title: Tamoxifen, CD8, aIgG, rep2

description: Library name: CnT_TMF_aIgG_rep2

data_processing:
FASTQ files were processed using nf-core/cutandrun pipeline v3.2.2. In this pipeline, Bowtie2 was used for alignment to mm10, and peaks were called with SEACR and MACS2 using IgG controls as reference.
Assembly: mm10
Supplementary files format and content: bigWig, bedGraph
Library strategy: CUT&Tag

source_name: CD8 T cells

growth_protocol: EXP-2: For KLF2 and ER-KLF3 CUT&Tag assays, naive P14 T cells were activated with 2 µg/mL anti-CD28 (clone 37.51) and 100 U/mL recombinant human IL-2  in 24-well plates pre-coated with 5 µg/mL anti-CD3e (clone 145-2C11) for 24 hours, then transduced with MIT-ERKLF3. On day 3, cells were subcultured with 10 ng/mL IL-7 and IL-15. Cells were cultured in complete media: RPMI-1640 media supplemented with 10% Hyclone fetal bovine serum, 1x penicillin-streptomycin-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 1x MEM amino acids, and 50 µM 2-mercaptoethanol.

extract_protocol:
EXP-1: For KLF2 CUT&Tag, P14 cells were sorted from day 8 post-infection spleens and lymph nodes.
Libraries were prepared following the Bench Top CUT&Tag v3 protocol by Steven Henikoff lab (Hatice S Kaya-Okur, 2020; Kaya-Okur et al., 2019) with minor modifications. Briefly, 200,000 live cells were lysed, nuclei isolated and bound to concanavalin A beads (EpiCypher). Nuclei were incubated overnight with anti-KLF2 antibody (Millipore #09-820) or Rabbit IgG (EpiCypher #13-0042). Anti-Rabbit Secondary Antibody (EpiCypher #13-0047) and pAG-Tn5 (EpiCypher) were added the next day. Tagmentation was performed in 50 μL of Tagmentation Buffer for 1 hour at 37°C in a thermocycler. The reaction was stopped by adding 3 μL of 0.5 M EDTA, 1 μL of 10% SDS, and 1 μL of Proteinase K (25 mg/10 mL in H2O), followed by incubation at 50°C for 1 hour. DNA was extracted using Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) and purified with the MinElute Reaction Cleanup Kit (Qiagen). Libraries were amplified with dual indexed adaptors.

treatment_protocol: EXP-2: On day 7, Thy1.1+ cells were sorted and treated with 0, 2, or 200 µM (Z)-4-hydroxytamoxifen for 3 hours, then immediately processed for CUT&Tag.

value: CD8 T cells

tag: antibody

value: Rabbit IgG (EpiCypher #13-0042)

value: Tamoxifen 3hour


title: CB42389 treatment C

status: Public on Apr 03 2026

type: SRA

city: Singapore

first: John

last: Ouyang

address: 8 College Rd

country: Singapore

institute: Duke-NUS Medical School

description: Library name: CB42389_C

library_source: transcriptomic single cell

data_processing:
The demultiplexed reads were aligned against GRCh38 reference genome using Alevin via salmon 1.5.2.53,54 The single-cell data were loaded into count matrices, and all 45 samples were merged using Seurat v4.1.55 Cells that expressed 500 to 6,702 (top 2% of entire dataset) genes and contained less than 20% of transcripts mapping to mitochondrial genes were retained. Subsequently, 5,000 cells were subsampled from each day and condition (13 day-conditions in total), with the exception of Day3-C, which is comprised of 2,151 cells, and Day0, where we subsampled 15,000 cells. This resulted in a total of 72,151 cells.
Assembly: GRCh38
Supplementary files format and content: Seurat Object containing all samples

molecule: polyA RNA

organism: Homo sapiens

source_name: Cord Blood

extract_protocol:
UCB-CD34+ were cultured respectively in Stemspan-AOF media supplemented in cytokine cocktails with and without C7 respectively as described previously for 3, 7 and 11 days. Harvested cells were then resuspended into single-cell suspensions into 1000-6000 cells/ul to be loaded into Chromium microfluidic chips with targeted cell recovery of 10,000 using the Chromium Controller (10x Genomics, USA).
Single-cell RNA-seq libraries were prepared according to the manufacturer’s instructions using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) (10 X Genomics, USA; PN-1000268). Captured libraries were sequenced on the Novaseq 6000 sequencer analyzer at PE150 and sequencing depth of 20,000 read pairs per cell (Novogene, China).

tag: tissue

value: Cord Blood

tag: cell line

value: Primary Cells

tag: cell type

value: HSPCs

tag: genotype

value: normal

tag: treatment

value: condition C

tag: batch


title: OM_B2_1d_Rep2

type: RNA

city: Seoup

first: Dong Woo

last: Lee

address: 174, Yulgok-ro, Jongno-gu

country: South Korea

institute: Seoul National University

description: Human PDLSC cultured in OM with BMP2 treatment for 1 day, replicate 2

hyb_protocol: Approximately 5.5 μg of labeled DNA target was hybridized to the Affymetrix GeneChip Array at 45°C for 16hour

scan_protocol: Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix).

data_processing: The probe cell intensity data computation and a CEL file generation was performed using Affymetrix® GeneChip Command Console® Software (AGCC).

label: biotin

molecule: total RNA

source_name: Human PDLSC (periodontal ligament stem cell)

label_protocol: The sense cDNA was then fragmented and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labeling kit

growth_protocol: PDLSCs were maintained in α-MEM supplemented with 10% FBS at 37°C in a 5% CO2 humidified incubator.  For the OM group, osteogenic medium (containing β-glycerophosphate, ascorbic acid, dexamethasone) was used.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions. cDNA was synthesized using the GeneChip WT (Whole Transcript) Amplification kit as described by the manufacturer.

treatment_protocol: NT: no additional treatment , B2: BMP2 (concentration 8nM) , B9: BMP9 (concentration 8nM) Cells were incubated for 1, 7, or 15 days depending on the group.

value: human periodontal ligament stem cells derived from adult donors

value: Normal

tag: age

value: 28 year old


title: OM_B9_1d_Rep2

description: Human PDLSC cultured in OM with BMP9 treatment for 1 day, replicate 2


title: WTR8

city: RTP

first: Thomas

last: Randall

state: NC

address: 101 TW Alexander

country: USA

institute: NIEHS

description: Library name: Sample 105

library_source: other

data_processing:
Paired-end sequencing of the hypervariable V3-V4 region of the 16S rRNA gene was performed with Read 1 (TATGGTAATTGTGTGCCAGCMGCCGCGGTAA), Read 2 (AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT), and Index Primers (ATTAGAWACCCBDGTAGTCCGGCTGACTGACT) using an Illumina MiSeq instrument (Illumina) to produce 250 base-paired reads.
Bioinformatics analysis of bacterial 16S rRNA amplicon data was conducted using the Quantitative Insights into Microbial Ecology (QIIME2 (qiime2-2022.11; 1) pipeline.
Statistical analysis done with DEseq2 (v 1.38.3)
The dada2 algorithm was used to generate amplicon sequence variants (ASVs) with empirically determined trimming settings for each analysis.
Taxonomic classification was done using the SILVA version 138 database formatted for QIIME2 (https://docs.qiime2.org/2024.10/data-resources/)
Assembly: mm10
Supplementary files format and content: OTU table in text format, sample IDs and readcounts for each sample
Library strategy: 16S rRNA-seq

molecule: other

organism: Mus musculus

source_name: lung

extract_protocol:
Lungs were excised from animals under sterile conditions and were collected into DNA extraction buffer containing ATL buffer and proteinase K (Qiagen) and shaken in a FastPrep Instrument (MP Biomedicals) at 5.0ms-1 for 30s. Following bead-beating, genomic DNA (gDNA) was extracted using the DNeasy Blood and Tissue Kit (Qiagen) in accordance with the manufacturer protocol.
The PCR amplification of the target region was performed using 515F (GTGYCAGCMGCCGCGGTAA) and 806R (GGACTACNVGGGTWTCTAAT) 16S primers. Amplicons were then purified using NGS clean-up and size-select (Macherey-Nagel) in accordance with the manufacturer protocol.

