title: E-K562-3

city: Xiamen

first: Huifen

last: Cao

email: hfcao_bnu@163.com, hfcao@hqu.edu.cn

state: Fujian

address: 668 Jimei Road

department: School of Biomedical Sciences

institute: Huaqiao University

data_processing:
The genomic and mitochondrial AP site mapping was performed the same way as in the SSiNGLe-ILM protocol (https://protocolexchange.researchsquare.com/article/pex-920/v2) with the two additional filtering steps. One, PCR duplicates were removed. Two, a read-pair was used only if the first base of the read 2 aligned to the reference sequence. The coordinate of one base upstream of the first aligning base of the read 2 was assigned as the position of the AP site. In the samples used for the chrM analysis, reads were aligned separately to the chrM genome sequences and processed in the same way. Then, the coordinates of AP sites mapping to chrM were added to the BED files containing AP sites mapping to the nuclear genome.
Genome_build: HG19(GRCh37) for human reads, mm10 (GRCm38) for the mouse reads and sequences corresponding to the bacterial spike-ins listed in the file spike-ins.fa for the reads from the spike-in control experiments.
Supplementary_files_format_and_content: The *spike-ins.bed files contain coordinates of AP sites in the sequences of spike-ins. All other BED files contain coordinates of AP sites mapped to either mouse or human genomes. The 4th columns show the number of reads representing the AP site.

growth_protocol: Human CML leukemia cell line K562 was obtained from Cell Bank of Chinese Academy of Sciences, and human cervical carcinoma HeLa cell line was obtained from National Infrastructure of Cell Line Resource. Cells were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, ExCell Bio) and 1% (v/v) pen-strep (HyClone) at 37 °C and 5% CO2.

extract_protocol:
12 male mice of C57BL/6J strain from 4 age groups (3-, 12-, 19- and 22-month-old) were used to extract brains, hearts, livers, bone marrow, PBMCs and sperms. Red cells in bone marrow were removed by Red Blood Cell Lysis Buffer (Beyotime). PBMCs were isolated from blood using Ficoll-Paque PLUS (GE Healthcare). The motile spermatozoa were isolated from the mouse epididymal tail by collecting cells floating in the upper layer of RPMI 1640 (Thermo Fisher Scientific). The isolated tissues were immediately put into liquid nitrogen for quick freezing. The whole organs were cut into small pieces and ground into powder by tissue homogenizer in presence of liquid nitrogen to avoid heating. All ground tissue samples were stored at -80C prior to DNA isolation. Genomic DNA from human cell lines and the mouse brain, bone marrow, heart, liver and PBMCs was isolated using TIANamp Genomic DNA kit (Tiangen, DP304) with RNaseA treatment according to the manufacturer's protocols. Genomic DNA from sperm cells was extracted using Sperm DNA Purification Kit (Simgen, 4202050) following the manufacturer?s instructions. All DNA samples were eluted in UltraPure? DNase/RNase-Free Distilled Water (Invitrogen), measured the concentration using SMA6000 (Merinton) and stored at -20C.
Library construction method: SSiNGLe-AP method

treatment_protocol: Two million K562 or HeLa cells were seeded at 1 million cells per ml of culture medium without penicillin-streptomycin solution in 6-well plates. After 16 hr, cells were treated with 1.5 mM MMS (Sigma) for 1 hr or grown without MMS as control. One microgram of the K562 or HeLa genomic DNA was used directly as input into the SSiNGLe-AP protocol. For the MX treatment, after DNase fragmentation, the purified genomic DNA was incubated at 37C for 30 min in 20 µl of 50 mM K3PO4 (pH 7.0) buffer with or without 33 mM MX (Abmole). The DNA was recovered by ethanol precipitation, dissolved in 15 µl water and subjected to SSiNGLe-AP procedure starting from the blocking step preceded by denaturation. All samples were processed with SSiNGLe-AP protocol based on APE1, with exception of some samples where Endo IV was used instead as indicated.

value: chronic myelogenous leukemia cell line

value: treated with Endo IV for AP site detection


title: K562, MMS, rep2

status: Public on Sep 15 2022

type: SRA

country: China

library_source: genomic

molecule: genomic DNA

organism: Homo sapiens

tag: cell line

tag: cell type

tag: treatment

value: treated with MMS for 1 h


title: K562, MMS, NT, rep1

value: untreated


title: Hela, MX, rep3

source_name: HeLa

value: HeLa

value: human cervical carcinoma cell lines

value: treated with MX for 30 min


title: K562, MMS, NT, rep2


title: K562, MMS, rep3


title: K562, MMS, rep1


title: K562, MMS, NT, rep3


title: Hela, MX, rep1


title: Hela, MX, NT, rep2


title: Hela, MX, rep2


title: Hela, MMS, rep3


title: Hela, MX, NT, rep1


title: Hela, MMS, rep1


title: Hela, MMS, rep2


title: Hela, MMS, NT, rep3


title: P-A-2, human genome

description: bacterial spike-in controls in background of human genome DNA

tag: spike-in

value: Leptotrichia wadei


title: P-A-1, human genome


title: Hela, MMS, NT, rep1


title: N-A-1, human genome


title: 3-L-3, rep2, Unblock

description: Unblocked control of corresponding deep-sequencing sample

organism: Mus musculus

source_name: Liver

tag: strain

tag: tissue

value: Liver

tag: age

value: 3 months


title: E-K562-1


title: P-A-3, human genome


title: Hela, MMS, NT, rep2


title: E-K562-2


title: CH12_LAM-HTGTS_WT_restored_rep1

city: Vienna

first: Tobias

last: Neumann

address: Campus-Vienna-Biocenter 1

country: Austria

institute: IMP

description:
Genomic DNA was extracted and c-Myc genomic region  was biotin isolated and PCR enriched
LAM-HTGTS

data_processing:
LAM-HTGTS
Base-calling and demultiplexing:
Raw nanopore fast5 files were basecalled using the Guppy basecaller from Oxford Nanopore Technologies (ONT) 3.6.0+98ff765.
Resulting reads were demultiplexed by detecting barcodes with a custom fork of qcat (https://github.com/nanoporetech/qcat) and splitting reads into chunks at every detected barcode site, assigning the resulting subreads to the majority vote of the detected barcode set.
Only reads >= 50 bp were used to remove linker-linker pairs.
Alignment and downsampling:
Reads were aligned using bwa mem v0.7.17-r1188 and primary alignments were retained with samtools -F 0x0100.
Reads mapping to the bait regions (Myc chr15:61794274-61844667; Irf2bp2 chr8:129089061-129140470) were extracted
Downsampling factors for matched replicates between conditions were calculated and downsampling was performed using picardTools MarkDuplicates (https://broadinstitute.github.io/picard/) v2.18.27.
Translocation and hotspot calling:
The pysam (https://github.com/pysam-developers/pysam)  module was used to extract reads from the bait regions and translocation breakpoints were extracted from all supplementary alignments in the SA bam tag outside the bait region.
Translocations also appearing the respective control samples were subtracted for the final translocation sets.
Hotspots on the resulting translocation bed files were called using hot_scan  (Version from October 17, 2013) with window width 10kb, significance level 0.05 and unadjusted p-values.
Assembly: NCBI mm9
Supplementary files format and content: Tab-delimited tables with translocation quantifications and log2-foldchanges on confident hotspot sets.
Library strategy: LAM-HTGTS

growth_protocol: CH12 cells cultured in complete RPMI medium wit IL4/CD40/TGF beta stimulation.

extract_protocol:
Genomic DNA was extracted and the c-Myc / Irf2bp2 genomic region was biotin isolated and PCR enriched
Libraries were prepared following the nanopore library preparation protocol Ligation Sequencing Kit SQK-LSK109

treatment_protocol: Cells were infected with pMXAIDER vector and electroporated with Cas9-sgRNA-Myc / Cas9-sgRNA-Irf2bp2

tag: genotype

value: WT restored


title: 3-L-3, rep1, Unblock


title: 3-H-3, rep2, Unblock

source_name: Heart

value: Heart


title: 19-L-2, rep2, Unblock

value: 19 months


title: Hela, MX, NT, rep3


title: 3-H-3, rep1, Unblock


title: 19-H-2, rep2, Unblock


title: 19-H-2, rep1, Unblock


title: 19-L-2-rep1

description: Deep-sequencing sample


title: 19-L-2, rep2

description: Deep-sequencing sample


title: 3-H-3, rep1

description: Deep-sequencing sample


title: 19-H-2, rep1

description: Deep-sequencing sample


title: N-A-2, human genome


title: 3-L-3, rep1

description: Deep-sequencing sample


title: 3-H-3, rep2

description: Deep-sequencing sample


title: 19-L-2-rep1, Unblock


title: 19-H-2, rep2

description: Deep-sequencing sample


title: N-A-3, human genome


title: E-3-L-3, rep2


title: E-19-L-2, rep1


title: H1-TKO mESCs, RNH MapR, replicate 1

status: Public on Sep 13 2022

city: Madrid

first: Maria

last: Gomez

address: Nicolas Cabrera, 1

country: Spain

institute: CBMSO(CSIC/UAM)

data_processing:
Alignment against mm10 genome using bwa mem
Peak-calling with MACS2 with standard parameters
Assembly: mm10
Supplementary files format and content: bigwig for browser compatible tracks, narrowPeak for MACS2 peak-calling
Supplementary files format and content: fastq for raw reads
Library strategy: MapR

source_name: mESCs

growth_protocol: Mouse embryonic stem cells were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Biosera), 1x non-essential aminoacids (Gibco), 1mM sodium piruvate (Gibco), 2mM L-glutamine (Gibco), 50 uM B-mercaptoethanol (Gibco), 103 U/mL LIF (ESGRO), 100 U/mL penicillin (Invitrogen) and 100 ug/mL streptomycin (Invitrogen), at 37ºC and 5% CO2

extract_protocol: GST-MNase and GST-RH-MNase protein expression and purification were carried out as described in Yan et al. (2019). MapR experiments were performed exactly as described in Yan et al. (2019) by exposing 5 millions of cells immobilized on Concavalin A-coated beads to 1uM of purified GST-MNase or GST-RH-MNase proteins. MNase-released DNA fragments ranging between 200-1000 bp were size- purified from agarose gels using QIAquick gel extraction kit (Qiagen). Libraries were generated with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) following the manufacturer’s recommendations and sequenced by 2x50 pair-end reads on a NovaSeq platform at Fundación Parque Científico de Madrid.

value: mESCs

value: GST-RH-MNase


title: WT mESCs, MNase MapR

value: WT

value: GST-MNase


title: H1-TKO mESCs, MNase MapR


title: E-19-H-2, rep1


title: E-19-L-2, rep2


title: E-3-L-3, rep1


title: E-3-H-3, rep2


title: 3-L-3, rep2

description: Deep-sequencing sample


title: WT mESCs, RNH MapR, replicate 2


title: WT mESCs, RNH MapR, replicate 1


title: E-3-H-3, rep1


title: E-19-H-2, rep2


title: 8849-5123

status: Public on Sep 12 2022

city: Toronto

first: Daniel

last: Schramek

state: ON

address: 600 University Ave, Room 1080

country: Canada

institute: Lunenfeld- Tanenbaum Research Institute

library_source: transcriptomic

data_processing:
Reads were aligned to mm10 using Hisat2 (2.1.0)
Raw reads obtained from bam files using featureCounts from subread R package; annotation: mm10  ensembl (Mus_musculus.GRCm38.102.gtf)
DESeq2 package in R was used to determine differentially expressed genes between samples
Assembly: mm10
Supplementary files format and content: tab-delimited raw read counts
Supplementary files format and content: tab-delimited DESEQ2 results: Healthy Brain Tissue (Ctrl) vs Type II Low Grade glioma Tumour Tissue

molecule: total RNA

source_name: rs55705857 mutant IDH mutant  brain tumour tissue

extract_protocol:
Mouse brains were harvested and embedded into paraffin, 10x 10 micron thick saggital paraffin sections were generated and tumour/tissue was scraped off the slide and RNA was extracted.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Brain Tumor

