP/RFP reads matrix

molecule: other

organism: Escherichia coli

source_name: bacterial cell

growth_protocol: Plasmid DNA purified from E. coli DH5a strain cultured overnight in LB at 37ºC

extract_protocol:
CAGEs containing plasmid DNA were generated with core solution contained 1ng/μL of plasmid DNA. After polymerization, CAGEs were washed three times with TI100 (pH 8.0), twice with TI10 (pH 8.0) and twice with TI (pH 8.0). Next, 14 reactions of CAGE dU PCR were performed in a 50 μL volumes using 2.5μL of 20μM GFP/RFP FW primer, 2.5μL of 20μM dU barcoded CMV [1-14] RV primer, and 27.5μL of packed CAGEs. The reaction was carried out by first incubating the tubes on ice for 5 mins, followed by cycling: 98ºC for 30s; 14 cycles of 98ºC for 15s, 68ºC for 20s, 72ºC for 4 min; a final extension of 72ºC for 2min and hold at 25ºC. After PCR, CAGEs were washed and pooled into a single tube. The CAGEs were then subject to a second PCR reaction performed in 50μL volumes as described above, except 2.5μL of 20μM GFP/RFP FW, 2.5μL of labeled dU universal RV primer and 30μL of packed CAGEs with no extra water were used with the same cycling conditions for 8 cycles. After the DNA amplification, CAGEs were washed and were subjected to split-and-pool barcoding. Following split-and-pool barcoding, CAGEs were split into 16x 1.5mL tubes and were dissolved as described above. DNA was purified using MinElute PCR purification kit (Qiagen, 28004) and barcoded DNA was eluted into 20μL of the elution buffer.
Barcoded and purified DNA was amplified using PCR. After the reaction, amplified barcoded DNA was purified using 0.8X AMPure XP beads (Beckman Coulter, A63881) and was eluted into a 15μL Elution Buffer. DNA concentration was measured using a Qubit™ dsDNA HS Assay (Thermo FIsher, Q32854). Final sequencing library index PCR was carried out using 20 ng of purified DNA product with 25 μL of 2x NEBNext® High-Fidelity 2X PCR Master Mix (NEB, M0543L) and 2.5 μL of 20μM P7_rv, 2.5μL of 20μM TruR1_x_p5 indexing primer that houses an 8bp library index and water to 50μL. PCR cycling program: 98 °C, 2 min; 3 cycles of 98 °C, 20 s; 55 °C, 30 s; 72 °C, 20 s and 9 cycles 98 °C, 20 s; 65 °C, 30 s; 72 °C, 20 s; with a 1 min final extension at 72 °C, and 4 °C, hold at the end.. Following final library indexing, DNA was double-side purified using 0.6x-0.8x SPRIselect magnetic beads (Beckman Coulter, B23318) and eluted into 15μL of water.

value: bacterial cell


title: LP03012

first: Reinhard

last: Gruber

address: Sensengasse 2a

institute: Medical University Vienna

data_processing:
Illumina bcl2fastq command line tool (v2.19.1.403)
Reads in fastq format were generated using the Illumina bcl2fastq command line tool (v2.19.1.403), incl. trimming of the sequencing adapters.
Reads in fastq format were aligned to the human reference genome version GRCh38 with Gencode 29 annotations using STAR aligner version 2.6.1a in 2-pass mode.
Raw reads per gene were counted by STAR. Batch correction was performed using ComBat from sva version 3.35.2.
Assembly: Reads in fastq format were aligned to the human reference genome version GRCh38 with Gencode 29 annotations using STAR aligner version 2.6.1a in 2-pass mode
Supplementary files format and content: ProcessedDataMatrix.txt.gz contains a matrix with raw gene counts, gene symbols and gene types for each sample

source_name: gingiva

growth_protocol: Gingival fibroblasts were grown in Minimum Essential Medium Eagle (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (Bio&Sell GmbH, Nuremberg, Germany) and 1% antibiotics (Sigma). The cells were seeded at 30,000 cells/cm2 onto culture dishes one day before being stimulated.

extract_protocol:
Total RNA was isolated utilizing the ExtractMe total RNA kit manufactured by Blirt S.A., Gdańsk, Poland.
Sequencing libraries were prepared at the Core Facility Genomics, Medical University of Vienna, using the NEBNext Poly(A) mRNA Magnetic Isolation Module and the NEBNext UltraTM II Directional RNA Library Prep Kit for Illumina according to the manufacturer's protocols (New England Biolabs, Ipswich, MA) with unique dual indices (UDI). Libraries were QCchecked on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) using a High Sensitivity DNA Kit for correct insert size and quantified using Qubit dsDNA H.S. Assay (Invitrogen, Waltham, MA).