treatment_protocol: Animals were treated with Bleomycin, TLR5-agonist/Bleomycin, or ABX/Bleomycin. Some animals receieved fecal microbiota transfer (FMT).

value: lung

tag: Sex

value: male

tag: strain

value: wild-type

value: FMT recipient


title: OM_NT_1d_Rep1

description: Human PDLSC cultured in OM with no treatment for 1 day, replicate 1


title: Tamoxifen, CD8, aIgG, rep1

city: Chicago

first: Weiguo

last: Cui

state: IL

address: 303 E Chicago Ave

department: Pathology

institute: Northwestern University

description: Library name: CnT_TMF_aIgG_rep1

library_source: genomic

molecule: genomic DNA


title: OM_B2_1d_Rep1

description: Human PDLSC cultured in OM with BMP2 treatment for 1 day, replicate 1


title: CB42389 treatment B

description: Library name: CB42389_B

value: condition B


title: OM_NT_7d_Rep1

description: Human PDLSC cultured in OM with no treatment for 7 day, replicate 1


title: WTR5

description: Library name: Sample 102


title: Tamoxifen 200uM, CD8, aKLF2, rep2

description: Library name: CnT_TMF_200uM_aKLF2_rep2

value: KLF2 (Millipore #09-820)

value: Tamoxifen 200uM 3hour


title: CB42389 treatment A

description: Library name: CB42389_A

value: condition A


title: GM_NT_1d_Rep1

description: Human PDLSC cultured in GM with no treatment for 1 day, replicate 1


title: Tripartite ETR targeting TGFBR2 with a truncated gRNA treated T cells, replicate2

city: Milano

first: Sara

last: Valsoni

state: MI

address: via Olgettina 60

department: TIGET

country: Italy

institute: SR-TIGET

description: Library name: Sil_TGFBR2_truncated_R2

library_source: transcriptomic

data_processing:
Image analysis and base calling were conducted by the NovaSeq Control Software v1.7 on the NovaSeq instrument and raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification.
Reads quality was controlled with Fastqc v0.11.9, while low quality reads and the adapters were removed using Trim Galore v0.6.6 according to following parameters: --quality 20, --length 25, --paired
High quality reads were aligned to the GRCh38 reference genome
Gene counts were quantified using the featureCounts function from Subread package v 2.0.1 and the corresponding annotation as gene model
Read distribution was estimated using the Negative Binomial Generalized log-linear model implemented in the R/Bioconductor package DESeq2 v1.30.0
Differential gene expression analysis was performed using the nbinomWaldTest function, and p-values were corrected using the Benjamini-Hochberg (BH) method
Transcripts Per Million (TPM) values displayed in scatter plots were calculated using the length from the featureCounts output
Assembly: GRCh38
Supplementary files format and content: text file with raw counts

source_name: Blood

growth_protocol: Activated T cells were cultured in RPMI supplemented with 1% penicillin/streptomyci, 2% L-glutamine, 10% FBS, and 5 ng/ml of each IL-7 and IL-15. Cells were cultured at 37°C in a 5% CO2 humidified incubator.

extract_protocol:
RNA exrtraction by RNeasy® Mini Kit (QIAGEN), following provider indications
RNA-seq libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations

treatment_protocol: After 2 days of Dynabeads activation, T cells were nucleofected with ETRs mRNA and gene-specific targeting gRNAs. Two weeks after T cells were restimulated with Dynabeads and kept in culture for additional 7 days before RNA extraction.

value: Blood

value: T cells

value: TGFBR2 epi-silencing


title: WTR2

description: Library name: Sample 99


title: WTR6

description: Library name: Sample 103


title: Tamoxifen 2uM, CD8, aKLF2, rep2

description: Library name: CnT_TMF_2uM_aKLF2_rep2

value: CD8 T cells

value: Tamoxifen 2uM 3hour


title: WTR7

description: Library name: Sample 104


title: WTR3

description: Library name: Sample 100


title: WTR4

description: Library name: Sample 101


title: Tripartite ETR targeting TGFBR2 with a truncated gRNA treated T cells, replicate1

description: Library name: Sil_TGFBR2_truncated_R1


title: WTD4

description: Library name: Sample 96

value: FMT donor


title: Tamoxifen 2uM, CD8, aKLF2, rep1

description: Library name: CnT_TMF_2uM_aKLF2_rep1


title: Tripartite ETR targeting TGFBR2 with a full-length gRNA treated T cells, replicate 2

description: Library name: Sil_TGFBR2_R2


title: WTD5

description: Library name: Sample 97


title: WTR1

description: Library name: Sample 98


title: WTD2

description: Library name: Sample 94


title: Tamoxifen 0uM, CD8, aKLF2, rep2

description: Library name: CnT_TMF_0uM_aKLF2_rep2

value: Tamoxifen 0uM 3hour


title: WTD3

description: Library name: Sample 95


title: KOR8

description: Library name: Sample 92


title: WTD1

description: Library name: Sample 93


title: Tripartite ETR targeting TGFBR2 with a full-length gRNA treated T cells, replicate 1

description: Library name: Sil_TGFBR2_R1


title: Tamoxifen 200uM, CD8, aKLF2, rep1

description: Library name: CnT_TMF_200uM_aKLF2_rep1


title: Tamoxifen 0uM, CD8, aKLF2, rep1

description: Library name: CnT_TMF_0uM_aKLF2_rep1


title: KOR7

description: Library name: Sample 91


title: Tripartite ETR targeting TET2 with a truncated gRNA treated T cells, replicate2

description: Library name: Sil_TET2_truncated_R2

value: TET2 epi-silencing


title: KOR6

description: Library name: Sample 90


title: Tamoxifen 200uM, CD8, aERKLF3, rep2

description: Library name: CnT_TMF_200uM_aERKLF3_rep2

value: ERalpha (C-terminal) (EpiCypher #13-2012)


title: KOR5

description: Library name: Sample 89

value: lung


title: KOR3

description: Library name: Sample 87

value: male


title: Tamoxifen 200uM, CD8, aERKLF3, rep1

description: Library name: CnT_TMF_200uM_aERKLF3_rep1


title: KOR4

description: Library name: Sample 88


title: KOD4

description: Library name: Sample 84


title: KOR2

description: Library name: Sample 86


title: Tripartite ETR targeting TET2 with a truncated gRNA treated T cells, replicate1