value: rs557+/+;Idh1R132H/+;p53mut; Cas9; Atrx KO


title: ADJ6, scRNAseq

city: Beijing

first: Kai

last: Chen

address: 8th Xishiku Street

department: General Surgery

institute: Peking University First Hospital

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
Raw data (BCL files) from Illumina Novaseq6000 platform was converted to fastq files using Illumina-implemented software bcl2fastq (v2.19.0.316). cDNA reads were aligned to human reference genome (GRCh38). Low-quality cells and genes filtering, barcode and UMI counting were performed with the cellranger software (v6.1.2) to obtain the filtered gene-cell matrixes. Next, gene-cell matrixes were imported into R software to further filtered out low-quality cells (< 500 genes/cell, > 25% mitochondria genes, < 1000 transcripts/cell) and genes (<10 cells/gene) using ‘Seurat’ R package (v3.2.3). Gene expression levels were normalized (LogNormalize) with “NormalizedData” function. A total of 2,000 highly variable genes were selected and used to conduct PCA reduction dimension. The t-distributed stochastic neighbor embedding (t-SNE) was performed. The ‘Soupx’ R package was applied to reduce the ambient mRNA contamination. Doublets were identified using the ‘DoubletFinder’ R package (v2.0.3), assuming that it was around 5% doublet formation rate to the loaded cells per specimen in a droplet channel. In addition, the ‘Harmony’ R package (https://github.com/immunogenomics/harmony) was used to integrate gene-cell matrixes derived from different specimens. We identified each cell cluster by matching the cluster-specific genes with known signatures of cell populations reported in previous studies and CellMarker database.
Assembly: GRCh38
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Adjacent noncancerous pancreatic tissue

extract_protocol:
All fresh resection specimens were preserved in the tissue storage solution (Miltenyi, Cat. no. 130-100-008) on ice and transported to the laboratory in CapitalBio Technology company within 1.5-2 hours. To ensure the success of tissue dissociation, multiple resection specimens from the same patients were digested. All specimens were cut into around 1 mm pieces and incubated in an optimal digestive solution, including enzyme cocktail, consisting of the Type VIII Collagenase (Sigma-Aldrich, Cat. no. C2139), DNase I (Sigma-Aldrich, Cat. no. D5025), trypsin inhibitor (Sigma-Aldrich, Cat. no. T6522), and Dispase II (neutral protease, grade II, Sigma-Aldrich, Cat. no. 4942078001). The single cell suspension was filtered with a 40 μm cell strainer (BD, Cat. no. 352340), then incubated with red blood cell lysis buffer (Roche, Cat. no. 11814389001) at 4°C for 10 minutes.
The concentration of single cell suspension was determined using the Count Star instrument and adjusted to 1000 cells/μl. The cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion using single cell 3’ Library and Gel Bead Kit v3.1 (10x Genomics, Cat. No. 1000121) and Chromium Next GEM Chip G Single Cell Kit (10x Genomics, Cat. No. 1000120) according to the manufacturer’s instructions. Briefly, cells were suspended in PBS containing 0.04% BSA. About 6,000 cells were loaded to each channel, and the target cell will be captured about 3000 cells per channel. Captured cells were lysed and the released RNA were barcoded by reverse transcription in each GEM. Reverse transcription was completed in 200 ul tubes (NEST Biotechnology, Cat. No. 401001) on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 minutes, then at 85°C for 5 min, and hold at 4°C. The cDNA libraries were generated, amplified, and quality assessed using the Agilent 4200. Finally, the cDNA libraries were sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with paired-end 150 bp (PE150) reading strategy.

value: Adjacent noncancerous pancreatic tissue

value: No treatment


title: PDAC5, scRNAseq

description: 10x Genomics

source_name: Pancreatic cancer

value: Pancreatic cancer


title: ADJ2, scRNAseq

description: 10x Genomics


title: PDAC6, scRNAseq

description: 10x Genomics


title: 2121-0387

value: rs55705857 mutant IDH mutant  brain tumour tissue

value: rs557AG+4bp;Idh1R132H/+;p53mut; Cas9; Atrx KO


title: PDAC2, scRNAseq

description: 10x Genomics


title: H1-TKO mESCs, RNH MapR, replicate 2


title: 9053-0389


title: ATAC_DN12_CD8_MED12

status: Public on Sep 09 2022

city: Stanford

first: Julia

last: Belk

state: California

address: 450 Serra Mall

country: USA

institute: Stanford University

data_processing:
reads were trimmed with fastp
reads were aligned to hg38 with hisat2
reads were deduplicated using picard
peaks were called using macs2
normalized bigwig files were generated using Rtracklayer
Assembly: hg38
Supplementary files format and content: bigwig genome wide signal track

source_name: primary T cells

extract_protocol:
ATACseq: Approximately 100k CAR-T cells were used from each sample. Nuclei were isolated with ATAC-LB (10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL) . ChIP: CAR-T cells (3 x 106) were double crosslinked by 50mM DSG (disuccinimidyl glutarate, #C1104 - ProteoChem) for 30 minutes followed by 10 minutes of 1% formaldehyde. Formaldehyde was quenched by the addition of glycine. Nuclei were isolated with ChIP lysis buffer (1% Triton x-100, 0.1% SDS, 150 mM NaCl, 1mM EDTA, and 20 mM Tris, pH 8.0). Nuclei were sheared with Covaris sonicator using the following setup: Fill level – 10, Duty Cycle – 5, PIP – 140, Cycles/Burst – 200, Time – 4 minutes). Sheared chromatin was immunoprecipitated overnight.
ATACseq libraries were prepared with Nextera DNA Library Preparation Kit (Illumina). ChIPseq libraries were prepared with Ovation Ultralow Library System V2 (Tecan).

treatment_protocol: cells were stimulated with anti-idiotype antibody for 3 hours

value: unstimulated

tag: chip antibody


title: PDAC3, scRNAseq

description: 10x Genomics


title: PDAC1, scRNAseq

description: 10x Genomics


title: ADJ1, scRNAseq

description: 10x Genomics


title: ATAC_DN12_CD4_MED12


title: ATAC_DN67_CD8_MED12


title: ATAC_DN67_CD4_MED12


title: ATAC_DN12_CD8_AAVS1


title: ATAC_DN67_CD8_AAVS1


title: ATAC_DN67_CD4_AAVS1


title: ATAC_DN59_CD8_MED12


title: ATAC_DN59_CD4_MED12


title: ATAC_DN59_CD4_AAVS1


title: ATAC_DN12_CD4_AAVS1


title: RNA_DN12_CD4_MED12

description:
TPM_genes_CD4_CD8.xlsx
KF122

data_processing:
Sequencing libraries were prepared by Novogene (Sacramento, CA) and 150 bp paired end sequencing at a depth of 3 x 107 reads per sample was obtained using the Illumina NovaSeq6000 platform
FASTQ files were generated by Novogene.
Transcripts were quantified with Salmon
DESeq2 was used to generate TPM (transcripts per million)
Assembly: hg38
Supplementary files format and content: Data transcripts per million summarized to gene level. Each column in the table is a sample. Each row is a gene.

extract_protocol:
Total RNA was extracted from fresh cells using the Qiagen Rneasy kit.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: cells were stimulated with anti-idiotye antibody for 3 hours


title: PDAC4, scRNAseq

description: 10x Genomics


title: 7103-1124


title: RNA_DN12_CD8_AAVS1

description:
TPM_genes_CD4_CD8.xlsx
KF121


title: RNA_DN67_CD8_MED12

description:
TPM_genes_CD4_CD8.xlsx
KF119


title: RNA_DN12_CD4_AAVS1

description:
TPM_genes_CD4_CD8.xlsx
KF120


title: RNA_DN67_CD4_MED12

description:
TPM_genes_CD4_CD8.xlsx
KF118


title: RNA_DN67_CD4_AAVS1

description:
TPM_genes_CD4_CD8.xlsx
KF116


title: RNA_DN67_CD8_AAVS1

description:
TPM_genes_CD4_CD8.xlsx
KF117


title: RNA_DN59_CD8_MED12

description:
TPM_genes_CD4_CD8.xlsx
KF115


title: Breast cancer cells - MCF7 and T47D multiomic/ATAC, si_Control_plus_Veh

status: Public on Sep 08 2022

city: Portland

first: Ellen

last: Langer

state: OR

address: 2720 SW Moody Ave

department: DOS (Department of Oncological Sciences)

institute: Oregon Health and Science University

description:
MCF7_multiome_ATAC_counts.tsv
MCF7_multiome_metadata.tsv
T47D_multiome_ATAC_counts.tsv
T47D_multiome_metadata.tsv

library_source: genomic single cell

data_processing:
Aligning and gene counting with CellRanger version 3.0.2
Filtering quality cells with Seurat (v 3.1.1)
Demultiplexing cell identity with Seurat (v 3.1.1)
Clustering and Differential Expression with Seurat (v 3.1.1)
Topic Modeling Analysis with TITAN
Enriched genes were tested for significance of overlap with the cistrome database ChIP-seq human factor binding for T4D, MCF7 and ZR-75-1
Assembly: hg38

source_name: Breast cancer cell line

growth_protocol: MCF-7, T-47D and ZR-75-1 cell lines were obtained from ATCC. MCF-7 cells were maintained in high glucose DMEM with L-glutamine and pyruvate (Gibco) supplemented with 10% FBS (HyClone) and 1% Penicillin-streptomycin (Gibco). T-47D and ZR-75-1 cells were maintained in RPMI 1640 (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were grown at 37oC with 5% CO2.

extract_protocol:
After treatment, cells were detached using phenol-red free TrypLE (Gibco) and collected in 1X DPBS (Gibco). Single cell suspensions from matrigel embedded PdXOs were obtained following dispase and TrypLE dissociation as described in their source publication. After one wash in 1X DPBS, cells were resuspended in 2% BSA-DPBS at a concentration of 10000 cells/ml. Following incubation with TruStain Fcx blocking solution (BioLegend), Totalseq A Hashtag antibodies were added per treatment group. Cells were washed 3 times with 1 ml 2%BSA-DPBS and resuspended to a volume of ~10000 cells/ml. Equal volumes of each treatment group were pooled and cell pool was assessed for cell concentration and viability.
Single-cell cDNA and HTO libraries were generated using Chromium Single Cell 3′ version 3 and 3.1 reagents (10x genomics and Biolegend) per manufacturer’s protocols. Chromium Single Cell Multiome ATAC + Gene Expression kit protocols were used per manufacturer's guidelines to generate the cDNA and ATAC libraries for the multiomic data.
Single-end sequencing of libraries was performed per manufacturer’s protocol on a Novaseq 6000 (Illumina) sequencer with HTO libraries constituting 5% of the sample.

treatment_protocol: Three days prior to hormone stimulation, cells or PdXO were transferred into their respective phenol-red free media supplemented with 5% charcoal-stripped FBS (Gibco) and 1% Penicillin-streptomycin (Gibco) and PdXO specific supplements. Culture media was refreshed 1 day prior to treatments. 17-β-estradiol (E2, Millipore Sigma), progesterone (PG, Millipore Sigma) or both hormones in combination were added at a final concentration of 100 nmol/l each for given time points.

value: vehicle

value: MCF-7 and T-47D

tag: tag

value: multiomic (scRNA and scATAC)


title: ATAC_DN59_CD8_AAVS1


title: Breast cancer organoid - HCI011, control

description:
HCI011_counts.txt
HCI011_metadata.txt

source_name: Patient derived organoid - HCI011

value: organoid


title: Breast cancer organoid - HCI011, ES

description:
HCI011_counts.txt
HCI011_metadata.txt

value: Estrogen Treatment


title: Breast cancer cells - MCF7 and T47D multiomic/GEX, si_Control_plus_E2

description:
MCF7_multiome_RNA_counts.tsv
MCF7_multiome_metadata.tsv
T47D_multiome_RNA_counts.tsv
T47D_multiome_metadata.tsv


title: Breast cancer cells - MCF7 and T47D multiomic/GEX, si_Control_plus_Veh


title: RNA_DN59_CD4_AAVS1

description:
TPM_genes_CD4_CD8.xlsx
KF112


title: Breast cancer cells - MCF7 and T47D multiomic/ATAC, si_Control_plus_E2


title: Breast cancer organoid - HCI003, HTO

description:
HCI003_counts.txt
HCI003_metadata.txt

source_name: Patient derived organoid - HCI003

value: ALL


title: RNA_DN12_CD8_MED12

description:
TPM_genes_CD4_CD8.xlsx
KF123


title: Breast cancer organoid - HCI003

description:
HCI003_counts.txt
HCI003_metadata.txt


title: siCTL-non-HUVEC rep1 (RNA-seq)

status: Public on Sep 06 2022

city: Seoul

first: GooTaeg

last: Oh

address: 52, Ewhayeodae-gil

department: Department of Life Sciences

country: South Korea

institute: Ewha Womans University

data_processing:
The sequence reads were counted by StringTie-2.1.3b and genes showing <10 read counts total were pre-filtered.
DESeq2 was used to obtain normalized count and identify differentially expressed genes in siCTL or siOASL transfected HUVECs (±TNFα/IFNγ).
For performing downstream analysis such as drawing heatmap, the count data was rlog-transformed using DESeq2
Assembly: GRCh38
Supplementary files format and content: RNA_normalized_count.txt.gz, RNA_transformed_count.txt.gz

source_name: HUVEC

growth_protocol: HUVECs were obtained from the American Type Culture Collection (Manassas, VA). HUVECs were cultured in EGM-2 BulletKit (Lonza, CC-3162) medium containing 10% FBS, 2mM L-glutamine, and penicillin (10,000 Units/mL)/streptomycin (10,000 ug/mL) and used at passage 7-10 for experiments. Cells were routinely maintained in a humidified chamber at 37℃ and 5% CO2.