treatment_protocol: Gingival fibroblasts were exposed to 4.5-fold diluted hyaDENT BG, resulting in a final concentration of a total of 4 mg/mL cross-linked and native HA for 6 hours in serum-free DMEM followed by isolation of total RNA.

value: gingiva

value: primary cells

value: gingival fibroblasts

value: wildtype

value: hyaluronic acid


title: HeLa_CCCP6h_ATF4_1

description: Library name: HeLa_CCCP6h_ATF4_1

value: HeLa

value: Human Cervical Carcinoma Cell


title: HeLa_DMSO6h_H3K27Ac_2

description: Library name: HeLa_DMSO6h_H3K27Ac_2

value: DMSO 6h


title: blood, day 23, group 7 #10

description: Library name: PR3287_140_b

value: conventional

value: group 7

value: day 23


title: Control, replicate 4, scRNAseq

description: 10x 3’ (v2)

value: Thymus

value: 8 weeks old

value: Male


title: HeLa_DMSO6h_ATF4_2

description: Library name: HeLa_DMSO6h_ATF4_2


title: LP03008


title: LP03004


title: R22312257-R22055332LR01-shNSUN2-5-1_combined_R1.fastq

city: wenzhou

first: bin

last: zhou

address: Nanbaixiang, Ouhai District, Wenzhou, Zhejiang Province

institute: Affiliated Hospital of Weifang Medical University

description: Library name: Sample 3

data_processing:
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.
Read count extraction and normalization were performed using CLC genomic benchwork
CLC Genomics Workbench v 11.0.1
Sequence reads were trimmed for adaptor sequence/low-quality sequence using CLC genomic benchwork (parameter- Quality limit: 0.05)
Trimmed sequence reads were mapped to GrCM38/hg38 using CLC genomic benchwork (parameters- mismath cost: 2 insertion cost: 3 deletion cost: 3 length fraction: 0.8 similarity fraction: 0.8)
Assembly: hg38
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample
Supplementary files format and content: csv files include RPKM values for each Sample

source_name: cell line

growth_protocol: NALM-6 cells were maintained in 1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
centrifuge to remove supernatant, add about 2ML PBS (2% FBS), with a total amount of about 2.5mL, transfer to flow cytometry, and sort 2 * 106 GFP+(NC/PLKO-NUSN2) cells,Add cells to trizol
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3ug of total RNA was used for the construction of sequencing libraries.RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

treatment_protocol: Comparative gene expression profiling analysis of RNA-seq data for NALM-6 cells and its KD derivatives (shNUSN2).

value: cell line

value: adipogenic differentiation

value: Separate ribosomal RNA

value: Day 0


title: R22312254-R22055329LR01-shNC-2_combined_R1.fastq

description: Library name: Sample 2

value: WT


title: blood, day 23, group 7 #9

description: Library name: PR3287_139_b

value: whole blood

value: NC/Nga

value: male


title: HeLa_DMSO6h_ATF4_1

description: Library name: HeLa_DMSO6h_ATF4_1

value: HeLa


title: blood, day 23, group 7 #8

description: Library name: PR3287_138_b


title: LP03009

value: gingiva

value: primary cells

value: gingival fibroblasts

value: wildtype

value: untreated


title: HeLa_Thap6h_rep2

description: Library name: HeLa_Thap6h_rep2

data_processing:
The raw reads were trimmed by Trim Galore (v0.6.10)
Trimmed reads were mapped to the mouse transcriptome (mm10) by Bowtie2
Assembly: hg38
Supplementary files format and content: bigwig

extract_protocol: ATAC-seq libraries were generated from approximately 1×106 cell nuclei with the TruePrep® DNA Library Prep Kit V2 for Illumina (Vazyme #TD501)

value: Human Cervical Carcinoma Cell

value: CCCP 6h


title: blood, day 23, group 7 #7

description: Library name: PR3287_137_b

value: conventional

value: group 7

value: day 23


title: Panc_TQ

city: Detroit

first: Yang

last: Shi

state: MI

address: 4100 John R St MM03BI

department: Karmanos Cancer Institute

institute: Wayne State University Schhol of Medicine

description:
Library name: Panc_TQ
Column name in processed data file: Panc_TQ_count