description: Library name: Sil_TET2_truncated_R1


title: Tripartite ETR targeting TET2 with a full-length gRNA treated T cells, replicate 1

description: Library name: Sil_TET2_R1


title: KOD3

description: Library name: Sample 83


title: Tamoxifen 2uM, CD8, aERKLF3, rep2

description: Library name: CnT_TMF_2uM_aERKLF3_rep2


title: Tamoxifen 2uM, CD8, aERKLF3, rep1

description: Library name: CnT_TMF_2uM_aERKLF3_rep1


title: Tripartite ETR targeting TET2 with a full-length gRNA treated T cells, replicate 2

description: Library name: Sil_TET2_R2


title: Tripartite ETR targeting B2M with a truncated gRNA treated T cells, replicate2

description: Library name: Sil_B2M_truncated_R2

value: B2M epi-silencing


title: Tripartite ETR targeting B2M with a truncated gRNA treated T cells, replicate1

description: Library name: Sil_B2M_truncated_R1


title: KOD1

description: Library name: Sample 81


title: KOR1

description: Library name: Sample 85

value: FMT recipient


title: 8137

description: Library name: Sample 80

value: wild-type


title: Tamoxifen 0uM, CD8, aERKLF3, rep2

description: Library name: CnT_TMF_0uM_aERKLF3_rep2


title: Tamoxifen 0uM, CD8, aERKLF3, rep1

description: Library name: CnT_TMF_0uM_aERKLF3_rep1


title: KOD2

description: Library name: Sample 82


title: OM_B9_1d_Rep1

description: Human PDLSC cultured in OM with BMP9 treatment for 1 day, replicate 1

value: human periodontal ligament stem cells derived from adult donors

value: Normal

value: 28 year old


title: Tripartite ETR targeting B2M with a full-length gRNA treated T cells, replicate 2

description: Library name: Sil_B2M_R2

value: Blood


title: 8136

description: Library name: Sample 79


title: 8135

description: Library name: Sample 78


title: LCMV Clone 13, Day8, CD8, aIgG

description: Library name: CnT_Cl13D8_CD8_aIgG

source_name: Spleen

value: Spleen

value: Rabbit IgG (EpiCypher #13-0042)


title: 8134

description: Library name: Sample 77


title: 8132

description: Library name: Sample 75


title: Tripartite ETR targeting B2M with a full-length gRNA treated T cells, replicate 1

description: Library name: Sil_B2M_R1

value: T cells


title: 8133

description: Library name: Sample 76


title: 8131

description: Library name: Sample 74


title: 8130

description: Library name: Sample 73


title: 8125

description: Library name: Sample 68


title: Tripartite ETR targeting CD3 with a truncated gRNA treated T cells, replicate 1

description: Library name: Sil_CD3D_truncated_R1

treatment_protocol: After 2 days of Dynabeads activation, T cells were nucleofected with ETRs mRNA and CD3D targeting gRNAs. Two weeks after T cells were restimulated with PMA/Iono and kept in culture for additional 7 days before RNA extraction.

value: CD3D epi-silencing


title: LCMV Clone 13, Day8, P14, aKLF2, rep2

description: Library name: CnT_Cl13D8_P14_aKLF2_rep2

value: P14 CD8 T cells

value: KLF2 (Millipore #09-820)


title: Tripartite ETR targeting CD3 with a truncated gRNA treated T cells, replicate 2

description: Library name: Sil_CD3D_truncated_R2


title: 8129

description: Library name: Sample 72


title: Tripartite ETR targeting with an unrelated gRNA treated T cells, replicate 1.

description: Library name: Sil_unrelated_R1

value: Epi-silencing with an unrelated  gRNA


title: 8127

description: Library name: Sample 70


title: testis.ip_Ctr1

city: Guangzhou

first: Xiaoyun

last: Wang

state: Guangdong

address: 190 Kaiyuan Avenue, Huangpu

department: Infection and Immunity

country: China

institute: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences

description: Library name: sample70

data_processing:
To retain high-quality sequencing reads for downstream analysis, trimming and filtering steps were applied using Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and fastp-0.23.4. Low-quality reads with a Phred score (Q-value) below 20, sequences shorter than 50 base pairs, and the first and last 5 bases of each read were removed to eliminate potential sequencing errors. Following these preprocessing steps, the data was reassessed using FastQC to verify that the quality met the required standards for downstream applications. The resultant clean sequencing reads were then used for downstream analysis.
Assembly: mm39
Library strategy: MeRIP-seq

source_name: testis

growth_protocol: Six-week-old male C57BL/6J mice were used for the experiment. There were four groups in animal experiments (3 mice for each group), including the control group (Con) and three vitamins (VB2, VB6 and VB12, respectively).

extract_protocol:
After 14 days of treatment, the mice were euthanized, brain, liver, and testis tissues were collected for subsequent library construction experiments, while the remaining tissues were stored in a -80°C freezer.
The m6A MeRIP-seq libraries were constructed using the m6A-seq2 protocol. Poly-A selected mRNA (1.8 μg per sample) was extracted using Oligo d(T)25 Magnetic Beads and fragmented to approximately 150 nucleotides (nt) using RNA fragmentation buffer. Fragmented RNA was DNase digestion, dephosphorylation, and purification using T4 PNK, TURBO DNase, FastAP (NEB, Thermo Scientific), and VAHTS RNA Clean Beads (Vazyme Biotech). A 3′ RNA barcode adapter was ligated using T4 RNA Ligase (NEB), and barcoded RNA samples were pooled, reserving 10% as input control. For m6A immunoprecipitation, RNA was heat-denatured and incubated with anti-m6A antibody (NEB)-coated Protein A/G magnetic beads (Thermo Scientific) in IPP buffer. Beads were washed with low- and high-salt IPP buffers to remove nonspecific binding. Enriched RNA was eluted with RLT buffer (Qiagen), purified, and reverse-transcribed to cDNA using SuperScript III (Thermo Scientific) with a specific rTd primer. cDNA synthesis was followed by RNA hydrolysis and Illumina 5′ adapter ligation. PCR enrichment was performed using the KAPA HiFi PCR Kit, with a universal forward primer and barcode-containing reverse primer. The amplified libraries were purified, quantified using a Qubit fluorometer, and assessed for quality using an Agilent Bioanalyzer 4200 TapeStation. RNA-seq and MeRIP-seq were conducted on the NovaSeq 6000 platform (Illumina) to generate paired-end 150 bp reads, producing ~10 GB of raw data per library.

treatment_protocol: Due to the differences in vitamin requirements among mice, the dosages for the study were based on previous studies. The dosage of VB2 was 50 mg/kg, VB6 was 50 mg/kg, and VB12 was 25 mg/kg.

value: testis

value: water


title: Tripartite ETR targeting with an unrelated gRNA treated T cells, replicate 2.