extract_protocol:
HUVECs were stimulated for 3hours and total RNA was manually extracted using Trizol reagent and purified by ethanol precipitation. TruSeq Stranded Total RNA LT Sample Prep Kit (Gold) was used with total RNA for the construction of sequencing libraries.
TruSeq Stranded Total RNA LT Sample Prep Kit (Gold) was used for the construction of sequencing libraries and total RNA libraries were processed following standard Illumina protocols.

treatment_protocol: Two replicates of siRNA-transfected HUVECs per group were treated with or without TNFα and IFNγ added in the serum-free EGM-2 for 3hours.

value: Human umbilical vein endothelial cells

value: non-treated

value: Control


title: siCTL-non-HUVEC rep2 (RNA-seq)


title: Octopus Adult Arm Left 1 Tip [OB2]

status: Public on Sep 07 2022

city: Abu Dhabi

first: Kirsten

last: Sadler Edepli

state: Abu Dhabi

department: Biology

country: United Arab Emirates

institute: New York University Abu Dhabi

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads where first assessed for quality using FastQC v0.11.5. Trimmomatic v0.36 was used for quality trimming and adapter sequence removal, with the parameters (ILLUMINACLIP: trimmomatic_adapter.fa:2:30:10 TRAILING:3 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36)
The dataset from hatchling was also processed with Fastp in order to remove poly-G tails and Novaseq/Nextseq specific artifacts. Following the Fastp quality trimming, the reads were assessed again using FastQC
Quality trimmed reads were used to produce psuedoalignments using Kallisto, and quantification was assessed with the --bias flag using the reference O. bimaculoides genome (PRJNA270931) and its corresponding annotation
The resulting transcripts per kilobase per million (TPMs) from the pseudo-counts were used for further downstream analysis
Assembly: PRJNA270931
Supplementary files format and content: .tsv file containing pseudo-counts (TPMs) for each transcript

organism: Octopus bimaculoides

source_name: Adult arm tip, collected till 1.5  cm from the arm tip

growth_protocol: Animals were catched from wild and manteined in husbandy accordingly to the current policy for the use of cephalopods at Marine Biology Laboratories (MBL, https://www.mbl.edu/policies/files/2018/07/J1.10-CEPH-Policies-Procedures-Jan-1-31-2020-fillable-form.pdf).

extract_protocol:
For each sample, tissues stored at -80°C were ground in liquid nitrogen using a mortar placed in dry ice. Resulting powder was aliquoted and 15 mg of this powder was used to extract  RNA. RNA was extracted using Trizol (Invitrogen) following the manufacturer’s instructions with some modifications. Briefly, during precipitation in isopropanol, 10 µg of Glycoblue (Thermo Fisher Scientific) was added and precipitation was performed overnight at -20°C followed by 1 hour centrifuge at 12000g at 4°C. RNA was resuspended in water . RNA was treated by DNAse I for 30 minutes at 37 °C followed by RNA purification (RapidOut DNA Removal Kit – Thermo Fisher Scientific).
RiboZero was used to remove ribosomal RNA and the remaining sample was used for library preparation according to manufacturer’s instructions (Illumina) from 250 ng of RNA. Libraries were sequenced on NextSeq550 (Illumina) to obtain 150 bp paired end reads.

treatment_protocol: Animals were euthanized by overdose of ethanol and tissues were immediately collected, flash frozen in liquid nitrogen, and stored at -80°C.

tag: Sex

value: Male

tag: developmental stage

value: Adult

value: arm tip


title: siOASL-non-HUVEC rep1 (RNA-seq)

value: OASL knockdown


title: Participant_8

city: Rochester

first: Juilee

last: Thakar

state: New York

address: 601 Elmwood Ave

department: Microbiology and Immunology

institute: University of Rochester

description: scRNA-seq data of PBMCs

data_processing:
Cell Ranger (version 2.1.1; 10x Genomics) was used for demultiplexing and alignment using default parameters
The Seurat R package was used to further process the gene counts obtained from the Cell Ranger pipeline.
Cells that express < 200 genes, > 2500 genes, and > 5% mitochondrial genes were filtered out.
Genes expressed in < 3 cells were filtered out.
Gene counts were per-cell normalized and log2-transformed.
Assembly: Human reference genome GRCh38 (Ensembl 93)
Supplementary files format and content: Output of the Cell Ranger count Analysis Pipeline (h5 format; unfiltered and unnormalized raw gene count matrices)
Supplementary files format and content: Comma-separated matrix files containing the output of the Seurat pipeline, ie,per-cell normalized log2-transformed counts and associated metadata containing per-cell annotations and clustering information (csv format). Contains processed data and metadata for all samples.

source_name: peripheral blood mononuclear cells

extract_protocol:
30 mls of blood per study participant was collected in ACD vacutainers and was processed within 2 - 3 hours of collection. Peripheral Blood Mononuclear Cells (PBMCs) were isolated using Ficoll density gradient centrifugation. 5 million PBMCs were preserved using RNAlater (Thermo Fisher) and were used for single-cell sequencing. Frozen vials containing cells in RNAlater were thawed quickly in a 37-degree water bath. Cell suspension was transferred to a 15ml conical tube. 10 ml PBS/2%FBS was slowly added. Samples were centrifuged at 1600rpm for 6 min. Washes were repeated for an additional 2 times for a total of 3 washes. Using the MACS Miltenyi Biotec Dead Cell removal kit (PN130-090-101), dead cells were removed using manufacturer’s recommendations. Cells were counted and cellular suspensions were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead-in-Emulsions (GEMs).
Single-cell RNA-Seq libraries were prepared using Chromium Single-Cell 3’ Library & Gel Bead Kit (10x Genomics). The beads were dissolved, and cells were lysed per manufacturer’s recommendations. GEM reverse transcription (GEM-RT) was performed to produce a barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken, and the pooled post-GEM-RT reaction mixtures were recovered, and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, PN37002D, ThermoFisher Scientific). The entire purified post GEM-RT product was amplified by PCR. This amplification reaction generated sufficient material to construct a 3’ cDNA library.  Enzymatic fragmentation and size selection was used to optimize the cDNA amplicon size and indexed sequencing libraries were constructed by End Repair, A-tailing, Adaptor Ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification.

tag: hiv status

value: HIV+

tag: atherosclerosis  (as) status

value: AS+

tag: cholesterol in mg/dl

tag: high-density lipids in mg/dl

value: peripheral blood mononuclear cells


title: Participant_6

description: scRNA-seq data of PBMCs


title: RNA_DN59_CD8_AAVS1

description:
TPM_genes_CD4_CD8.xlsx
KF113

value: unstimulated


title: Participant_5

description: scRNA-seq data of PBMCs


title: RNA_DN59_CD4_MED12

description:
TPM_genes_CD4_CD8.xlsx
KF114


title: Breast cancer cells - TAMR

description:
TAMR_E2_counts.txt
TAMR_E2_metadata.txt

value: TAMR


title: Octopus Adult Arm Left 1 Tip [OB9]

value: Female


title: Participant_2

description: scRNA-seq data of PBMCs

value: AS-


title: Participant_4

description: scRNA-seq data of PBMCs


title: Participant_1

description: scRNA-seq data of PBMCs


title: siOASL-non-HUVEC rep2 (RNA-seq)


title: Participant_3

description: scRNA-seq data of PBMCs


title: Participant_7

description: scRNA-seq data of PBMCs


title: DNA mehtylation of human gastric cancer (S44TP)

type: genomic

city: Shinagawa-ku

first: Hideyuki

last: Takeshima

state: Tokyo

department: Institute for Advanced Life Sciences

country: Japan

institute: Hoshi University

hyb_protocol: Standard Illumina Protocol

scan_protocol: The array was scanned with an iScan System

data_processing: The data were analyzed using GenomeStudio Methylation Module Software.

label: Cy5 and Cy3

label_protocol: Standard Illumina Protocol

extract_protocol: Genomic DNA was extracted by standard phenol/chloroform method, and was quantified using a Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies).

tag: disease state

value: Gastric cancer

tag: stomach location

value: Body

tag: phenotype

value: Mucinous adenocarcinoma (poorly differenciated adenoca)

tag: ethnicity

value: Asian


title: DNA mehtylation of human gastric cancer (S42TP)

value: poorly differenciated adenoca


title: DNA mehtylation of human gastric cancer (S47TP)

value: poorly differenciated adenoca and sinnet cell carcinoma


title: DNA mehtylation of human gastric cancer (S51TP)


title: DNA mehtylation of human gastric cancer (S43TP)

value: poorly to well adenoca


title: DNA mehtylation of human gastric cancer (S53TP)

value: moderately to poorly adenoca


title: human gastric cancer (S152TP)

source_name: stomach


title: human gastric cancer (S131TP)

value: Poorly differentiated adenocarcinoma, solid type / moderately differentiated


title: human gastric cancer (S150TP)

value: Moderately differentiated adenocarcinoma / Poorly differentiated adenocarcinoma


title: human gastric cancer (S124TP)

value: Poorly differentiated adenocarcinoma / Mucinous adenocarcinom


title: DNA mehtylation of human gastric cancer (S45TP)

value: well to moderate adenoca


title: DNA mehtylation of human gastric cancer (S37TP)

value: Poorly to moderately differenciated adenoca


title: DNA mehtylation of human gastric cancer (S39TP)


title: human gastric cancer (S137TP)

value: Papillary adenocarcinoma / moderately differentiated


title: human gastric cancer (S25TP)

description: SAMPLE 23

value: stomach

value: Poorly to moderately differentiated adenoca


title: human gastric cancer (S23TP)

description: SAMPLE 21

value: moderately differentiated adenoca


title: human gastric cancer (S141TP)

value: Gastric cancer


title: human gastric cancer (S54TP)

value: Body


title: DNA mehtylation of human gastric cancer (S35TP)

value: papillary and moderately adenoca


title: human gastric cancer (S26TP)

description: SAMPLE 24

value: Mucinous adenocarcinoma (poorly differentiated adenoca)

value: Asian


title: human gastric cancer (S151TP)


title: DNA mehtylation of human gastric cancer (S40TP)

value: moderately differenciated adenoca and papillary


title: human gastric cancer (S31TP)

description: SAMPLE 25

value: poorly differentiated adenoca


title: human gastric cancer (S154TP)


title: DNA mehtylation of human gastric cancer (S36TP)

value: papillary and well differenciated adenoca


title: DNA mehtylation of human gastric cancer (S33TP)

value: moderately to poorly adenoca


title: siOASL-non-HUVEC rep2 (miRNA-seq)

data_processing:
The sequence reads were counted by miRDeep2 and miRNA showing <10 read counts total were pre-filtered.
DESeq2 was used to obtain normalized count and identify differentially expressed miRNA in siCTL or siOASL transfected HUVECs (±TNFα/IFNγ).
For performing downstream analysis such as drawing heatmap, the count data was rlog-transformed using DESeq2
Assembly: GRCh38
Supplementary files format and content: miRNA_normalized_count.txt.gz, miRNA_transformed_count.txt.gz

growth_protocol: HUVECs obtained from the American Type Culture Collection (Manassas, VA). HUVECs were cultured in EGM-2 BulletKit (Lonza, CC-3162) medium containing 10% FBS, 2mM L-glutamine, and penicillin (10,000 Units/mL)/streptomycin (10,000 ug/mL) and used at passage 7-10 for experiments. Cells were routinely maintained in a humidified chamber at 37℃ and 5% CO2.

extract_protocol:
HUVECs were stimulated  for 3hours and total RNA was manually extracted using Trizol reagent. TruSeq Stranded Small RNA Library Prep Kit was used for the construction of small RNA sequencing libraries.
RNA libraries were prepared by TruSeq Small RNA Library Prep kit for sequencing using standard Illumina protocols.