data_processing:
Cutadapt were used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC. Bowtie2 and Tophat2 were used to map reads to the human genome GRCh38.p13. Remaining reads (unmapped reads) were still mapped to genome using tophat-fusion. CIRCExplorer was used to de novo assemble the mapped reads to circular RNAs at first; Then, back splicing reads were identified in unmapped reads by tophat-fusion and CIRCExplorer. Circular RNA expressions from different samples or groups were caculated by scripts in house. The differential expression analyses were performed using R package EdgeR and those circRNAs with absolute value of log2 (fold change) > 1 and p-value < 0.05 were regarded as showing differential expression.
Assembly: GRCh38
Supplementary files format and content: The processed data file is a tab-delimited text file include read counts (unnormalized) for each sample generated as described in the data processing step.

growth_protocol: Pancreatic cancer cell lines, MiaPaCa-2 and PANC-1 were obtained ATCC (Manassas, VA, USA). MiaPaCa-2 and PANC-1 were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin.

extract_protocol:
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number>7.0.
Approximately 10 ug of total RNA was used to remove ribosomal RNA according to the manuscript of the Epicentre Ribo-Zero Gold Kit (Illumina, San Diego, USA). The linear RNA was digested according to the manuscript of the Epicentre Ribonuclease R (Illumina, San Diego, USA). Following purification, the linear RNA fractions is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with a strand-specific library preparation by dUTP method, the average insert size for the paired-end libraries was 300±50 bp. And then we performed the paired-end 2×150bp sequencing on an Illumina Hiseq 4000 platform housed in the LC Sciences following the vendor's recommended protocol.

treatment_protocol: MiaPaCa-2 cells were exposed to 20 µM TQ for 24 hours, while PANC-1 cells were treated with 10 µM TQ for the same duration.

value: human pancreatic ductal adenocarcinoma cell line

value: TQ treatment


title: HeLa_Thap6h_rep1

description: Library name: HeLa_Thap6h_rep1

value: WT


title: LP03005


title: blood, day 23, group 7 #6

description: Library name: PR3287_136_b

data_processing:
rRNA was filtered using RiboPicker, and mapped to the mouse genome GRCm39 using the DRAGEN Bio-IT Platform (Illumina).
Assembly: GRCm39
Supplementary files format and content: txt file including raw counts for each sample
Supplementary files format and content: txt file including TPM for each sample

extract_protocol:
Blood samples were collected from the tail vein (approximately 0.1 mL) using EDTA-2K-coated capillary tubes without anesthesia. Total RNA was isolated from blood using a NucleoSpin RNA Blood Kit (Macherey-Nagel, Easton, PA, USA), according to the manufacturer’s instructions.
Libraries were prepared using TruSeq Stranded Total RNA with RiboZero Globin (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.

value: whole blood


title: Panc_Control

description:
Library name: Panc_C
Column name in processed data file: Panc_C_count

value: No treatment (Control)


title: LP03001


title: HeLa_DMSO6h_rep2

description: Library name: HeLa_DMSO6h_rep2

value: DMSO 6h


title: Mia_TQ

description:
Library name: Mia_TQ
Column name in processed data file: Mia_TQ_count