description: Library name: Sil_unrelated_R2


title: LCMV Clone 13, Day8, P14, aKLF2, rep3

description: Library name: CnT_Cl13D8_P14_aKLF2_rep3


title: 8124

description: Library name: Sample 67

value: lung

value: male


title: testis.ip_Ctr3

description: Library name: sample72


title: 8128

description: Library name: Sample 71


title: 8126

description: Library name: Sample 69


title: LCMV Clone 13, Day8, P14, aKLF2, rep1

description: Library name: CnT_Cl13D8_P14_aKLF2_rep1


title: testis.ip_Ctr2

description: Library name: sample71


title: Tripartite ETR targeting CD3D full-length gRNAs treated T cells, replicate 2

description: Library name: Sil_CD3D_R2


title: 8123

description: Library name: Sample 66

value: ABX/Bleomycin


title: 8122

description: Library name: Sample 65

value: wild-type


title: Tripartite ETR targeting CD3D full-length gRNAs treated T cells, replicate 1

description: Library name: Sil_CD3D_R1


title: 8121

description: Library name: Sample 64


title: testis.ip_B6_3

description: Library name: sample69

value: vitamin VB6


title: 8120

description: Library name: Sample 63


title: 8118

description: Library name: Sample 61


title: Tripartite ETR targeting with an unrelated gRNA treated T cells, replicate 1

description: Library name: Sil_unrelated_R1


title: Tripartite ETR targeting with an unrelated gRNA treated T cells, replicate 2

description: Library name: Sil_unrelated_R2


title: 8116

description: Library name: Sample 59


title: 8119

description: Library name: Sample 62


title: testis.ip_B2_3

description: Library name: sample66

value: vitamin VB2


title: testis.ip_B6_1

description: Library name: sample67


title: 8112

description: Library name: Sample 55


title: 8115

description: Library name: Sample 58


title: testis.ip_B2_2

description: Library name: sample65


title: 8114

description: Library name: Sample 57


title: testis.ip_B6_2

description: Library name: sample68


title: 8110

description: Library name: Sample 53


title: 8111

description: Library name: Sample 54


title: 25C/TB

city: Poznan

first: Marzena

last: Gajecka

address: Strzeszynska 32

country: Poland

institute: Institute of Human Genetics, Polish Academy of Sciences

description:
Library name: D3
Fresh frozen Spatial transcriptome sequencing

data_processing:
The raw FASTQ files and histology images were processed for each sample using the 10x Genomics Space Ranger software (version 2.1.0).
Reads were aligned to the human Cell Ranger hg38 reference genome (available at https://cf.10xgenomics.com/supp/spatial-exp/refdata-gex-GRCh38-2020-A.tar.gz) using the STAR method.
The spatial feature counts were generated using the spaceranger count function.
Assembly: hg38
Supplementary files format and content: aligned_fiducials.jpg: aligned fiducial of the tissue image
Supplementary files format and content: barcodes.tsv.gz: list of spatial barcodes
Supplementary files format and content: detected_tissue_image.jpg: image of tissue and spots
Supplementary files format and content: features.tsv.gz: list of gene IDs
Supplementary files format and content: matrix.mtx.gz: gene expression count data in Matrix Market Exchange Format
Supplementary files format and content: scalefactors_json.json: scalefactors in json format
Supplementary files format and content: tissue_hires_image.png: high resolution image of tissue
Supplementary files format and content: tissue_lowres_image.png: low resolution image of tissue
Supplementary files format and content: tissue_positions.csv: list of barcodes and pixel coordinates of each Capture Area
Library strategy: Spatial transcriptomics

source_name: Cornea

extract_protocol:
The full-thickness corneas were submersed in Optimal Cutting Temperature (O.C.T.) compound (Tissue Tek, Sakura) and immediately frozen on dry ice with the use of Cryospray (Engelbrecht GmbH). The 10 µm-thick two or three consecutive cryosections of one cornea were placed in the Capture Area of a Visium Slide. The slides were stored at -80°C until further analysis.
After tissue fixation in methanol, H&E staining, and microscopic imaging the Visium Spatial Gene Expression Libraries were constructed according to the manufacturer’s protocol.

value: Cornea

tag: disease status

value: non-affected


title: 12C/TB

description:
Library name: D1
Fresh frozen Spatial transcriptome sequencing


title: 8080

description: Library name: Sample 52

value: Tlr5fx/fx SPC-Cre

value: Bleomycin


title: 10WUM/KTCN

description:
Library name: D4
Fresh frozen Spatial transcriptome sequencing

value: Keratoconus


title: 8117

description: Library name: Sample 60


title: 8079

description: Library name: Sample 51


title: 8113

description: Library name: Sample 56


title: 8078

description: Library name: Sample 50


title: testis.ip_B12_1

description: Library name: sample61

value: vitamin VB12


title: 20C/TB

description:
Library name: D2
Fresh frozen Spatial transcriptome sequencing


title: testis.ip_B2_1

description: Library name: sample64


title: 8077

description: Library name: Sample 49


title: testis.ip_B12_3

description: Library name: sample63


title: testis.ip_B12_2

description: Library name: sample62


title: 8074

description: Library name: Sample 46


title: 8075

description: Library name: Sample 47


title: 14WUM/KTCN

description:
Library name: C2
Fresh frozen Spatial transcriptome sequencing


title: 26C/TB

description:
Library name: C4
Fresh frozen Spatial transcriptome sequencing


title: 8076

description: Library name: Sample 48


title: 8072

description: Library name: Sample 44


title: 28C/TB

description:
Library name: C3
Fresh frozen Spatial transcriptome sequencing


title: 8071

description: Library name: Sample 43


title: testis.input_Ctr1

description: Library name: sample58


title: 6WUM/KTCN

description:
Library name: B3
Fresh frozen Spatial transcriptome sequencing


title: Input, cardiomyocytes, Mouse 87907, Lmna CKO rep 2

city: Cincinnati

first: Kohta

last: Ikegami

state: OH

address: 240 Albert Sabin Way, Cincinnati, OH 45229

department: Division of Molecular Cardiovascular Biology

institute: Cincinnati Children's Hospital Medical Center

description: Library name: AE87422

data_processing:
Pol II ChIP-seq reads (paired-end 100 bp) were processed by fastp to remove duplicated reads and then aligned to the mouse and human reference genomes individually using Bowtie2 (ver 2.4.2). Reads with mapping quality (MAPQ) greater than 20 were used in subsequent analyses.
We scaled per-base mouse ChIP-seq read coverage linearly by multiplying with a scaling factor, which was computed based on the spike-in reads. More specifically, assuming that the human spike-in chromatin was exactly proportionally added to experimental mouse chromatin across ChIP reactions, the scaling factor would be hChIPi^-1, where hChIPi is the total ChIP-seq read count aligned to the human genome in ChIP reaction i. In practice, however, the spike-in chromatin fraction might vary between ChIP reactions due to pipetting and chromatin quantification errors, which would proportionally affect hChIPi. Therefore, we estimate the spike-in chromatin fraction using reads from the “input” fraction withdrawn from the reaction, by computing hINPUTi /(hINPUTi + mINPUTi), where hINPUTi and mINPUTi are the total input read counts aligned to the human and mouse genome, respectively, in reaction i. Together, we computed a scaling factor (SF) for each ChIP-seq reaction (i) as: SFi = hChIPi^-1 * {hINPUTi /(hINPUTi + mINPUTi)} * k, where k is a constant, for which we used 1*10^6 for convenience.
Assembly: mm39 and GRCh39
Supplementary files format and content: *xPEn.bw reports paired-end fragment coverage per base, normalized by sequencing depth for the target genome.
Supplementary files format and content: *_spnorm.bw reports paired-end fragment coverage per base, normalized by spike-in controls and sequencing depth.