tag: cell type

value: Human umbilical vein endothelial cells (HUVEC)

tag: treatment

value: not treated

value: OASL knockdown


title: DNA mehtylation of human gastric cancer (S34TP)

value: Antrum

value: poorly differenciated adenoca


title: human gastric cancer (S24TP)

description: SAMPLE 22

tag: tissue

value: stomach


title: siCTL-non-HUVEC rep1 (miRNA-seq)

value: Control


title: sci-Thp1-M.tb [V300067419_L02_9]

status: Public on Sep 05 2022

city: ShangHai

first: Da

last: Lin

address: No. 650 Songjiang Road

institute: Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

description:
V300067419_L02_9
sci_ra_pairs.tar.gz

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
In PETs extraction step, the parameter “--fq1 [R1_file] --fq2 [R2-file] –linker GTCGGANNNNNNNNGCTAGCNNNNNNNNTCCGAC --enzyme T^TA^A” were used.
For cell split step, the default parameters were used: 1, 2, 4 differences of hamming distance were allowed for the comparison between “read barcode VS library barcode”, “barcode1 VS barcode2 (within same reads)”, “barcode R1 VS barcode R2 (barcode within R1 and R2).”
Single cell embedding using the scHiCTools package, all cells matrix was generated with 1 Mb resolution.
Assembly: hg19
Supplementary files format and content: Sorted pairs file for each single cell
Library strategy: sciDLO Hi-C

source_name: Peripheral blood

extract_protocol:
After multiple displacement amplification (MDA) reaction, 1 μl 10% SDS and 79 μl ddH2O were added to the sample, and the DNA was purified by 200 μl VAHTS DNA Clean Beads
Single-cell-indexed DLO Hi-C (sciDLO Hi-C) was based on DLO Hi-C and two rounds of molecular barcoding to capture the chromatin conformation of individual cells

value: macrophage

value: Peripheral blood


title: human gastric cancer (S162TP)

value: Gastric cancer

value: Tubular adenocarcinoma


title: sci-Thp1-M.tb [V300067419_L02_8]

description:
V300067419_L02_8
sci_ra_pairs.tar.gz


title: DNA mehtylation of human gastric cancer (S32TP)

value: Body


title: siCTL-non-HUVEC rep2 (miRNA-seq)


title: sci-Thp1-M.tb [V300067419_L02_7]

description:
V300067419_L02_7
sci_ra_pairs.tar.gz


title: sci-Thp1-M.tb [V300067419_L02_60]

description:
V300067419_L02_60
sci_ra_pairs.tar.gz


title: sci-Thp1-M.tb [V300067419_L02_58]

description:
V300067419_L02_58
sci_ra_pairs.tar.gz


title: sci-Thp1-M.tb [V300067419_L02_4]

description:
V300067419_L02_4
sci_ra_pairs.tar.gz


title: sci-Thp1-M.tb [V300067419_L02_5]

description:
V300067419_L02_5
sci_ra_pairs.tar.gz


title: sci-Thp1-M.tb [V300067419_L02_57]

description:
V300067419_L02_57
sci_ra_pairs.tar.gz


title: sci-Thp1-M.tb [V300067419_L02_2]

description:
V300067419_L02_2
sci_ra_pairs.tar.gz


title: sci-Thp1-M.tb [V300067419_L02_6]

description:
V300067419_L02_6
sci_ra_pairs.tar.gz


title: sci-Thp1-macro [V300067419_L04_9]

description:
V300067419_L04_9
sci_thpp_pairs.tar.gz


title: sci-Thp1-macro [V300067419_L04_8]

description:
V300067419_L04_8
sci_thpp_pairs.tar.gz


title: sci-Thp1-M.tb [V300067419_L02_59]

description:
V300067419_L02_59
sci_ra_pairs.tar.gz


title: sci-Thp1-M.tb [V300067419_L02_1]

description:
V300067419_L02_1
sci_ra_pairs.tar.gz


title: sci-Thp1-macro [V300067419_L04_7]

description:
V300067419_L04_7
sci_thpp_pairs.tar.gz


title: sci-Thp1-macro [V300067419_L04_6]

description:
V300067419_L04_6
sci_thpp_pairs.tar.gz


title: human gastric cancer (S21TP)

description: SAMPLE 19

value: Asian


title: human gastric cancer (S15TP)

description: SAMPLE 13

value: Mucinous adenocarcinoma (poorly differentiated adenoca and signet)


title: human gastric cancer (S17TP)

description: SAMPLE 15

value: well differentiated adeno and papillary


title: human gastric cancer (S18TP)

description: SAMPLE 16

value: poorly differentiated adenoca


title: human gastric cancer (S20TP)

description: SAMPLE 18


title: human gastric cancer (S10TP)

description: SAMPLE 8

value: moderately to poorly adenoca


title: sci-Thp1-macro [V300067419_L04_59]

description:
V300067419_L04_59
sci_thpp_pairs.tar.gz


title: sci-Thp1-macro [V300067419_L04_58]

description:
V300067419_L04_58
sci_thpp_pairs.tar.gz


title: human gastric cancer (S14TP)

description: SAMPLE 12

value: well and poorly differentiated adenoca


title: sci-Thp1-macro [V300067419_L04_57]

description:
V300067419_L04_57
sci_thpp_pairs.tar.gz


title: human gastric cancer (S19TP)

description: SAMPLE 17

value: Poorly to moderately differentiated adenoca


title: human gastric cancer (S1TP)

description: SAMPLE 1

value: poorly differentiated adenoca and signet cell carcinoma


title: human gastric cancer (S16TP)

description: SAMPLE 14

value: Antrum


title: sci-Thp1-macro [V300067419_L04_4]

description:
V300067419_L04_4
sci_thpp_pairs.tar.gz


title: sci-Thp1-macro [V300067419_L04_3]

description:
V300067419_L04_3
sci_thpp_pairs.tar.gz


title: sci-Thp1-macro [V300067419_L04_2]

description:
V300067419_L04_2
sci_thpp_pairs.tar.gz


title: human gastric cancer (S2TP)

description: SAMPLE 2

value: moderately differentiated adenoca


title: human gastric cancer (S11TP)

description: SAMPLE 9

value: stomach

value: Anturm

value: Endocrine carcinoma (poorly differentiated adenoca)


title: sci-Thp1-mono [V300067419_L03_9]

description:
V300067419_L03_9
sci_thp_pairs.tar.gz

value: Peripheral blood


title: sci-Thp1-M.tb [V300067419_L02_10]

description:
V300067419_L02_10
sci_ra_pairs.tar.gz

value: macrophage


title: human gastric cancer (S22TP)

description: SAMPLE 20

value: Gastric cancer


title: human gastric cancer (S12TP)

description: SAMPLE 10


title: sci-Thp1-mono [V300067419_L03_8]

description:
V300067419_L03_8
sci_thp_pairs.tar.gz


title: sci-Thp1-macro [V300067419_L04_10]

description:
V300067419_L04_10
sci_thpp_pairs.tar.gz


title: sci-Thp1-macro [V300067419_L04_5]

description:
V300067419_L04_5
sci_thpp_pairs.tar.gz


title: human gastric cancer (S5TP)

description: SAMPLE 5

value: Body

value: well differentiated adeno


title: sci-Thp1-mono [V300067419_L03_7]

description:
V300067419_L03_7
sci_thp_pairs.tar.gz


title: human gastric cancer (S4TP)

description: SAMPLE 4

value: Asian


title: sci-Thp1-mono [V300067419_L03_60]

description:
V300067419_L03_60
sci_thp_pairs.tar.gz


title: sci-Thp1-mono [V300067419_L03_5]

description:
V300067419_L03_5
sci_thp_pairs.tar.gz


title: sci-Thp1-mono [V300067419_L03_59]

description:
V300067419_L03_59
sci_thp_pairs.tar.gz


title: human gastric cancer (S13TP)

description: SAMPLE 11


title: sci-Thp1-M.tb [V300067419_L02_3]

description:
V300067419_L02_3
sci_ra_pairs.tar.gz


title: sci-Thp1-mono [V300067419_L03_6]

description:
V300067419_L03_6
sci_thp_pairs.tar.gz


title: sci-Thp1-mono [V300067419_L03_4]

description:
V300067419_L03_4
sci_thp_pairs.tar.gz


title: sci-Thp1-mono [V300067419_L03_58]

description:
V300067419_L03_58
sci_thp_pairs.tar.gz


title: sci-Thp1-mono [V300067419_L03_57]

description:
V300067419_L03_57
sci_thp_pairs.tar.gz


title: sci-Thp1-mono [V300067419_L03_3]

description:
V300067419_L03_3
sci_thp_pairs.tar.gz


title: sci-Thp1-mono [V300067419_L03_2]

description:
V300067419_L03_2
sci_thp_pairs.tar.gz


title: sci-Thp1-mono [V300067419_L03_10]

description:
V300067419_L03_10
sci_thp_pairs.tar.gz


title: human gastric cancer (S9TP)

description: SAMPLE 7


title: sci-Thp1-macro [V300067419_L04_60]

description:
V300067419_L04_60
sci_thpp_pairs.tar.gz


title: sci-Thp1-macro [V300067419_L04_1]

description:
V300067419_L04_1
sci_thpp_pairs.tar.gz


title: sci-Thp1-mono [V300067419_L03_1]

description:
V300067419_L03_1
sci_thp_pairs.tar.gz


title: siOASL-non-HUVEC rep1 (miRNA-seq)

value: Human umbilical vein endothelial cells (HUVEC)

value: not treated

value: OASL knockdown


title: Thp1-M.tb-rep1

data_processing:
Trimmomatic (version: 0.33) was used to filter out the low quality bases and adapter sequences. Clean reads longer than 36 bp at both ends were kept for further processing.
TopHat (version: v2.1.1) with bowtie2 (version: 2.3.5.1) was used to align the paired end RNA-Seq reads to human reference genome (hg19).
HTSeq (version: 0.11.2) was used to count the mapped reads on the transcripts corresponding to each gene.
DESeq2 was used for normalization and differential expression analysis.
Assembly: hg19
Supplementary files format and content: tab-delimited text files include raw reads counts for each Sample

extract_protocol:
RNA was extracted using the RNAiso Plus (Takara, Catalog code: 9109) according to manufacturer’s protocol.
Sequencing libraries were prepared using the VAHTS Stranded mRNA-Seq Library Prep Kit (Vazyme, Catalog code: NR602-02) according to manufacturer's protocol.

tag: cell line

value: WT

value: H37Ra infection


title: hMDMs-control-rep3

source_name: hMDMs primary cells

value: hMDMs primary cells

tag: genotype


title: Human hMDMs cells infected by H37Rv_4

data_processing:
ChIA-PET Tool was modified and used for linker filtering.
Two end of interacted fragments were seperated by the command awk.
Two end of interacted fragments were separately mapped to hg19 by bwa v0.7.12-r1039 with the parameter -n 0, and then unique mapping reads were kept.
Samtools v1.2 was used to transfer the sam format to bed format.
Two end bed format files were combined to bedpe file by a perl script.
Redundancies and same fragment interactions were discard by perl scripts.
The bedpe format file sorted by the command sort.
The sorted file transferred to matrix by a python script.
The interactions were convered to .hic files by Juicer tools and loops were called with default paremeters.
Assembly: hg19
Supplementary files format and content: Sorted pairs file containing a list of valid contacts
Library strategy: DLO Hi-C

growth_protocol: THP-1 cells were and hMDMs maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
DNA was precipitated at room temperature with 10µl of Dr. GenTLE Precipitation Carrier (Takara), 100µl of 3 M sodium acetate (pH 5.2), and an equal volume of isopropanol
In situ DLO Hi-C library construction strategies refer to previously published articles: “Lin, D. et al. Digestion-ligation-only Hi-C is an efficient and cost-effective method for chromosome conformation capture. Nature genetics 50, 754 (2018).”

treatment_protocol: To differentiate the monocytes to macrophages, cells were treated with PMA (phorbol 12-myristate 13-acetate) (final concentration = 40 ng/µl) for 48 h at 37 °C in a 5% CO2 atmosphere, and then washed with pre-warmed PBS and incubated with fresh culture medium for another 24 h. For M.tb infection experiment, H37Ra were pelleted (4,000 rpm, RT, 10 min), washed twice with RPMI-1640, resuspended in 1 ml THP-1 culture medium, and dispersed using BD insulin syringes (BD, 328421). Monocyte derived macrophages (Thp1-macro) were infected with H37Ra at MOI (multiplicity of infection) 20. Four hour later the infected cells were washed twice with pre-warmed PBS and incubated with fresh culture medium for another 8 h.