title: blood, day 23, group 7 #5

description: Library name: PR3287_135_b

value: NC/Nga

value: male


title: R22312258-R22055333LR01-shNSUN2-5-2_combined_R1.fastq

description: Library name: Sample 4

value: cell line

value: adipogenic differentiation

value: Separate ribosomal RNA

value: Day 0


title: 16_V_3

description:
Library name: V_16h_rep3
Vehicle heart, 16h rep3

value: heart

value: vehicle


title: blood, day 23, group 7 #4

description: Library name: PR3287_134_b

value: conventional


title: Mia_Control

description:
Library name: Mia_C
Column name in processed data file: Mia_C_count


title: Single-Cell RNA-seq: Symptomatic SMA Infant #2

city: Atlanta

first: Manoj

middle: K

last: Bhasin

state: GA

address: 1750 Haygood Dr NE, N320

department: Aflac Cancer and Blood Disorders Center

institute: Children's Healthcare of Atlanta

description: Library name: SMA_P4

data_processing:
Bulk RNA-seq: The resulting FastQ files underwent comprehensive quality control using FastQC (version 0.12.0), followed by trimming of low-quality reads and adapters using Skewer (version 0.2.2). Subsequently, Salmon (version 1.10.1) was employed to align the trimmed FastQ files to the hg38 reference genome and generate transcript counts for each sample. These were then summarized into gene-level counts using tximport.
Single-cell RNA-seq: Reads were aligned to the hg19 reference genome and quantified with CellRanger (version 3.0.2). Quality control and preprocessing were performed using the Seurat package (version 4.4.0) to ensure the inclusion of high-quality cells based on the percentage of mitochondrial genes and the number of genes/molecules. Doublets were identified and removed using DoubletFinder (version 2.0.4), and normalization and variance stabilization were performed with the SCTransform algorithm (version 0.4.1).
Assembly: GRCh38 (bulk RNA-seq), GRCh37 (single-cell RNA-seq)
Supplementary files format and content: tab-delimited text file with raw gene counts for each healthy/SMA sample in bulk RNA data
Supplementary files format and content: RDS object file with seurat object containing all healthy and SMA single-cell samples

source_name: Peripheral Blood

extract_protocol:
Bulk RNA-seq: RNA was extracted from whole blood using a Quick-RNA Whole Blood kit (Cat #R1201, Zymo Research) following the manufacturer’s protocol. The quality and integrity of the extracted RNA were assessed using an Agilent Bioanalyzer to ensure sufficient RNA quality for sequencing. Library preparation was then performed using the Illumina TruSeq RNA Sample Preparation Kit, converting the RNA into cDNA for sequencing.
Single-cell RNA-seq: All samples were collected in lavender blood collection tubes, transported to the lab on ice, and cryopreserved using the SENSE protocol. PBMCs were isolated using an EasySep human CD15 positive selection kit (Cat #18651, STEMCELL), and viability was confirmed to exceed 80% with the TC20 Automated Cell Counter (Cat #1450102, Bio-Rad).
Bulk RNA-seq: The raw sequencing libraries were sequenced on an Illumina NovaSeq 6000 platform (Novogene, Sacramento, CA), generating paired-end 150 bp reads.
Single-cell RNA-seq: Single-cell RNA sequencing (scRNA-seq) was performed on viably thawed samples using the Drop-seq approach, which encapsulates single cells and uniquely barcoded primer beads in oil droplets for high-throughput transcriptome analysis. Single-cell suspensions were processed with the 10x Genomics workflow, generating digitally barcoded droplets for cell lysis and library preparation using a 10x Chromium Controller. Libraries were prepared with the 10x Genomics Single Cell 5’ v2 and Chromium Next GEM Single Cell 5’ v2 reagent kits. Sequencing was conducted on the NovaSeq 6000 platform, achieving an average detection of ~1,000–2,000 genes per cell.

value: Peripheral Blood

value: PBMC


title: 16_V_2

description:
Library name: V_16h_rep2
Vehicle heart, 16h rep2


title: blood, day 23, group 7 #3

description: Library name: PR3287_133_b

value: group 7

value: day 23


title: 15700004, CD4+ T cells, asthma case

city: Aurora

first: Randi

last: Johnson

state: Colorado

address: 1890 N. Revere Court

institute: University of Colorado

description: Library name: 15700004

data_processing:
CoCo was used to adjust the Ensembl annotation file for alignment and assembly.
FastQC was used to detect technical issues, then adapter trimming was performed using BBDuk.
FastQC was then run again to confirm trimming was successful. HISAT2 was used for alignment to GRCh38 using the default options.
Sorting and flagging of reads was performed using SAMtools prior to transcript assembly with StringTie and applying the CoCo correct_count function using the corrected Ensembl annotation file to account for overlapping genes and multi-mapping reads.
Raw counts were converted to transcripts per million (TPM) values for each gene and each sample.
Within-sample TPM distributions were used to detect and exclude samples where data generation was not successful. Additional sample-level metrics such as ribosomal contamination were calculated using Picard CollectRnaSeqMetrics. Transcripts with inadequate variability for detection of differential expression were filtered using the DESeq2 theta value. The DESeq2 varianceStabilizingTransformation function was used to transform the count data (normalized by division by the size factors or normalization factors), to be approximately homoscedastic and normalized with respect to library size.
Technical variability present in the cleaned and transformed TPM values due to sequencing batch was removed with the removeBatchEffect function in limma.
Assembly: GRCh38
Supplementary files format and content: tab-delimited text file includes transformed, batch-adjusted TPM values

source_name: CD4+ T cells

extract_protocol:
CD4+ T helper cells were isolated from PBMCs by negative selection using magnetic depletion of non-target cells (CD4 + T Cell Isolation Kit, Miltenyi Biotec Inc) and then lysed immediately. RNA and DNA were extracted at the same time (AllPrep protocol, Qiagen Inc).
Libraries were prepared using the TruSeq total RNA Ribo Zero kit (Illumina Inc) and sequenced on the HISEQ 4000 using 151bp paired-end reads.