source_name: Cardiomyocyte

growth_protocol:
Mice were housed in cages with up to four animals per cage and maintained with free access to standard chow and water. Mice were administered with heparin (100 Units, NDC 25021–400-10), anesthetized with Isoflurane, and euthanized by cervical dislocation. Hearts were perfused with 100 mM KCl in PBS and moved to Potassium buffer (4.65 mM 2,3-butanedione monoxime, 0.5 mM EGTA, 11.1 mM glucose, 40.2 mM KCl, 16.2 mM KH2PO4, 70 mM L-Asp potassium salt, 10 mM Na-pyruvate, 10 mM taurine, 10.1 mM HEPES, pH 7.4). Excised hearts were cannulated to the Langendorff retrograde perfusion system through the aorta and perfused with prewarmed Digestion buffer (1 mg/ml Collagenase Type II (Worthington, LS004177), 10 mM glucose, 5 mM HEPES, 5.4 mM KCl, 1.2 mM MgCl2, 150 mM NaCl and 2 mM sodium pyruvate, 10 mM taurine and 12 mM 2,3-butanedione monoxime, pH 7.35) for 20 min at 37°C. Digested hearts were perfused with 10 ml of 1% paraformaldehyde (PFA; Electron Microscopy Slides, #15710) in PBS prior to cell dissociation. Cardiomyocytes were then dissociated by gently triturating in 1% PFA in PBS. Cell suspension was filtrated through a 240 μm mesh and incubated with 1% PFA for a total 10 min (from perfusion to this step) and quenched by 125 mM glycine. Cardiomyocytes were sedimented at the bottom of conical tubes by gravity, and the resulting cell pellet was further purified in PBS using gravity.
BJ-5ta human fibroblasts, used as spike-in controls, were cultured in high-glucose DMEM (Gibco, 11965-092) containing 9% fetal bovine serum (FBS) and penicillin-streptomycin. BJ-5ta cells were crosslinked in 1% formaldehyde for 15 min and the reaction was quenched by 125 mM glycine.

extract_protocol:
Fixed cardiomyocytes and BJ-5ta cells were separately thawed on ice and sequentially lysed in Lysis Buffer 1 (LB1: 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 50 mM HEPES-KOH, pH 7.5) and Lysis Buffer 2 (LB2: 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0), each for 10 min with gentle rotation. Permeabilized cells were then resuspended in Lysis Buffer 3 (LB3: 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroyl sarcosine, 10 mM Tris-HCl, pH 8.0), and chromatin was fragmented by sonication using a Bioruptor (high setting, 10 min). Triton X-100 was added to a final concentration of 1%, and insoluble material was removed by centrifugation at 14,000 g for 10 min. For each immunoprecipitation, chromatin corresponding to 2.6 µg of mouse DNA was mixed with 0.15 µg of human spike-in DNA in LB3 supplemented with protease inhibitor cocktail (Sigma-Aldrich, 539131) and phosphatase inhibitors (Abcam, ab201113). After clarification by centrifugation, 10% of the supernatant was reserved as input. Extracts were incubated overnight at 4 °C with rotation using rabbit anti-RNA polymerase II N-terminal domain antibody (Cell Signaling Technology, 14958). Immunocomplexes were captured by incubation with Dynabeads Protein G beads (Invitrogen, 10004D) for 3 h at 4 °C. Beads were washed sequentially with LB3 containing 1% Triton X-100 (twice), LB3 with 1% Triton X-100 and 1 M NaCl, LB3 with 1% Triton X-100 and 0.5 M NaCl, LB3 with 1% Triton X-100, and twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) containing 0.1% Tween-20. After the first wash, beads were transferred to fresh tubes. Chromatin was eluted twice in elution buffer (1% SDS, 250 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) at 65 °C with continuous agitation, and eluates were combined. Input samples were adjusted to the same final volume. Samples were treated with RNase A (0.08 mg ml⁻¹) for 1 h at room temperature and proteinase K (0.08 mg ml⁻¹) for 2 h at 55 °C, followed by reversal of crosslinks at 65 °C for 14 h. DNA was purified using a Qiagen PCR purification kit and eluted in 100 µl of 10 mM Tris-HCl (pH 8.0).
DNA libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, E7645L)

treatment_protocol: All mice used in experiments were administered with tamoxifen (Sigma, T5648) at 6-8 weeks of age via intraperitoneal injections (100 μL of 4 mg/mL solution per day for 4 consecutive days) dissolved in corn oil (Sigma, C8267)

value: Cardiomyocyte

value: Lmna F/F; Myh6MerCreMer-Tg/0

value: 2 weeks post tamoxifen


title: 8WUM/KTCN

description:
Library name: B4
Fresh frozen Spatial transcriptome sequencing


title: Input, cardiomyocytes, Mouse 87989, Lmna WT rep 3

description: Library name: AE87424

value: Lmna +/+; Myh6MerCreMer-Tg/0


title: Input, cardiomyocytes, Mouse 87920, Lmna CKO rep 3

description: Library name: AE87423


title: testis.input_B6_2

description: Library name: sample56

value: testis


title: 8069

description: Library name: Sample 41

value: lung


title: 8070

description: Library name: Sample 42

value: male


title: testis.input_B6_3

description: Library name: sample57


title: 5WUM/KTCN

description:
Library name: B2
Fresh frozen Spatial transcriptome sequencing


title: 8067

description: Library name: Sample 39


title: 8068

description: Library name: Sample 40


title: testis.input_Ctr2

description: Library name: sample59

value: water


title: Input, cardiomyocytes, Mouse 87903, Lmna WT rep 2

description: Library name: AE87421


title: testis.input_B6_1

description: Library name: sample55


title: testis.input_Ctr3

description: Library name: sample60


title: 2WUM/KTCN

description:
Library name: B1
Fresh frozen Spatial transcriptome sequencing


title: testis.input_B2_3

description: Library name: sample54


title: 4WUM/KTCN

description:
Library name: A3
Fresh frozen Spatial transcriptome sequencing


title: Input, cardiomyocytes, Mouse 87902, Lmna WT rep 1

description: Library name: AE87420


title: Input, cardiomyocytes, Mouse 88137, Lmna CKO rep 1

description: Library name: AE87419


title: hHGF-NSG, alectinib treated, six weeks, replicate 1

city: Tampa

first: Bina

last: Desai

state: Florida

address: 12902 USF Magnolia Drive

institute: H. Lee Moffitt Cancer Center

description: Library name: h_6W_A1

data_processing:
Reads were mapped to the human - GRCh38 reference genome, normalized reads were obtained from DeSeq2
Reads were mapped to the mm10 (Mus musculus) mouse reference genome, normalized reads were obtained.
Assembly: hg38, MM10
Supplementary files format and content: FPKM normalized gene counts: fpkm_hg38.txt, fpkm_mm10.txt (samples N_ctrl2 and N_6W_A4 are not represented in fpkm_mm10.txt.).