value: H37Rv infection


title: Thp1-mono-rep2

value: monocyte


title: hMDMs-control-rep1


title: hMDMs-control-rep2


title: hMDMs-M.tb-rep2


title: Human hMDMs cells infected by H37Rv_1


title: Human hMDMs cells infected by H37Rv_3


title: Thp1-mono-rep1


title: hMDMs-M.tb-rep1


title: Thp1-macro-rep1

value: PAM stimulate


title: hMDMs-M.tb-rep3


title: Human hMDMs cells WT control_1


title: Human hMDMs cells WT control_4


title: Thp1-macro-rep2


title: Human hMDMs cells infected by H37Rv_2


title: Human hMDMs cells infected by H37Rv_undecoded


title: Human hMDMs cells WT control_2


title: Human hMDMs cells WT control_undecoded


title: Sample 106_HeLa-WT_polyA-RNA_Input_rep3

city: Chicago

first: Li-Sheng

last: Zhang

state: IL

address: 929 E 57th St Rm E313

department: Department of Chemistry

institute: The University of Chicago

description: Processed data file: GSE179798_HeLa_mRNA_WT_BID-seq.xlsx

data_processing:
The sequencing data were all trimmed with cutadapt tool to remove adapters and low-quality reads. PCR duplicates were removed with BBMap tool (v.38.73), 5-mer random barcodes at reads ends were trimmed and low-quality or short reads (less than 20 nt) were removed using cutadapt tool (v.1.15). Remaining reads were aligned to hg38 or mm10 genome using Tophat2 (v.2.1.1) and bowtie2 (v.2.4.0) allowing, at most, three mismatches. The generated.bam files were split into positive and negative strands and sorted using Samtools (v.1.9). Sequence variants were identified by measuring the base composition at each position using bam-readcount software (v.0.8.0). The generated bam-readcount results were parsed and analyzed by inhouse scripts. Internal deletion ratio at each Ψ candidate site suggested by Tophat2, was further calculated by data output from bam-readcount pipeline and confirmed by direct IGV visualization (v.2.8.0). In summary, one Ψ candidate site needs to satisfy the following criteria in its deletion profile: (1) deletion rate above 5% (with deletion count above five) in BID-seq libraries; (2) deletion rate below 1% in ‘Input’ libraries; (3) total reads coverage depth above 20 in both BID-seq and ‘Input’ libraries; (4) deletion rate above 1.5-fold over background in any given sequence motif (defined as the deletion rates detected from RNA probes containing 0% Ψ, as in Supplementary Fig. 1c); (5) we excluded uridine sites at the neighboring nucleotide 3′ or 5′ to known Ψ sites; (6) all deletion signatures must be from ‘U’ sites marked by hg38 or mm10 FASTA file, instead of from A or C or G. Under the current sequencing depth (around 80 M reads per library), we set RPKM = 1.5 as the lowest expression level for mRNA Ψ detection.
The ‘input’ samples of BID-seq, for both human cell lines and mouse tissues, are equivalent to regular RNA-seq; therefore, we quantified the gene-level read counts of input samples that aligned to hg38 or mm10 for gene expression analysis with Cufflinks software (v.2.2.1). GO analysis was performed using the online analysis software DAVID 2021 (https://david.ncifcrf.gov).

molecule: polyA RNA

growth_protocol: HeLa, 293T, and A549 cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured at 37 °C with 5.0% CO2 in a Heracell™ VIOS 160i incubator (Thermo Scientific). All cell lines were grown in DMEM medium (GIBCO, 11995) supplemented with 10% v/v FBS and 1% penicillin/streptomycin (GIBCO). The percentage of surviving cells after treatment was assessed by the SRB assay. Cell cycle distribution and cell size determination were assessed by flow cytometry.

extract_protocol:
(Note RNA 3′ SR Adapter: 5’rApp NNNNN AGATCGGAAGAGCACACGTCT-3SpC3; 5′ SR Adapter: 5′-GUUCAGAGUUCUACAGUCCGACGAUC-3′)
Wild-type HeLa, HEK293T or A549 cells (three replicates for each sample, one 3.5-cm plate per replicate); HeLa shControl or PUS knockdown cells were prepared as described (two replicates for each sample, one 10-cm plate per replicate); mouse tissues, except immune cells, were collected from two replicates (one wild-type mouse per replicate), age- and sex-matched. To harvest enough material, mouse immune cells such as B cells, CD4 T cells and CD8 T cells were collected and combined from two replicates (eight wild-type mice per replicate), again age- and sex-matched. After extracting the polyA+ RNA from HeLa cells or mouse tissues, around 10–20 ng RNA was fragmented using RNA Fragmentation Reagents (catalog no. AM8740, Invitrogen) and heated at 70 °C for 14 min, followed by purification with RNA Clean and Concentrator (Zymo Research). 3′-End repair and 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL 10× T4 PNK reaction buffer (catalog no. B0201S, NEB) and 3 µl T4 PNK, diluted to a final volume of 30 µl, and incubated at 37 °C for 45 min; then 1.5 µl T4 PNK and 1.5 µl 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by RNA Clean and Concentrator (Zymo Research) purification eluting with 10 µl RNase-free water. To perform 3′-adapter ligation, 10 µl 3′-repaired and 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (sequences listed above) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′-deadenylase (catalog no. M0331S, NEB) at 30 °C for 30 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 30 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 9.3 µl RNase-free water.
The purified RNA was incubated with 1.2 µl 10 µM 5′ SR Adapter (5′-sequences listed above-3′) at 70 °C for 2 mins and placed immediately on ice. Then 2.5 µl 10× T4 RNA ligase reaction buffer, 1.0 µl 25 mM ATP, 10 µl PEG8000 (50%) and 1 µl T4 RNA Ligase 1 (high concentration, catalog no. M0437M, NEB) were added to the RNA–adapter mixture. The reaction was mixed well and incubated at 25 °C for 8 h, followed by RNA Clean and Concentrator (Zymo Research) purification, eluting with 10 µl RNase-free water.
A 1.5 µl aliquot of the purified RNA was saved for ‘Input’ library construction, 8.5 µl was subjected to BID-seq optimized bisulfite treatment, as the ‘Treated’ sample. The 8.5 µl RNA was mixed with 45 µl freshly prepared BID-seq BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, prepared by dissolving 270 mg Na2SO3 and 34 mg NaHSO3 in 900 µl RNase-free water) and incubated at 70 °C for 3 h. Then, 75 µl RNase-free H2O, 270 µl RNA binding buffer (RNA Clean and Concentrator), and 400 µl ethanol were added to the reaction mixture, which was mixed well and loaded on a RNA Clean and Concentrator-5 column. After spinning and washing once with 200 µl RNA wash buffer (RNA Clean and Concentrator), 200 µl RNA Desulphonation Buffer (catalog no. R5001-3-40, Zymo Research) was added to the column and incubated at room temperature for 1 h. This was followed by spinning and washing twice with 700 µl RNA wash buffer (RNA Clean and Concentrator), followed by eluting RNA with 10.5 µl RNase-free water. Then the ‘Input’ was diluted to 10.5 µl with RNase-free water. Both ‘Input’ and ‘Treated’ samples were mixed with 1.0 µl 2.0 µM SR RT primer (5′-AGACGTGTGCTCTTCCGATCT -3′) at 65 °C for 2 min and moved immediately onto ice. To this was added 4 µl 5× SSIV Buffer, 2 µl 10 mM dNTP Solution Mix (NEB), 1 µl 100 mM dithiothreitol, 0.5 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 1 µl SuperScript IV Reverse Transcriptase (SSIV, catalog no. 18090050, Thermo Scientific). The reaction was mixed well and incubated at 50 °C for 1 h, followed by adding 1 µl RNase H (catalog no. M0297L, NEB) and incubating at 37 °C for 20 min. The reaction mixture was heated at 70 °C for 5 min and then cDNA was purified using DNA Clean and Concentrator (Zymo Research). The eluted cDNAs (20 µl) were stored at –80 °C; 4 µl was used for each 15-cycle PCR amplification reaction, which was performed with the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina Nova-seq 6000 with single-end 100 bp read length.

treatment_protocol: Wild-type cells are used for these RNA-seq libraries.

value: HeLa

tag: genotype/variation

value: Input


title: ATAC-seq_Thp1-M.tb-rep2

data_processing:
Trimmomatic (version: 0.33) was used to filter out the low quality bases and adapter sequences.
Trimmed reads were aligned to the human genome assembly (hg19) with the Burrows-Wheeler Aligner-MEM (version: 0.7.17-r1188)
For each sample, SAMtools (version: 1.7) was used to sort the mapped reads by position.
After marking the duplication reads by Picard toolkit (version: 1.119), uniquely mapped reads and non-redundant sequences were kept for further analysis.
F-Seq (version: 3) was used to call ATAC-Seq peaks.
Assembly: hg19
Supplementary files format and content: bigWig

growth_protocol: THP-1 cells (ATCC, TIB-202) were grown in RPMI-1640 containing 10 % FBS

extract_protocol: 100,000 cells were centrifugation at 800 g for 5 min and then washed once using 500 µl of cold 1× PBS and centrifugation at 800 g for another 5 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl (pH 8.0 at 25 °C), 10 mM NaCl, 0.3% Igepal CA-630). After lysis, nuclei were spun at 800g in 4 °C for 10 min. The pellet was resuspended in the transposase reaction mix (10 µl 5× TTBL buffer (Vazyme, TD501-02), 5 µl TTE Mix V50 buffer (Vazyme, TD501-02) and 35 µl ddH2O water). Tagmentation was carried out for 10 min at 55 °C. Immediately following transposition, The DNA was purified using a Qiagen MinElute PCR Purification Kit (Qiagen, 28004) and eluted with 10 μl elution buffer. The ATAC-Seq library was amplified by the primers in TruePrepTM Index Kit V2 for Illumina® (Vazyme, TD202).

value: Peripheral blood

value: infected with H37Ra


title: Thp1-M.tb-rep2

value: macrophage


title: ATAC-seq_Thp1-M.tb-rep1


title: Human hMDMs cells WT control_3


title: Sample 88_Mouse_Thymus_polyA-RNA_Treated_rep2

description: Processed data file: GSE179798_Mouse_mRNA_12-Tissues_BID-seq.xlsx

growth_protocol: Mice were housed in a virus-free facility at 21 ± 1 °C with a controlled 12-hour light cycle (Individually Ventilated Caging System (GM500)). The animals have access to standard chow and water ad libitum. The relative humidity was controlled at 55% ± 10%. The mice (Mus musculus) were fed an SD with 5.5% fat content (Rat and Mouse No. 3 breeding diet, Special Diet Services). All mice in this study were C57BL6/N background strain wild-type mice. All data were obtained from 7-week-old male mice.