value: CD4+ T cells

value: female

tag: genetic pc1

tag: asthma

value: case

tag: geo loc_name

value: Barbados


title: Single-Cell RNA-seq: Symptomatic SMA Infant #1

description: Library name: SMA_P3

tag: tissue


title: R22312253-R22055328LR01-shNC-1_combined_R1.fastq

description: Library name: Sample 1

tag: cell line

value: WT


title: 16_V_1

description:
Library name: V_16h_rep1
Vehicle heart, 16h rep1


title: Single-Cell RNA-seq: Asymptomatic SMA Infant #2

description: Library name: SMA_P2

tag: cell type


title: blood, day 23, group 7 #2

description: Library name: PR3287_132_b

value: whole blood

value: NC/Nga

value: male

tag: genotype


title: 16_H_3

description:
Library name: HES_16h_rep3
Hesperitin-treated heart, 16h rep3

tag: treatment

value: hsperitin


title: 15600009, CD4+ T cells, control

description: Library name: 15600009

value: control


title: 15700003, CD4+ T cells, asthma case

description: Library name: 15700003


title: Single-Cell RNA-seq: Asymptomatic SMA Infant #1

description: Library name: SMA_P1


title: blood, day 40, group 6 #10

description: Library name: PR3287_130_b

tag: strain

value: group 6

value: day 40


title: 15600001, CD4+ T cells, control

description: Library name: 15600001

tag: Sex


title: 16_H_2

description:
Library name: HES_16h_rep2
Hesperitin-treated heart, 16h rep2

value: heart


title: 16_H_1

description:
Library name: HES_16h_rep1
Hesperitin-treated heart, 16h rep1


title: 16_DOXH_3

description:
Library name: DOXH_16h_rep3
DOX plus hes-treated heart, 16h replicate 3

value: doxorubicin plus hsperitin


title: 15556004, CD4+ T cells, control

description: Library name: 15556004


title: HeLa_DMSO6h_rep1

description: Library name: HeLa_DMSO6h_rep1

value: HeLa

value: Human Cervical Carcinoma Cell

value: DMSO 6h


title: blood, day 40, group 6 #9

description: Library name: PR3287_129_b

tag: condition

value: conventional


title: blood, day 40, group 6 #8

description: Library name: PR3287_128_b

tag: group


title: 15551002, CD4+ T cells, asthma case

description: Library name: 15551002

value: CD4+ T cells

value: female


title: Bulk RNA-seq: SMA Infant #4, Post-Treatment

description: Library name: S44PO

value: Peripheral Blood

value: PBMC


title: Bulk RNA-seq: SMA Infant #4, Pre-Treatment

description: Library name: S44PR


title: Bulk RNA-seq: SMA Infant #3, Post-Treatment

description: Library name: S25PO


title: 16_DOXH_2

description:
Library name: DOXH_16h_rep2
DOX plus hes-treated heart, 16h replicate 2

value: WT


title: Bulk RNA-seq: SMA Infant #3, Pre-Treatment

description: Library name: S25PR


title: 15548001, CD4+ T cells, asthma case

description: Library name: 15548001

value: case

value: Barbados


title: blood, day 40, group 6 #6

description: Library name: PR3287_126_b

value: whole blood

value: NC/Nga

value: male

tag: dermatitis score


title: blood, day 23, group 7 #1

description: Library name: PR3287_131_b

value: group 7

tag: time

value: day 23


title: 16_DOXH_1

description:
Library name: DOXH_16h_rep1
DOX plus hes-treated heart, 16h replicate 1