source_name: xenograft tumor tissue

extract_protocol:
Total RNA was isolated using TRIzol reagent (Invitrogen, 15596026) according to the manufacturer’s protocol. The concentration, quality, and integrity for total RNA was measured using NanoDrop and TapeStation system.
Library preparation and quality assessment were accomplished using Novogene’s library guidelines.

value: xenograft tumor tissue

value: ALK+ NSCLC

tag: host mouse

value: hHGF-NSG

value: six weeks


title: testis.input_B2_1

description: Library name: sample52

value: vitamin VB2


title: 8064

description: Library name: Sample 36


title: 3WUM/KTCN

description:
Library name: A1
Fresh frozen Spatial transcriptome sequencing

value: Cornea


title: hHGF-NSG, alectinib treated, six weeks, replicate 3

description: Library name: h_6W_A3


title: 8066

description: Library name: Sample 38

value: Tlr5fx/fx SPC-Cre

value: Bleomycin


title: 8063

description: Library name: Sample 35


title: testis.input_B12_3

description: Library name: sample51

value: vitamin VB12


title: hHGF-NSG, alectinib treated, two weeks, replicate 4

description: Library name: h_2W_A4

value: two weeks


title: 8073

description: Library name: Sample 45


title: testis.input_B2_2

description: Library name: sample53


title: 8062

description: Library name: Sample 34


title: Pol II ChIP, cardiomyocytes, Mouse 87920, Lmna CKO rep 3

description: Library name: AE87405

value: Cardiomyocyte

value: Lmna F/F; Myh6MerCreMer-Tg/0


title: Pol II ChIP, cardiomyocytes, Mouse 87989, Lmna WT rep 3

description: Library name: AE87406

value: Lmna +/+; Myh6MerCreMer-Tg/0

value: 2 weeks post tamoxifen


title: hHGF-NSG, alectinib treated, six weeks, replicate 2

description: Library name: h_6W_A2


title: hHGF-NSG, alectinib treated, two weeks, replicate 3

description: Library name: h_2W_A3


title: testis.input_B12_2

description: Library name: sample50

value: testis


title: 8065

description: Library name: Sample 37

value: lung

value: male


title: testis.input_B12_1

description: Library name: sample49


title: 8061

description: Library name: Sample 33


title: Pol II ChIP, cardiomyocytes, Mouse 87903, Lmna WT rep 2

description: Library name: AE87403


title: Pol II ChIP, cardiomyocytes, Mouse 87907, Lmna CKO rep 2

description: Library name: AE87404


title: Pol II ChIP, cardiomyocytes, Mouse 87902, Lmna WT rep 1

description: Library name: AE87402


title: hHGF-NSG, alectinib treated, two weeks, replicate 2

description: Library name: h_2W_A2

value: xenograft tumor tissue


title: liver.ip_Ctr3

description: Library name: sample48

source_name: liver

value: liver

value: water


title: Pol II ChIP, cardiomyocytes, Mouse 88137, Lmna CKO rep 1

description: Library name: AE87401


title: liver.ip_Ctr2

description: Library name: sample47


title: liver.ip_Ctr1

description: Library name: sample46


title: 8058

description: Library name: Sample 30


title: 8060

description: Library name: Sample 32


title: hHGF-NSG, Therapy naïve, replicate 2

description: Library name: h_ctrl2

value: ALK+ NSCLC

value: hHGF-NSG

value: Therapy naïve


title: liver.ip_B6_3

description: Library name: sample45

value: vitamin VB6


title: 10X,Su/Hx+LNA-224,mouse lung,LN9

city: New York

first: Yifei

last: Sun

address: 1468 Madison Ave

institute: Icahn School of Medicine at Mount Sinai

description: Library name: LN9

data_processing:
For analysis of the raw data, cellranger was used to generate the matrix files.
Assembly: mm10
Supplementary files format and content: Text based tab separated coverage file

molecule: nuclear RNA

extract_protocol:
Single nuclei were collected from snap frozen mouse lung
!0X 3'DE(V3) scRNA-seq


title: 8057

description: Library name: Sample 29

value: Bleomycin


title: hHGF-NSG, alectinib treated, two weeks, replicate 1

description: Library name: h_2W_A1


title: hHGF-NSG, Therapy naïve, replicate 1

description: Library name: h_ctrl1


title: 8059

description: Library name: Sample 31


title: 10X,Su/Hx+LNA-224,mouse lung,LN7

description: Library name: LN7


title: NSG, alectinib treated, six weeks, replicate 4

description: Library name: N_6W_A4

value: NSG

value: six weeks


title: hHGF-NSG, Therapy naïve, replicate 3

description: Library name: h_ctrl3


title: liver.ip_B6_2

description: Library name: sample44


title: NSG, alectinib treated, six weeks, replicate 3

description: Library name: N_6W_A3


title: 10X,Su/Hx+LNA-ctrl,mouse lung,LN6

description: Library name: LN6

value: Su/Hx+LNA-ctrl


title: liver.ip_B2_2

description: Library name: sample41

value: vitamin VB2


title: 8055

description: Library name: Sample 27


title: liver.ip_B6_1

description: Library name: sample43


title: 10X,Su/Hx+LNA-ctrl,mouse lung,LN4

description: Library name: LN4


title: 10X,Su/Hx+LNA-ctrl,mouse lung,LN5

description: Library name: LN5


title: 8056

description: Library name: Sample 28


title: liver.ip_B2_3

description: Library name: sample42


title: liver.ip_B2_1

description: Library name: sample40


title: 8053

description: Library name: Sample 25


title: 8054

description: Library name: Sample 26


title: NSG, alectinib treated, six weeks, replicate 1

description: Library name: N_6W_A1


title: NSG, alectinib treated, six weeks, replicate 2

description: Library name: N_6W_A2


title: 10X,Su/Hx+LNA-224,mouse lung,LN8

description: Library name: LN8


title: 8051

description: Library name: Sample 24

value: wild-type


title: NSG, alectinib treated, two weeks, replicate 4

description: Library name: N_2W_A4

value: two weeks


title: 10X,Nox,mouse lung,LN2

description: Library name: LN2

value: NOX


title: 8050

description: Library name: Sample 23

value: lung


title: liver.ip_B12_3

description: Library name: sample39

value: vitamin VB12


title: NSG, alectinib treated, two weeks, replicate 3

description: Library name: N_2W_A3

value: xenograft tumor tissue


title: 10X,Nox,mouse lung,LN1

description: Library name: LN1


title: NSG, alectinib treated, two weeks, replicate 2

description: Library name: N_2W_A2


title: Biofilm, C6706∆toxR-pOueS-C6706, replicate 3

city: Memphis

first: Deepak

last: Balasubramanian

state: TN

address: 262 Danny Thomas Pl Rm D2037

department: Host-Microbe Interactions

institute: St. Jude Children's Research Hospital

data_processing:
Demultiplexing, quality control and adapter trimming was performed with bcl-convert (v3.9.3). CLC Genomics Workbench (Qiagen) was used for subsequent data processing. Briefly, paired Illumina reads were mapped to the El Tor reference genome (GenBank AE003852.1- Chromosome I; AE003853.1- chromosome II) using the following settings to generate the transcripts per million bases (TPM) values: mismatch cost 2, insertion / deletion cost 3, length / similarity fraction 0.8, global strand specific alignment. Subsequent pairwise analysis between two datasets of interest (absolute fold change ≥ 2.0 filtered on average expression for FDR correction ≤ 0.01) were used for downstream analysis.
Assembly: Vibrio cholerae El Tor reference genome (GenBank AE003852.1- Chromosome I; AE003853.1- chromosome II)
Supplementary files format and content: Excel files containing read counts and TPM data

organism: Vibrio cholerae O1 str. C6706

source_name: O1 biovar El Tor

growth_protocol: AKI broth was used for virulence-inducing conditions (15 g l-1 BactoTM peptone, 4 g l-1 yeast extract, 5 g l-1 NaCl, 0.03% w/v sodium bicarbonate). Cells were grown for at 37 ˚C, initially static for 3.5 hrs followed by 4 hrs at 200 RPM before RNA isolation.