extract_protocol:
(Note RNA 3′ SR Adapter: 5’rApp NNNNN ATCACG AGATCGGAAGAGCACACGTCT-3SpC3; 5′ SR Adapter: 5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′)
Wild-type HeLa, HEK293T or A549 cells (three replicates for each sample, one 3.5-cm plate per replicate); HeLa shControl or PUS knockdown cells were prepared as described (two replicates for each sample, one 10-cm plate per replicate); mouse tissues, except immune cells, were collected from two replicates (one wild-type mouse per replicate), age- and sex-matched. To harvest enough material, mouse immune cells such as B cells, CD4 T cells and CD8 T cells were collected and combined from two replicates (eight wild-type mice per replicate), again age- and sex-matched. After extracting the polyA+ RNA from HeLa cells or mouse tissues, around 10–20 ng RNA was fragmented using RNA Fragmentation Reagents (catalog no. AM8740, Invitrogen) and heated at 70 °C for 14 min, followed by purification with RNA Clean and Concentrator (Zymo Research). 3′-End repair and 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL 10× T4 PNK reaction buffer (catalog no. B0201S, NEB) and 3 µl T4 PNK, diluted to a final volume of 30 µl, and incubated at 37 °C for 45 min; then 1.5 µl T4 PNK and 1.5 µl 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by RNA Clean and Concentrator (Zymo Research) purification eluting with 10 µl RNase-free water. To perform 3′-adapter ligation, 10 µl 3′-repaired and 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (sequences listed above) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′-deadenylase (catalog no. M0331S, NEB) at 30 °C for 30 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 30 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 9.3 µl RNase-free water.
The purified RNA was incubated with 1.2 µl 10 µM 5′ SR Adapter (5′-sequences listed above-3′) at 70 °C for 2 mins and placed immediately on ice. Then 2.5 µl 10× T4 RNA ligase reaction buffer, 1.0 µl 25 mM ATP, 10 µl PEG8000 (50%) and 1 µl T4 RNA Ligase 1 (high concentration, catalog no. M0437M, NEB) were added to the RNA–adapter mixture. The reaction was mixed well and incubated at 25 °C for 8 h, followed by RNA Clean and Concentrator (Zymo Research) purification, eluting with 10 µl RNase-free water.
A 1.5 µl aliquot of the purified RNA was saved for ‘Input’ library construction, 8.5 µl was subjected to BID-seq optimized bisulfite treatment, as the ‘Treated’ sample. The 8.5 µl RNA was mixed with 45 µl freshly prepared BID-seq BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, prepared by dissolving 270 mg Na2SO3 and 34 mg NaHSO3 in 900 µl RNase-free water) and incubated at 70 °C for 3 h. Then, 75 µl RNase-free H2O, 270 µl RNA binding buffer (RNA Clean and Concentrator), and 400 µl ethanol were added to the reaction mixture, which was mixed well and loaded on a RNA Clean and Concentrator-5 column. After spinning and washing once with 200 µl RNA wash buffer (RNA Clean and Concentrator), 200 µl RNA Desulphonation Buffer (catalog no. R5001-3-40, Zymo Research) was added to the column and incubated at room temperature for 1 h. This was followed by spinning and washing twice with 700 µl RNA wash buffer (RNA Clean and Concentrator), followed by eluting RNA with 10.5 µl RNase-free water. Then the ‘Input’ was diluted to 10.5 µl with RNase-free water. Both ‘Input’ and ‘Treated’ samples were mixed with 1.0 µl 2.0 µM SR RT primer (5′-AGACGTGTGCTCTTCCGATCT -3′) at 65 °C for 2 min and moved immediately onto ice. To this was added 4 µl 5× SSIV Buffer, 2 µl 10 mM dNTP Solution Mix (NEB), 1 µl 100 mM dithiothreitol, 0.5 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 1 µl SuperScript IV Reverse Transcriptase (SSIV, catalog no. 18090050, Thermo Scientific). The reaction was mixed well and incubated at 50 °C for 1 h, followed by adding 1 µl RNase H (catalog no. M0297L, NEB) and incubating at 37 °C for 20 min. The reaction mixture was heated at 70 °C for 5 min and then cDNA was purified using DNA Clean and Concentrator (Zymo Research). The eluted cDNAs (20 µl) were stored at –80 °C; 4 µl was used for each 15-cycle PCR amplification reaction, which was performed with the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina Nova-seq 6000 with single-end 100 bp read length.

treatment_protocol: Wild-type mice are used for these RNA-seq libraries.

value: Thymus

tag: strain

value: Male

tag: age

value: 7 weeks


title: ATAC-seq_Thp1-macro-rep2

value: uninfected


title: ATAC-seq_Thp1-macro-rep1


title: human gastric cancer (S6TP)

description: SAMPLE 6

value: Gastric cancer

value: stomach

value: Body

value: Mucinous adenocarcinoma (poorly differentiated adenoca)

value: Asian


title: ATAC-seq_Thp1-mono-rep2


title: Sample 86_Mouse_Thymus_polyA-RNA_Input_rep2

value: WT

tag: Sex


title: human gastric cancer (S3TP)

description: SAMPLE 3

value: Antrum

value: poorly differentiated adenoca


title: Thp1-M.tb-H3K4me1-rep1

data_processing:
FastQC (version: v0.11.8) was used to assess the quality of raw reads.
Trimmomatic (version: 0.33) was used to filter out the low quality bases and adapter sequences.
Burrows-Wheeler Aligner-MEM (version: 0.7.17-r1188) was applied to map the reads to the human genome assembly (hg19).
Mapped reads were sorted by position and duplications were discarded by SAMtools (version: 1.3.1).
Peaks were called by MACS2 (version: 2.1.1.20160309). For broad peaks, the parameterswere-B --broad -q 0.05. For narrow peaks, the parameter was -B.
bigWig files were generated using deeptools (-p 30; --normalizeUsing RPKM)
Assembly: hg19
Supplementary files format and content: bigWig

source_name: Thp1-M.tb cells

extract_protocol:
Cells were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin was sonicated using a ultrasonicator (Bioruptor Pico).
4×10^6 cells and 3 μg antibody were used per immunoprecipitation. Antibody of H3K4me1 (Abcam, ab8895), H3K4me3 (Abcam, ab8580), H3K9me3 (Abcam, ab8898), H3K27ac (Abcam, ab4729), and H3K27me3 (Millipore, 07-449) were used in this study. The ChIP DNA was obtained using SimpleChIP® Enzymatic Chromatin IP Kit (CST, #9003). After immunoprecipitation, ChIP-seq library was constructed using NEBNext® Ultra™ DNA Library Prep Kit (NEB, E7370S).

tag: chip antibody

value: H3K4me1 (Abcam, ab8895)


title: Thp1-M.tb-Input


title: Thp1-M.tb-H3K27ac-rep1

value: H3K27ac (Abcam, ab4729)


title: Thp1-M.tb-H3K9me3-rep1

value: H3K9me3 (Abcam, ab8898)


title: Thp1-M.tb-H3K4me3-rep2

value: H3K4me3 (Abcam, ab8580)

value: infected with H37Ra


title: Sample 87_Mouse_Thymus_polyA-RNA_Treated_rep1

value: Thymus


title: Thp1-M.tb-H3K27me3-rep1

value: macrophage

value: H3K27me3 (Millipore, 07-449)


title: Thp1-macro-H3K4me1-rep1

source_name: Thp1-macro cells

value: uninfected


title: Thp1-M.tb-H3K9me3-rep2


title: Thp1-M.tb-H3K27me3-rep2


title: Thp1-macro-H3K4me1-rep2


title: Thp1-M.tb-H3K4me1-rep2


title: Thp1-M.tb-H3K27ac-rep2


title: Thp1-M.tb-H3K4me3-rep1


title: Thp1-macro-Input


title: Thp1-macro-H3K9me3-rep2


title: Thp1-macro-H3K4me3-rep1


title: Thp1-macro-H3K4me3-rep2


title: Thp1-macro-H3K27ac-rep2


title: Thp1-macro-H3K27me3-rep2


title: Thp1-macro-H3K27ac-rep1


title: Thp1-macro-H3K27me3-rep1


title: ATAC-seq_Thp1-mono-rep1

value: Peripheral blood

value: monocyte


title: Thp1-macro-H3K9me3-rep1


title: Thp1-mono-H3K9me3-rep2

source_name: Thp1-mono cells


title: Thp1-mono-Input


title: Thp1-mono-H3K9me3-rep1


title: Thp1-mono-H3K27ac-rep2


title: Sample 104_HeLa-WT_polyA-RNA_Input_rep1

value: HeLa

value: Input


title: Sample 105_HeLa-WT_polyA-RNA_Input_rep2


title: Thp1-M.tb-DLO-HiC_4

value: H37Ra infection


title: Thp1-mono-H3K27me3-rep1


title: Sample 102_HeLa-WT_polyA-RNA_Treated_rep2

value: WT

value: Treated


title: Sample 101_HeLa-WT_polyA-RNA_Treated_rep1


title: Thp1-mono-H3K4me1-rep1


title: Sample 103_HeLa-WT_polyA-RNA_Treated_rep3


title: Thp1-mono-H3K27me3-rep2


title: Sample 99_HEK293T-WT_polyA-RNA_Input_rep2

description: Processed data file: GSE179798_HEK293T_mRNA_WT_BID-seq.xlsx


title: Sample 98_HEK293T-WT_polyA-RNA_Input_rep1


title: Thp1-macro-DLO-HiC_4

value: PAM stimulate


title: Thp1-M.tb-DLO-HiC_1


title: Thp1-macro-DLO-HiC_2


title: Thp1-M.tb-DLO-HiC_3


title: Sample 100_HEK293T-WT_polyA-RNA_Input_rep3


title: Thp1-M.tb-DLO-HiC_2


title: Sample 97_HEK293T-WT_polyA-RNA_Treated_rep3


title: Thp1-macro-DLO-HiC_1


title: Thp1-mono-DLO-HiC_4


title: Sample 95_HEK293T-WT_polyA-RNA_Treated_rep1


title: Sample 96_HEK293T-WT_polyA-RNA_Treated_rep2


title: Thp1-mono-DLO-HiC_3


title: Thp1-mono-H3K4me1-rep2

value: H3K4me1 (Abcam, ab8895)


title: Thp1-mono-H3K4me3-rep2


title: Thp1-mono-H3K4me3-rep1


title: Thp1-mono-H3K27ac-rep1

value: H3K27ac (Abcam, ab4729)


title: Thp1-macro-DLO-HiC_3


title: Sample 94_A549-WT_polyA-RNA_Input_rep3

description: Processed data file: GSE179798_A549_mRNA_WT_BID-seq.xlsx


title: Sample 91_A549-WT_polyA-RNA_Treated_rep3


title: Thp1-mono-DLO-HiC_2


title: Thp1-mono-DLO-HiC_1


title: Sample 90_A549-WT_polyA-RNA_Treated_rep2


title: Sample 20_HeLa_polyA-RNA_shDKC1_Treated_rep2

description: processed data file: GSE179798_HeLa_mRNA_shControl_BID-seq.xlsx

growth_protocol: HeLa and 293TN cell lines were purchased from the American Type Culture Collection (ATCC). Cells were grown in DMEM medium (11995, high glucose, Gibco) supplemented with 10% v/v FBS and 1% penicillin/streptomycin (GIBCO). Cells were cultured at 37 °C with 5.0% CO2 in a Heracell™ VIOS 160i incubator (Thermo Scientific).

extract_protocol:
(Note RNA 3′ SR Adapter: 5’rApp NNNNN TTAGGC AGATCGGAAGAGCACACGTCT-3SpC3; 5′ SR Adapter: 5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′)
Wild-type HeLa, HEK293T or A549 cells (three replicates for each sample, one 3.5-cm plate per replicate); HeLa shControl or PUS knockdown cells were prepared as described (two replicates for each sample, one 10-cm plate per replicate); mouse tissues, except immune cells, were collected from two replicates (one wild-type mouse per replicate), age- and sex-matched. To harvest enough material, mouse immune cells such as B cells, CD4 T cells and CD8 T cells were collected and combined from two replicates (eight wild-type mice per replicate), again age- and sex-matched. After extracting the polyA+ RNA from HeLa cells or mouse tissues, around 10–20 ng RNA was fragmented using RNA Fragmentation Reagents (catalog no. AM8740, Invitrogen) and heated at 70 °C for 14 min, followed by purification with RNA Clean and Concentrator (Zymo Research). 3′-End repair and 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL 10× T4 PNK reaction buffer (catalog no. B0201S, NEB) and 3 µl T4 PNK, diluted to a final volume of 30 µl, and incubated at 37 °C for 45 min; then 1.5 µl T4 PNK and 1.5 µl 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by RNA Clean and Concentrator (Zymo Research) purification eluting with 10 µl RNase-free water. To perform 3′-adapter ligation, 10 µl 3′-repaired and 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (sequences listed above) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′-deadenylase (catalog no. M0331S, NEB) at 30 °C for 30 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 30 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 9.3 µl RNase-free water.
The purified RNA was incubated with 1.2 µl 10 µM 5′ SR Adapter (5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′) at 70 °C for 2 mins and placed immediately on ice. Then 2.5 µl 10× T4 RNA ligase reaction buffer, 1.0 µl 25 mM ATP, 10 µl PEG8000 (50%) and 1 µl T4 RNA Ligase 1 (high concentration, catalog no. M0437M, NEB) were added to the RNA–adapter mixture. The reaction was mixed well and incubated at 25 °C for 8 h, followed by RNA Clean and Concentrator (Zymo Research) purification, eluting with 10 µl RNase-free water.
A 1.5 µl aliquot of the purified RNA was saved for ‘Input’ library construction, 8.5 µl was subjected to BID-seq optimized bisulfite treatment, as the ‘Treated’ sample. The 8.5 µl RNA was mixed with 45 µl freshly prepared BID-seq BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, prepared by dissolving 270 mg Na2SO3 and 34 mg NaHSO3 in 900 µl RNase-free water) and incubated at 70 °C for 3 h. Then, 75 µl RNase-free H2O, 270 µl RNA binding buffer (RNA Clean and Concentrator), and 400 µl ethanol were added to the reaction mixture, which was mixed well and loaded on a RNA Clean and Concentrator-5 column. After spinning and washing once with 200 µl RNA wash buffer (RNA Clean and Concentrator), 200 µl RNA Desulphonation Buffer (catalog no. R5001-3-40, Zymo Research) was added to the column and incubated at room temperature for 1 h. This was followed by spinning and washing twice with 700 µl RNA wash buffer (RNA Clean and Concentrator), followed by eluting RNA with 10.5 µl RNase-free water. Then the ‘Input’ was diluted to 10.5 µl with RNase-free water. Both ‘Input’ and ‘Treated’ samples were mixed with 1.0 µl 2.0 µM SR RT primer (5′-AGACGTGTGCTCTTCCGATCT -3′) at 65 °C for 2 min and moved immediately onto ice. To this was added 4 µl 5× SSIV Buffer, 2 µl 10 mM dNTP Solution Mix (NEB), 1 µl 100 mM dithiothreitol, 0.5 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 1 µl SuperScript IV Reverse Transcriptase (SSIV, catalog no. 18090050, Thermo Scientific). The reaction was mixed well and incubated at 50 °C for 1 h, followed by adding 1 µl RNase H (catalog no. M0297L, NEB) and incubating at 37 °C for 20 min. The reaction mixture was heated at 70 °C for 5 min and then cDNA was purified using DNA Clean and Concentrator (Zymo Research). The eluted cDNAs (20 µl) were stored at –80 °C; 4 µl was used for each 15-cycle PCR amplification reaction, which was performed with the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina Nova-seq 6000 with single-end 100 bp read length.