value: heart

value: doxorubicin plus hsperitin


title: Bulk RNA-seq: SMA Infant #2, Post-Treatment

description: Library name: S14PO


title: 15547003, CD4+ T cells, control

description: Library name: 15547003

tag: age

value: control


title: 16_DOX_3

description:
Library name: DOX_16h_rep3
DOX-treated heart, 16h replicate 3

tag: time point

value: doxorubicin


title: Bulk RNA-seq: SMA Infant #2, Pre-Treatment

description: Library name: S14PR


title: 15547002, CD4+ T cells, control

description: Library name: 15547002


title: 16_DOX_2

description:
Library name: DOX_16h_rep2
DOX-treated heart, 16h replicate 2

tag: batch


title: blood, day 40, group 6 #4

description: Library name: PR3287_124_b

value: conventional

value: group 6

value: day 40


title: blood, day 40, group 6 #5

description: Library name: PR3287_125_b


title: 15543008, CD4+ T cells, control

description: Library name: 15543008

value: CD4+ T cells

value: female

tag: genetic pc1


title: 16_DOX_1

description:
Library name: DOX_16h_rep1
DOX-treated heart, 16h replicate 1

value: WT


title: 15543001, CD4+ T cells, asthma case

description: Library name: 15543001

tag: asthma

value: case


title: blood, day 40, group 6 #3

description: Library name: PR3287_123_b


title: 15539008, CD4+ T cells, control

description: Library name: 15539008

tag: geo loc_name

value: Barbados


title: HCT116-siPUS7-mRNA-seq-rp3

city: Chicago

first: Chuan

last: He

state: IL

address: 929 E 57th St

department: Chemistry Department

institute: University of Chicago

description:
Library name: HCT116-mRNA-seq-9
HCT116-siPUS7_VS_siCtrl-mRNA-seq.DEresults.xlsx

data_processing:
The code for sequencing data analysis has been publicly available on GitHub (https://github.com/YutaoZhao1224/PUS7-project). BID-seq: Only R1 reads of paired-end reads were processed for BID-seq data analysis. R1 was trimmed by cutadapt (version 4.8) with following parameters “-a ATCACGAGATCGGAAGAGCA -g TTCTACAGTCCGACGATC -e 0.1 -q 20 -m 32 -n 3 -O 8” to remove adaptors and low-quality reads. PCR duplicates were removed with BBMap tool (v.38.73.). 5-mer random barcodes at reads ends were trimmed by cutadapt (version 4.8). Remaining reads were aligned to hg38 by STAR mapping software (2.7.11b). The generated bam file was sorted by samtools (1.21) and subjected to samtools mpileup for searching deletion signature. The Ψ candidate needs to satisfy the following criteria:  1) deletion ratio above 0.05 (with deletion count above four) in bisulfite-treated library; 2) deletion ratio below 0.02 in input libraries; 3) total read coverage depth above 20 for bisulfite-treated and input libraries; 4) all deletion signatures must be from ‘U’ sites marked by hg38; 5) all deletion signatures should be identified in each biological replicates. The differentially regulated Ψ sites were identified by two-tailed Student’s t-test (p < 0.05).  PAR-CLIP:  Only R1 reads of paired-end reads were processed for PAR-CLIP data analysis. R1 was trimmed by cutadapt (version 4.8) with following parameters -a ATCACGAGATCGGAAGAGCA -g TTCTACAGTCCGACGATC -e 0.1 -q 20 -m 32 -n 3 -O 8” to remove adaptors and low-quality reads. PCR duplicates were removed with BBMap tool (v.38.73.). 5-mer random barcodes at reads ends were trimmed by cutadapt (version 4.8). Remaining reads were aligned to hg38 by bowtie2 mapping software (2.4.4). The aligned sam files were subjected to PARalyzer (v1.5) for peak identification. The parameters in the “.ini” file used as the input for PARazyler are as follows: BANDWIDTH=3 CONVERSION=T>C MINIMUM_READ_COUNT_PER_GROUP=5 MINIMUM_READ_COUNT_PER_CLUSTER=2 MINIMUM_READ_COUNT_FOR_KDE=3 MINIMUM_CLUSTER_SIZE=11 MINIMUM_CONVERSION_LOCATIONS_FOR_CLUSTER=2 MINIMUM_CONVERSION_COUNT_FOR_CLUSTER=2 MINIMUM_READ_COUNT_FOR_CLUSTER_INCLUSION=1 MINIMUM_READ_LENGTH=20 MAXIMUM_NUMBER_OF_NON_CONVERSION_MISMATCHES=1  KAS-seq: The paired-end reads were trimmed by cutadapt (version 4.8) with following parameters “-a AGATCGGAAGAGCACACGTCTG -A AGATCGGAAGAGCGTCGTGT -n 3 --pair-filter=any -n 3 -O 8” to remove adaptors and low-quality reads. PCR duplicates were removed with BBMap tool (v.38.73). Remaining reads were aligned to hg38 by bowtie2 mapping software (2.4.4). The sorted bam files were converted to bigwig files by bamCoverage (deeptools 3.5.5). The peaks were identified by macs2 (version 2.2.6) and visualized by IGV (version 11.0.13). The read coverage on promoter (from TSS-100 to TSS+300 bp) and genebody (from TSS+300 to TSS+2000 bp) was calculated by bedtools coverage (v2.30.0). The Pol II release ratio was calculated as read coverage of genebody over promoter. Those genes with promoter read coverage less than 20 were discarded for data analysis.  mRNA-seq: The paired-end reads were trimmed by cutadapt (version 4.8) with following parameters “-a AGATCGGAAGAGCACACGTCTG -A AGATCGGAAGAGCGTCGTGT -e 0.15 -q 20 --nextseq-trim=20 -O 6 -n 3 --pair-filter=any” to remove adaptors and low-quality reads. PCR duplicates were removed with BBMap tool (v.38.73). Remaining reads were aligned to hg38 by STAR mapping software (2.7.11b). The generated bam file was sorted by samtools (version 1.21). FeatureCounts software (version 2.0.3) was used to count reads mapped to protein-coding genes from hg38, and differentially expressed gene analysis was conducted by DEseq2 software. Differentially expressed genes were identified with cutoff p < 0.01, |log2FC|≥1.
Assembly: hg38
Supplementary files format and content: "HCT116-siPUS7_VS_siCtrl_BID-seq.xlsx" shows the pseudorudine profile changes on nucleus RNA by BID-seq; "HCT116_PARclip_1.bed" and "HCT116_PARclip_2.bed" are bed files for PUS7 binding RNA peaks; "HCT116-KAS-seq-PRR.values.xlsx" shows "pol II release ratio" for each gene in PUS7 KD, 7SK KD and control groups; All bigwig files show the genomic coverage of KAS-seq in PUS7 KD, 7SK KD and control groups; "HCT116-si7SK_VS_siCtrl-mRNA-seq.DEresults.xlsx" and "HCT116-siPUS7_VS_siCtrl-mRNA-seq.DEresults.xlsx" show mRNA expression level change after 7SK KD or PUS7 KD, respectively.