extract_protocol:
Total RNA was extracted using the DirectZol RNA miniprep kit, Zymo Research, following manufacturer instructions
DNase treatment, rRNA depletion, library preparation and Illumina NovaSeq 6000 sequencing (2x51 bp reads) was performed at SeqCenter, LLC (Pittsburgh, PA).

value: O1 biovar El Tor

value: Strain C6706

value: {delta}toxR

value: Biofilm growth


title: 8048

description: Library name: Sample 21

value: male


title: 10X,Nox,mouse lung,LN3

description: Library name: LN3


title: 8049

description: Library name: Sample 22


title: Biofilm, C6706∆toxR-pOueS-C6706, replicate 1


title: liver.input_Ctr2

description: Library name: sample35

value: liver

value: water


title: NSG, Therapy naïve, replicate 3

description: Library name: N_ctrl3

value: ALK+ NSCLC

value: NSG

value: Therapy naïve


title: 8045

description: Library name: Sample 20

value: Bleomycin


title: liver.ip_B12_1

description: Library name: sample37


title: 8042

description: Library name: Sample 19


title: NSG, alectinib treated, two weeks, replicate 1

description: Library name: N_2W_A1


title: NSG, Therapy naïve, replicate 4

description: Library name: N_ctrl4


title: liver.input_Ctr3

description: Library name: sample36


title: NSG, Therapy naïve, replicate 2

description: Library name: N_ctrl2


title: liver.ip_B12_2

description: Library name: sample38


title: Biofilm, C6706∆toxR, replicate 3


title: Biofilm, C6706∆toxR-pOueS-C6706, replicate 2


title: 8041

description: Library name: Sample 18


title: Biofilm, C6706∆toxR, replicate 2


title: liver.input_B6_3

description: Library name: sample33

value: vitamin VB6


title: liver.input_Ctr1

description: Library name: sample34


title: liver.input_B6_2

description: Library name: sample32


title: NSG, Therapy naïve, replicate 1

description: Library name: N_ctrl1


title: Biofilm, C6706∆toxR, replicate 1


title: 8039

description: Library name: Sample 16


title: 8035

description: Library name: Sample 14


title: liver.input_B2_3

description: Library name: sample30

value: vitamin VB2


title: 8032

description: Library name: Sample 12


title: liver.input_B2_2

description: Library name: sample29


title: GFP cells, rep2

first: Joanna

last: Zyprych-Walczak

address: Wojska Polskiego 28

department: Department of Mathematical and Statistical Methods

institute: Poznan University of Life Sciences

description: Library name: GFP_II

data_processing:
The pre-processing was performed by removing low-quality reads (Q<30) and sequencing adapters using Trim Galore (v0.6.10).
Differential expression analysis: The pre-processed RNA-seq reads were mapped to the human reference genome (GRCh38/hg38, Gencode v44) using STAR (v2.7.11a). Read counts were then normalized by DESeq2 R package. Low expressed genes (with less than 0.1 mean CPM in each condition i.e. RBMXL3, GFP or WT) were excluded from further analysis. Next, we performed the differential expression analysis comparing the RBMXL3 vs. GFP and the RBMXL3 vs. WT conditions using DESeq2. The set of significant differentially expressed genes (DEGs) was determined based on adjusted p-value (FDR) < 0.05 and |log2FC|≥1. As the final list of genes we intersected the significant DEGs of the two comparisons (RBMXL3 vs. GFP, RBMXL3 vs. WT), and we retrieved only those genes with the same trend (upregulation or downregulation) in both comparisons.
Differential splicing analysis: To quantify transcripts expression (TPM) in each condition (RBMXL3, GFP and WT) we employed the Salmon tool (v1.10.2) and aligned the pre-processed RNA-seq reads to the reference transcriptome (Gencode v44). The alternative splicing analysis was then performed using the SUPPA2 tool (v2.3).
Assembly: GRCh38/hg38, Gencode v44
Supplementary files format and content: tab-separated file with TPM values for each sample
Supplementary files format and content: tab-separated file with normalized counts (CPM) for each sample

growth_protocol: TCam-2 cells were cultured at 37 °C and 5% CO2 in Roswell Park Memorial Institute 1640 Medium (RPMI 1640 Medium, GlutaMAX™ Supplement Thermo Fisher Scientific, 61870044) supplemented with 10% heat-inactivated Fetal Bovine Serum (Cytiva, HYCLSV30160.03) and 1% Antibiotic-Antimycotic solution (100X) (Thermo Fisher Scientific, 15240062).

extract_protocol:
RNA was isolated with RNeasy Plus Mini Kit (Qiagen, 74136) and residual DNA was removed by TURBO DNA-free™ kit (Thermo Fisher Scientific, AM1907) treatment according to the manufacturer’s protocol.
RNA libraries for RNA-seq were prepared using TruSeq Stranded mRNA LT kit (Illumina).

value: human testicular cancer cell line

value: GFP overexpression


title: RBMXL3 cells, rep2

description: Library name: RBMXL3_II

value: RBMXL3 overexpression


title: liver.input_B2_1

description: Library name: sample28


title: Biofilm, C6706∆ompU-pOueS-GBE1114, replicate 2

value: {delta}ompU


title: 8040

description: Library name: Sample 17

value: lung


title: liver.input_B6_1

description: Library name: sample31


title: WT cells, rep2

description: Library name: WT_II

value: WT


title: 8038

description: Library name: Sample 15


title: GFP cells, rep1

description: Library name: GFP_I


title: Biofilm, C6706∆ompU-pOueS-GBE1114, replicate 1

value: O1 biovar El Tor

value: Strain C6706


title: Biofilm, C6706∆ompU-pOueS-C6706, replicate 2

value: Biofilm growth


title: 8034

description: Library name: Sample 13

value: male


title: Biofilm, C6706∆ompU-pOueS-C6706, replicate 1


title: 8031

description: Library name: Sample 11

value: wild-type


title: RBMXL3 cells, rep1

description: Library name: RBMXL3_I


title: Biofilm, C6706∆rpoE, replicate 2

value: {delta}rpoE


title: WT cells, rep1

description: Library name: WT_I


title: Biofilm, C6706∆rpoE, replicate 1


title: 8030

description: Library name: Sample 10


title: liver.input_B12_2

description: Library name: sample26

value: liver

value: vitamin VB12


title: liver.input_B12_3

description: Library name: sample27


title: Biofilm, C6706∆ompU-pOmpU-C6706, replicate 2


title: liver.input_B12_1

description: Library name: sample25


title: 8029

description: Library name: Sample 9

value: Bleomycin


title: brain.ip_Ctr2

description: Library name: sample23

source_name: brain

value: brain

value: water


title: RCA adipose biopsy - RC12 is identifier of sample

city: St. Louis

first: Dakota

middle: R

last: Kamm

state: Missouri

address: 1 Brookings Dr

institute: Washington University in St. Louis

description:
Library name: rc12_rca
gene counts

data_processing:
All gene counts were imported into the R/Bioconductor package EdgeR5 and TMM normalization size factors were calculated to adjust for samples for differences in library size.
Assembly: GRCh38
Supplementary files format and content: xlsx file includes raw counts for each sample. Sample name is top row

source_name: Adipose Tissue

extract_protocol:
RNA was harvested using a Rneasy Mini Kit - Qiagen - Catalogue #74104. Between 500ng to 1 ug of total RNA was used for construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using the Ensembl release 101 primary assembly with STAR version 2.7.9a1.