treatment_protocol: For lentivirus production in preparing shControl and shPUS cell lines, a lentiviral construct (pLKO-Tet-On for inducible knockdown of pseudouridine synthetases),  together with pMD2.G (Addgene 12259) and psPAX2 (Addgene 12260) were cotransfected into 293TN cells (System Biosciences) as previously described (Mol Cell. 2020 Nov 19;80(4):633-647.e7). Viruses were concentrated by the PEG-it Virus Precipitation Solution and used for infecting cells in the presence of TransDux (System Biosciences). Pools of stable transfectants were selected by antibiotics or sorted by flow cytometry. Doxycycline (0.5 mg/mL) was used to induce shRNA.


title: Sample 85_Mouse_Thymus_polyA-RNA_Input_rep1

value: Male

value: 7 weeks


title: Sample 83_Mouse_Testis_polyA-RNA_Treated_rep1

value: Testis


title: Sample 81_Mouse_Testis_polyA-RNA_Input_rep1


title: Sample 80_Mouse_Small-intestine_polyA-RNA_Treated_rep2

value: Small intestine


title: Sample 76_Mouse_Liver_polyA-RNA_Treated_rep2

value: Liver


title: Sample 79_Mouse_Small-intestine_polyA-RNA_Treated_rep1


title: Sample 93_A549-WT_polyA-RNA_Input_rep2


title: Sample 18_HeLa_polyA-RNA_shTRUB2_Treated_rep2

extract_protocol:
(Note RNA 3′ SR Adapter: 5’rApp NNNNN TTAGGC AGATCGGAAGAGCACACGTCT-3SpC3; 5′ SR Adapter: 5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′)
Wild-type HeLa, HEK293T or A549 cells (three replicates for each sample, one 3.5-cm plate per replicate); HeLa shControl or PUS knockdown cells were prepared as described (two replicates for each sample, one 10-cm plate per replicate); mouse tissues, except immune cells, were collected from two replicates (one wild-type mouse per replicate), age- and sex-matched. To harvest enough material, mouse immune cells such as B cells, CD4 T cells and CD8 T cells were collected and combined from two replicates (eight wild-type mice per replicate), again age- and sex-matched. After extracting the polyA+ RNA from HeLa cells or mouse tissues, around 10–20 ng RNA was fragmented using RNA Fragmentation Reagents (catalog no. AM8740, Invitrogen) and heated at 70 °C for 14 min, followed by purification with RNA Clean and Concentrator (Zymo Research). 3′-End repair and 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL 10× T4 PNK reaction buffer (catalog no. B0201S, NEB) and 3 µl T4 PNK, diluted to a final volume of 30 µl, and incubated at 37 °C for 45 min; then 1.5 µl T4 PNK and 1.5 µl 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by RNA Clean and Concentrator (Zymo Research) purification eluting with 10 µl RNase-free water. To perform 3′-adapter ligation, 10 µl 3′-repaired and 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (sequences listed above) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′-deadenylase (catalog no. M0331S, NEB) at 30 °C for 30 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 30 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 9.3 µl RNase-free water.
The purified RNA was incubated with 1.2 µl 10 µM 5′ SR Adapter (5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′) at 70 °C for 2 mins and placed immediately on ice. Then 2.5 µl 10× T4 RNA ligase reaction buffer, 1.0 µl 25 mM ATP, 10 µl PEG8000 (50%) and 1 µl T4 RNA Ligase 1 (high concentration, catalog no. M0437M, NEB) were added to the RNA–adapter mixture. The reaction was mixed well and incubated at 25 °C for 8 h, followed by RNA Clean and Concentrator (Zymo Research) purification, eluting with 10 µl RNase-free water.
A 1.5 µl aliquot of the purified RNA was saved for ‘Input’ library construction, 8.5 µl was subjected to BID-seq optimized bisulfite treatment, as the ‘Treated’ sample. The 8.5 µl RNA was mixed with 45 µl freshly prepared BID-seq BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, prepared by dissolving 270 mg Na2SO3 and 34 mg NaHSO3 in 900 µl RNase-free water) and incubated at 70 °C for 3 h. Then, 75 µl RNase-free H2O, 270 µl RNA binding buffer (RNA Clean and Concentrator), and 400 µl ethanol were added to the reaction mixture, which was mixed well and loaded on a RNA Clean and Concentrator-5 column. After spinning and washing once with 200 µl RNA wash buffer (RNA Clean and Concentrator), 200 µl RNA Desulphonation Buffer (catalog no. R5001-3-40, Zymo Research) was added to the column and incubated at room temperature for 1 h. This was followed by spinning and washing twice with 700 µl RNA wash buffer (RNA Clean and Concentrator), followed by eluting RNA with 10.5 µl RNase-free water. Then the ‘Input’ was diluted to 10.5 µl with RNase-free water. Both ‘Input’ and ‘Treated’ samples were mixed with 1.0 µl 2.0 µM SR RT primer (5′-AGACGTGTGCTCTTCCGATCT -3′) at 65 °C for 2 min and moved immediately onto ice. To this was added 4 µl 5× SSIV Buffer, 2 µl 10 mM dNTP Solution Mix (NEB), 1 µl 100 mM dithiothreitol, 0.5 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 1 µl SuperScript IV Reverse Transcriptase (SSIV, catalog no. 18090050, Thermo Scientific). The reaction was mixed well and incubated at 50 °C for 1 h, followed by adding 1 µl RNase H (catalog no. M0297L, NEB) and incubating at 37 °C for 20 min. The reaction mixture was heated at 70 °C for 5 min and then cDNA was purified using DNA Clean and Concentrator (Zymo Research). The eluted cDNAs (20 µl) were stored at –80 °C; 4 µl was used for each 15-cycle PCR amplification reaction, which was performed with the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina Nova-seq 6000 with single-end 100 bp read length.


title: Sample 74_Mouse_Liver_polyA-RNA_Input_rep2


title: Sample 82_Mouse_Testis_polyA-RNA_Input_rep2


title: Sample 73_Mouse_Liver_polyA-RNA_Input_rep1


title: Sample 17_HeLa_polyA-RNA_shTRUB2_Treated_rep1


title: Sample 78_Mouse_Small-intestine_polyA-RNA_Input_rep2


title: Sample 89_A549-WT_polyA-RNA_Treated_rep1


title: Sample 92_A549-WT_polyA-RNA_Input_rep1


title: Sample 84_Mouse_Testis_polyA-RNA_Treated_rep2


title: Sample 19_HeLa_polyA-RNA_shDKC1_Treated_rep1


title: Sample 71_Mouse_Kidney_polyA-RNA_Treated_rep1

value: Kidney


title: Sample 72_Mouse_Kidney_polyA-RNA_Treated_rep2


title: Sample 16_HeLa_polyA-RNA_shTRUB1_Treated_rep2


title: Sample 15_HeLa_polyA-RNA_shTRUB1_Treated_rep1

extract_protocol:
(Note RNA 3′ SR Adapter: 5’rApp NNNNN CGATGT AGATCGGAAGAGCACACGTCT-3SpC3; 5′ SR Adapter: 5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′)
Wild-type HeLa, HEK293T or A549 cells (three replicates for each sample, one 3.5-cm plate per replicate); HeLa shControl or PUS knockdown cells were prepared as described (two replicates for each sample, one 10-cm plate per replicate); mouse tissues, except immune cells, were collected from two replicates (one wild-type mouse per replicate), age- and sex-matched. To harvest enough material, mouse immune cells such as B cells, CD4 T cells and CD8 T cells were collected and combined from two replicates (eight wild-type mice per replicate), again age- and sex-matched. After extracting the polyA+ RNA from HeLa cells or mouse tissues, around 10–20 ng RNA was fragmented using RNA Fragmentation Reagents (catalog no. AM8740, Invitrogen) and heated at 70 °C for 14 min, followed by purification with RNA Clean and Concentrator (Zymo Research). 3′-End repair and 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL 10× T4 PNK reaction buffer (catalog no. B0201S, NEB) and 3 µl T4 PNK, diluted to a final volume of 30 µl, and incubated at 37 °C for 45 min; then 1.5 µl T4 PNK and 1.5 µl 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by RNA Clean and Concentrator (Zymo Research) purification eluting with 10 µl RNase-free water. To perform 3′-adapter ligation, 10 µl 3′-repaired and 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (sequences listed above) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′-deadenylase (catalog no. M0331S, NEB) at 30 °C for 30 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 30 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 9.3 µl RNase-free water.
The purified RNA was incubated with 1.2 µl 10 µM 5′ SR Adapter (5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′) at 70 °C for 2 mins and placed immediately on ice. Then 2.5 µl 10× T4 RNA ligase reaction buffer, 1.0 µl 25 mM ATP, 10 µl PEG8000 (50%) and 1 µl T4 RNA Ligase 1 (high concentration, catalog no. M0437M, NEB) were added to the RNA–adapter mixture. The reaction was mixed well and incubated at 25 °C for 8 h, followed by RNA Clean and Concentrator (Zymo Research) purification, eluting with 10 µl RNase-free water.
A 1.5 µl aliquot of the purified RNA was saved for ‘Input’ library construction, 8.5 µl was subjected to BID-seq optimized bisulfite treatment, as the ‘Treated’ sample. The 8.5 µl RNA was mixed with 45 µl freshly prepared BID-seq BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, prepared by dissolving 270 mg Na2SO3 and 34 mg NaHSO3 in 900 µl RNase-free water) and incubated at 70 °C for 3 h. Then, 75 µl RNase-free H2O, 270 µl RNA binding buffer (RNA Clean and Concentrator), and 400 µl ethanol were added to the reaction mixture, which was mixed well and loaded on a RNA Clean and Concentrator-5 column. After spinning and washing once with 200 µl RNA wash buffer (RNA Clean and Concentrator), 200 µl RNA Desulphonation Buffer (catalog no. R5001-3-40, Zymo Research) was added to the column and incubated at room temperature for 1 h. This was followed by spinning and washing twice with 700 µl RNA wash buffer (RNA Clean and Concentrator), followed by eluting RNA with 10.5 µl RNase-free water. Then the ‘Input’ was diluted to 10.5 µl with RNase-free water. Both ‘Input’ and ‘Treated’ samples were mixed with 1.0 µl 2.0 µM SR RT primer (5′-AGACGTGTGCTCTTCCGATCT -3′) at 65 °C for 2 min and moved immediately onto ice. To this was added 4 µl 5× SSIV Buffer, 2 µl 10 mM dNTP Solution Mix (NEB), 1 µl 100 mM dithiothreitol, 0.5 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 1 µl SuperScript IV Reverse Transcriptase (SSIV, catalog no. 18090050, Thermo Scientific). The reaction was mixed well and incubated at 50 °C for 1 h, followed by adding 1 µl RNase H (catalog no. M0297L, NEB) and incubating at 37 °C for 20 min. The reaction mixture was heated at 70 °C for 5 min and then cDNA was purified using DNA Clean and Concentrator (Zymo Research). The eluted cDNAs (20 µl) were stored at –80 °C; 4 µl was used for each 15-cycle PCR amplification reaction, which was performed with the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina Nova-seq 6000 with single-end 100 bp read length.