source_name: Colon tissue

growth_protocol: HCT116 cells were grown in RPMI-1640, with 10% FBS and 1% penicillin-streptomycin. Cells were maintained at 37°C with 5% CO2.

extract_protocol:
RNA was isolated using Trizol method and precipitated using ethanol.
BID-seq for nucleus RNA: Nucleus fraction was isolated based on cell fractionation protocol. Nucleus RNA was purified by TRIzol reagent (Invitrogen) and ethanol precipitation. rRNA was further removed from Nucleus RNA by RiboMinus™ Eukaryote System v2 (Invitrogen). 50 ng nucleus RNA from PUS7KD and control HCT116 cells were used for BID-seq. The BID-seq was performed as previously described. 3 biological replicates were used for both the PUS7 KD and control conditions. PAR-CLIP: We performed PAR-CLIP based on a previously reported protocol. Briefly, four 15 cm plates of HCT116 cells were seeded per replicate and grown to 80% confluency before adding 4 μL of 1M 4SU to each plate. After 14 hours of incubation, the media was aspirated, and the cells were washed once with 5 mL of ice-cold PBS per plate. The cells were crosslinked on ice using 0.15 J cm⁻² of 365 nm UV light, applied twice. Crosslinked cells were collected with cell lifters, and four volumes of lysis buffer (50 mM HEPES, pH 7.5; 150 mM KCl; 2 mM EDTA; 0.5% [v/v] NP-40; freshly added 1:100 protease inhibitor (Roche)) were added to the pellet. After 10 minutes of incubation on ice with periodic mixing, the lysate was centrifuged at 15,000 g for 15 minutes, and the supernatant was collected. RNase T1 (Thermo Scientific) was added to a final concentration of 0.1 U μL⁻¹, followed by a 15-minute incubation at room temperature, which was then quenched on ice. 5 µg of anti-PUS7 were added to the lysate and incubated at 4 °C for 2 hours with periodic rotation. 100 µL Protein A or G beads (Thermo Scientific) were washed twice with IP wash buffer (50 mM HEPES, pH 7.5; 300 mM KCl; 0.05% [v/v] NP-40; freshly added 1:100 protease inhibitor (Roche) and 40 U mL⁻¹ RNasin®) and resuspended in 50 μL lysis buffer before being added to the antibody-lysate mixture. Another 2-hour incubation at 4 °C with low-speed rotation followed. After incubation, the beads were washed three times with IP wash buffer, resuspended in 200 μL of IP wash buffer, and subjected to a second RNase T1 digestion (10 U μL⁻¹ final concentration) for 15 minutes at room temperature. The reaction was quenched with 10 μL SUPERase-In, followed by a 5-minute incubation on ice. Beads were washed three times with high-salt wash buffer (50 mM HEPES, pH 7.5; 500 mM KCl; 0.05% [v/v] NP-40; freshly added 1:100 protease inhibitor (Roche)) and twice with 1X PNK buffer (NEB). They were resuspended in 200 μL of 1X PNK buffer and underwent T4 PNK end repair (Thermo Fisher Scientific) at 37 °C using standard procedures. After incubation, beads were washed once with 1X PNK buffer, followed by proteinase K digestion. RNA was recovered using RNA Clean & Concentrator (Zymo Research) and prepared for library construction using the NEBNext Small RNA Library Prep Set for Illumina (NEB). Alternative adaptors were used for small RNA library preparation. 3′ SR Adapter: 5′App-NNNNNATCACGAGATCGGAAGAGCACACGTCT-3SpC3; 5′ SR Adapter: 5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′. The libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina Nova-seq 6000 platform. KAS-seq: KAS-seq was performed following a previously reported protocol. Briefly, HCT116 cells were transfected with siPUS7, 7SK ASO, or siCtrl and harvested into 1.5 mL tubes after 3 days. Cells were suspended in 1 mL medium containing 500 mM N3-kethoxal and incubated on a shaker at 37 °C for 10 minutes to label genomic ssDNA. The medium was then discarded, and genomic DNA was extracted. The N3-kethoxal-labeled ssDNA was biotinylated, enriched, and fragmented according to the established KAS-seq protocol. Dual-index libraries were prepared for high-throughput sequencing using the Accel-NGS Methyl-seq DNA Library Kit and sequenced on Illumina Nova-seq 6000 platform. mRNA-seq: HCT116 cells were transfected with siPUS7, 7SK ASO, or siCtrl and harvested into 1.5 mL tubes after 3 days. mRNA was extracted and applied to mRNA-seq using NEBNext® Ultra™ II RNA Library Prep Kit (NEB). The libraries were sequenced on Illumina Nova-seq 6000 platform.