title: Sample 14_HeLa_polyA-RNA_shPUSL1_Treated_rep2


title: Sample 69_Mouse_Kidney_polyA-RNA_Input_rep1


title: Sample 77_Mouse_Small-intestine_polyA-RNA_Input_rep1


title: Sample 75_Mouse_Liver_polyA-RNA_Treated_rep1


title: Sample 13_HeLa_polyA-RNA_shPUSL1_Treated_rep1


title: Sample 12_HeLa_polyA-RNA_shPUS7L_Treated_rep2


title: Sample 68_Mouse_Heart_polyA-RNA_Treated_rep2

value: Heart


title: Sample 70_Mouse_Kidney_polyA-RNA_Input_rep2


title: Sample 9_HeLa_polyA-RNA_shPUS7_Treated_rep1


title: Sample 65_Mouse_Heart_polyA-RNA_Input_rep1


title: Sample 66_Mouse_Heart_polyA-RNA_Input_rep2


title: Sample 67_Mouse_Heart_polyA-RNA_Treated_rep1


title: Sample 7_HeLa_polyA-RNA_shPUS3_Treated_rep1


title: Sample 6_HeLa_polyA-RNA_shPUS1_Treated_rep2

extract_protocol:
(Note RNA 3′ SR Adapter: 5’rApp NNNNN TTAGGC AGATCGGAAGAGCACACGTCT; 5′ SR Adapter: 5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′)
Wild-type HeLa, HEK293T or A549 cells (three replicates for each sample, one 3.5-cm plate per replicate); HeLa shControl or PUS knockdown cells were prepared as described (two replicates for each sample, one 10-cm plate per replicate); mouse tissues, except immune cells, were collected from two replicates (one wild-type mouse per replicate), age- and sex-matched. To harvest enough material, mouse immune cells such as B cells, CD4 T cells and CD8 T cells were collected and combined from two replicates (eight wild-type mice per replicate), again age- and sex-matched. After extracting the polyA+ RNA from HeLa cells or mouse tissues, around 10–20 ng RNA was fragmented using RNA Fragmentation Reagents (catalog no. AM8740, Invitrogen) and heated at 70 °C for 14 min, followed by purification with RNA Clean and Concentrator (Zymo Research). 3′-End repair and 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL 10× T4 PNK reaction buffer (catalog no. B0201S, NEB) and 3 µl T4 PNK, diluted to a final volume of 30 µl, and incubated at 37 °C for 45 min; then 1.5 µl T4 PNK and 1.5 µl 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by RNA Clean and Concentrator (Zymo Research) purification eluting with 10 µl RNase-free water. To perform 3′-adapter ligation, 10 µl 3′-repaired and 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (sequences listed above) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′-deadenylase (catalog no. M0331S, NEB) at 30 °C for 30 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 30 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 9.3 µl RNase-free water.
The purified RNA was incubated with 1.2 µl 10 µM 5′ SR Adapter (5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′) at 70 °C for 2 mins and placed immediately on ice. Then 2.5 µl 10× T4 RNA ligase reaction buffer, 1.0 µl 25 mM ATP, 10 µl PEG8000 (50%) and 1 µl T4 RNA Ligase 1 (high concentration, catalog no. M0437M, NEB) were added to the RNA–adapter mixture. The reaction was mixed well and incubated at 25 °C for 8 h, followed by RNA Clean and Concentrator (Zymo Research) purification, eluting with 10 µl RNase-free water.
A 1.5 µl aliquot of the purified RNA was saved for ‘Input’ library construction, 8.5 µl was subjected to BID-seq optimized bisulfite treatment, as the ‘Treated’ sample. The 8.5 µl RNA was mixed with 45 µl freshly prepared BID-seq BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, prepared by dissolving 270 mg Na2SO3 and 34 mg NaHSO3 in 900 µl RNase-free water) and incubated at 70 °C for 3 h. Then, 75 µl RNase-free H2O, 270 µl RNA binding buffer (RNA Clean and Concentrator), and 400 µl ethanol were added to the reaction mixture, which was mixed well and loaded on a RNA Clean and Concentrator-5 column. After spinning and washing once with 200 µl RNA wash buffer (RNA Clean and Concentrator), 200 µl RNA Desulphonation Buffer (catalog no. R5001-3-40, Zymo Research) was added to the column and incubated at room temperature for 1 h. This was followed by spinning and washing twice with 700 µl RNA wash buffer (RNA Clean and Concentrator), followed by eluting RNA with 10.5 µl RNase-free water. Then the ‘Input’ was diluted to 10.5 µl with RNase-free water. Both ‘Input’ and ‘Treated’ samples were mixed with 1.0 µl 2.0 µM SR RT primer (5′-AGACGTGTGCTCTTCCGATCT -3′) at 65 °C for 2 min and moved immediately onto ice. To this was added 4 µl 5× SSIV Buffer, 2 µl 10 mM dNTP Solution Mix (NEB), 1 µl 100 mM dithiothreitol, 0.5 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 1 µl SuperScript IV Reverse Transcriptase (SSIV, catalog no. 18090050, Thermo Scientific). The reaction was mixed well and incubated at 50 °C for 1 h, followed by adding 1 µl RNase H (catalog no. M0297L, NEB) and incubating at 37 °C for 20 min. The reaction mixture was heated at 70 °C for 5 min and then cDNA was purified using DNA Clean and Concentrator (Zymo Research). The eluted cDNAs (20 µl) were stored at –80 °C; 4 µl was used for each 15-cycle PCR amplification reaction, which was performed with the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina Nova-seq 6000 with single-end 100 bp read length.


title: Sample 10_HeLa_polyA-RNA_shPUS7_Treated_rep2


title: Sample 5_HeLa_polyA-RNA_shPUS1_Treated_rep1


title: Sample 3_HeLa_polyA-RNA_shControl_Treated_rep1

extract_protocol:
(Note RNA 3′ SR Adapter: 5’rApp NNNNN CGATGT AGATCGGAAGAGCACACGTCT; 5′ SR Adapter: 5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′)
Wild-type HeLa, HEK293T or A549 cells (three replicates for each sample, one 3.5-cm plate per replicate); HeLa shControl or PUS knockdown cells were prepared as described (two replicates for each sample, one 10-cm plate per replicate); mouse tissues, except immune cells, were collected from two replicates (one wild-type mouse per replicate), age- and sex-matched. To harvest enough material, mouse immune cells such as B cells, CD4 T cells and CD8 T cells were collected and combined from two replicates (eight wild-type mice per replicate), again age- and sex-matched. After extracting the polyA+ RNA from HeLa cells or mouse tissues, around 10–20 ng RNA was fragmented using RNA Fragmentation Reagents (catalog no. AM8740, Invitrogen) and heated at 70 °C for 14 min, followed by purification with RNA Clean and Concentrator (Zymo Research). 3′-End repair and 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL 10× T4 PNK reaction buffer (catalog no. B0201S, NEB) and 3 µl T4 PNK, diluted to a final volume of 30 µl, and incubated at 37 °C for 45 min; then 1.5 µl T4 PNK and 1.5 µl 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by RNA Clean and Concentrator (Zymo Research) purification eluting with 10 µl RNase-free water. To perform 3′-adapter ligation, 10 µl 3′-repaired and 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (sequences listed above) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′-deadenylase (catalog no. M0331S, NEB) at 30 °C for 30 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 30 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 9.3 µl RNase-free water.
The purified RNA was incubated with 1.2 µl 10 µM 5′ SR Adapter (5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′) at 70 °C for 2 mins and placed immediately on ice. Then 2.5 µl 10× T4 RNA ligase reaction buffer, 1.0 µl 25 mM ATP, 10 µl PEG8000 (50%) and 1 µl T4 RNA Ligase 1 (high concentration, catalog no. M0437M, NEB) were added to the RNA–adapter mixture. The reaction was mixed well and incubated at 25 °C for 8 h, followed by RNA Clean and Concentrator (Zymo Research) purification, eluting with 10 µl RNase-free water.
A 1.5 µl aliquot of the purified RNA was saved for ‘Input’ library construction, 8.5 µl was subjected to BID-seq optimized bisulfite treatment, as the ‘Treated’ sample. The 8.5 µl RNA was mixed with 45 µl freshly prepared BID-seq BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, prepared by dissolving 270 mg Na2SO3 and 34 mg NaHSO3 in 900 µl RNase-free water) and incubated at 70 °C for 3 h. Then, 75 µl RNase-free H2O, 270 µl RNA binding buffer (RNA Clean and Concentrator), and 400 µl ethanol were added to the reaction mixture, which was mixed well and loaded on a RNA Clean and Concentrator-5 column. After spinning and washing once with 200 µl RNA wash buffer (RNA Clean and Concentrator), 200 µl RNA Desulphonation Buffer (catalog no. R5001-3-40, Zymo Research) was added to the column and incubated at room temperature for 1 h. This was followed by spinning and washing twice with 700 µl RNA wash buffer (RNA Clean and Concentrator), followed by eluting RNA with 10.5 µl RNase-free water. Then the ‘Input’ was diluted to 10.5 µl with RNase-free water. Both ‘Input’ and ‘Treated’ samples were mixed with 1.0 µl 2.0 µM SR RT primer (5′-AGACGTGTGCTCTTCCGATCT -3′) at 65 °C for 2 min and moved immediately onto ice. To this was added 4 µl 5× SSIV Buffer, 2 µl 10 mM dNTP Solution Mix (NEB), 1 µl 100 mM dithiothreitol, 0.5 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 1 µl SuperScript IV Reverse Transcriptase (SSIV, catalog no. 18090050, Thermo Scientific). The reaction was mixed well and incubated at 50 °C for 1 h, followed by adding 1 µl RNase H (catalog no. M0297L, NEB) and incubating at 37 °C for 20 min. The reaction mixture was heated at 70 °C for 5 min and then cDNA was purified using DNA Clean and Concentrator (Zymo Research). The eluted cDNAs (20 µl) were stored at –80 °C; 4 µl was used for each 15-cycle PCR amplification reaction, which was performed with the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina Nova-seq 6000 with single-end 100 bp read length.

value: shControl


title: Sample 63_Mouse_Cerebral-cortex_polyA-RNA_Treated_rep1

value: Cerebral cortex


title: Sample 58_Mouse_Cerebellum_polyA-RNA_Input_rep2

value: Cerebellum


title: Sample 2_HeLa_polyA-RNA_shControl_Input_rep2

value: HeLa

value: Input


title: Sample 60_Mouse_Cerebellum_polyA-RNA_Treated_rep2

value: WT


title: Sample 61_Mouse_Cerebral-cortex_polyA-RNA_Input_rep1


title: Sample 62_Mouse_Cerebral-cortex_polyA-RNA_Input_rep2


title: Sample 1_HeLa_polyA-RNA_shControl_Input_rep1


title: Sample 4_HeLa_polyA-RNA_shControl_Treated_rep2

value: Treated


title: Sample 56_Mouse_CD8-T_polyA-RNA_Treated_rep2

value: CD8 T cell


title: Sample 57_Mouse_Cerebellum_polyA-RNA_Input_rep1


title: Sample 53_Mouse_CD8-T_polyA-RNA_Input_rep1


title: Sample 55_Mouse_CD8-T_polyA-RNA_Treated_rep1


title: Sample 54_Mouse_CD8-T_polyA-RNA_Input_rep2


title: Sample 11_HeLa_polyA-RNA_shPUS7L_Treated_rep1


title: Sample 8_HeLa_polyA-RNA_shPUS3_Treated_rep2


title: Sample 52_Mouse_CD4-T_polyA-RNA_Treated_rep2

value: CD4 T cell


title: Sample 51_Mouse_CD4-T_polyA-RNA_Treated_rep1


title: Sample 49_Mouse_CD4-T_polyA-RNA_Input_rep1


title: Sample 64_Mouse_Cerebral-cortex_polyA-RNA_Treated_rep2


title: Sample 59_Mouse_Cerebellum_polyA-RNA_Treated_rep1


title: Sample 48_Mouse_Bone-marrow_polyA-RNA_Treated_rep2

value: Bone marrow


title: Sample 47_Mouse_Bone-marrow_polyA-RNA_Treated_rep1

value: Male