treatment_protocol: HCT116 cells were transfected with siPUS7, 7SK ASO or siCtr and harvested 72 h post transfection.

tag: tissue

value: Colon tissue

value: CRC

value: PUS7 knockdown


title: 15537023, CD4+ T cells, control

description: Library name: 15537023

value: male


title: HCT116-siPUS7-mRNA-seq-rp2

description:
Library name: HCT116-mRNA-seq-8
HCT116-siPUS7_VS_siCtrl-mRNA-seq.DEresults.xlsx

tag: cell line


title: blood, day 40, group 6 #2

description: Library name: PR3287_122_b

value: whole blood

value: NC/Nga


title: blood, day 40, group 6 #1

description: Library name: PR3287_121_b


title: MCD54

city: HANGZHOU

first: junxia

last: min

state: zhejiang province

address: zhejiang university zijingang campus medcine school research building C155

department: school of medicine

institute: zhejiang university

data_processing:
RNA-seq reads were mapped to the mouse reference genome using TopHat.
For sequencing read alignment, a set of possible 17- and 18-bp tags was created from all CCGG sites in the Mus musculus genome assembly; Only uniquely aligned reads were used for gene and exon quantification.
The locations of the CCGG sites were annotated as the gene promoter (-2000 to -1 bp), coding sequence (CDS), exon, intron, 5’ UTR, 3’ UTR, or intergenic region.
The Cufflinks tool was used to quantify isoform expression. Differentially methylated sites were analyzed using a one-way ANOVA (R package) and mapped to signaling pathways using the KEGG pathway mapping tool.
Assembly: mm10

source_name: mice tissue

extract_protocol:
For RNA sequencing, mice livers were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent.
Sequencing libraries were generated using VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina ® （NR604-01/02）following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample.

treatment_protocol: Where indicated, 8-weeks-old male control mice and Slc7a11-LTG mice were fed MCD for 1week (Research Diets, A02082002BR) ,isolated mice liver and extracted RNA.

value: liver


title: MCD53


title: MCD78


title: blood, day 40, group 6 #7

description: Library name: PR3287_127_b


title: HCT116-si7SK-mRNA-seq-rp3

description:
Library name: HCT116-mRNA-seq-6
HCT116-si7SK_VS_siCtrl-mRNA-seq.DEresults.xlsx

tag: cell type

value: 7SK knockdown