value: Adipose Tissue


title: brain.ip_Ctr3

description: Library name: sample24


title: SQ adipose biopsy - RC12 is identifier of sample

description:
Library name: rc12_sq
gene counts


title: 8028

description: Library name: Sample 8


title: 8026

description: Library name: Sample 7


title: Biofilm, C6706∆ompU-pOmpU-C6706, replicate 1


title: RCA adipose biopsy - RC14 is identifier of sample

description:
Library name: rc14_rca
gene counts


title: SQ adipose biopsy - RC14 is identifier of sample

description:
Library name: rc14_sq
gene counts


title: Biofilm, C6706∆ompU, replicate 1


title: Biofilm, WT C6706, replicate 2


title: SQ adipose biopsy - RC10 is identifier of sample

description:
Library name: rc10_sq
gene counts


title: 8025

description: Library name: Sample 6


title: brain.ip_Ctr1

description: Library name: sample22


title: brain.ip_B6_3

description: Library name: sample21

value: vitamin VB6


title: RCA adipose biopsy - RC10 is identifier of sample

description:
Library name: rc10_rca
gene counts


title: 8023

description: Library name: Sample 5


title: Biofilm, C6706∆ompU, replicate 2


title: Biofilm, WT C6706, replicate 1


title: SQ adipose biopsy - RC09 is identifier of sample

description:
Library name: rc09_sq
gene counts


title: brain.ip_B6_2

description: Library name: sample20


title: AKI, C6706∆rpoE, replicate 1

value: AKI growth


title: 8022

description: Library name: Sample 4


title: 8018

description: Library name: Sample 1


title: SQ adipose biopsy - RC08 is identifier of sample

description:
Library name: rc08_sq
gene counts


title: brain.ip_B6_1

description: Library name: sample19


title: RCA adipose biopsy - RC09 is identifier of sample

description:
Library name: rc09_rca
gene counts


title: brain.ip_B2_3

description: Library name: sample18

value: vitamin VB2


title: AKI, C6706∆toxR-pOueS-GBE1114, replicate 2

value: {delta}toxR


title: AKI, C6706∆rpoE, replicate 2


title: brain.ip_B2_2

description: Library name: sample17


title: 8021

description: Library name: Sample 3

value: lung


title: brain.ip_B2_1

description: Library name: sample16


title: 8020

description: Library name: Sample 2


title: RCA adipose biopsy - RC08 is identifier of sample

description:
Library name: rc08_rca
gene counts


title: SQ adipose biopsy - RC16 is identifier of sample

description:
Library name: rc_16_sq
gene counts


title: input

city: Shanghai

first: Yu

last: Li

address: Minhang Dongchuan Road No.500

institute: East China Normal University

data_processing:
Illumina Casava1.7 software used for basecalling.
Reads were filtered by Skewer to exclude reads with low quality, adapter and short (less than 50bp) sequence fragments
ChIP-seq reads were aligned to the mm10 genome using bowtie2
Peaks were called using MACS2 callpeak (v2.1.1) with the following setting: p value < 0.01, FD > 1.5
Genome_build: mm10
Supplementary_files_format_and_content: bigWig

source_name: input

growth_protocol: For C3H10T1/2 cells differentiation procedure, confluent cells were induced to differentiate to beige/brown adipocytes in differentiation medium supplemented with 5 μg/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 5 μM dexamethasone, 0.125 mM indomethacin, 50 nM T3, and 1μM rosiglitazone for 48 h and subsequently cultured in maintenance medium supplemented with 5 μg/ml insulin, 50 nM T3 and 1 μM rosiglitazone. The maintenance medium was changed every 2 days. The cells were collected on the 7th day.

extract_protocol:
The extracted chromatin from differentiated C3H10T1/2 cells was digested and fragmented into 200 to 1000 bp which was then immunoprecipitated by ChIP grade antibodies against HSF1 or a normal IgG using Magnetic Beads. After that, the DNA and transcriptional factor complexes were uncross-linked to obtain the pure DNA fragment.
DNA libraries were prepared for sequencing using the TruSeq® chip DNA LT Sample Prep Kit accroding the manufacturer's protocal.

treatment_protocol: ChIP assays were performed using a SimpleChIP® Plus Enzymatic Chromatin IP Kit (9003, Cell signaling) according to manufacturer’s instructions. For ChIP-seq, DNA libraries were prepared for sequencing using the TruSeq® chip DNA LT Sample Prep Kit (illumina, USA) and performed by Genergy Biotechnology (shanghai, China) using illumina Hiseq3000.For peak calling, MACS2 callpeak (v2.1.1) were used for the enrichment regions of reads on the genome for each ChIP (treatment) and Input (control) pair, using p value < 0.01.

tag: chip antibody

value: differentiate to beige adipocytes


title: brain.ip_B12_3

description: Library name: sample15


title: AKI, C6706∆toxR-pOueS-GBE1114, replicate 1

value: O1 biovar El Tor

value: Strain C6706


title: ip

source_name: ip

value: Anti-HSF1 antibody (CST)


title: AKI, C6706∆toxR-pOueS-C6706, replicate 3


title: RCA adipose biopsy - RC16 is identifier of sample

description:
Library name: rc_16_rca
gene counts


title: AKI, C6706∆toxR-pOueS-C6706, replicate 2


title: AKI, C6706∆toxR-pOueS-C6706, replicate 1


title: Tripartite ETR targeting B2M with a 16nt gRNA treated K-562 B2M tdTomato cells, replicate 3

description: Library name: Tripartite_B4F4_16_R3

data_processing:
Image analysis and base calling were conducted by the NovaSeq Control Software v1.7 on the NovaSeq instrument and raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification.
Reads quality was controlled with Fastqc v0.11.9, while low quality reads and the adapters were removed using Trim Galore v0.6.6 according to following parameters: --quality 20, --length 25, --paired
A custom reference was created using the reform function  by inserting the Tomato sequence inside the B2M gene into the human reference genome GRCh38 and adding a new transcript containing the first B2M exon along with the Tomato inside the Gencode v35 annotation. High quality remaining reads were aligned to the custom reference genome using STAR v2.7.6a  with default parameters
Gene counts were quantified using the featureCounts function from Subread package v 2.0.1 and the custom annotation as gene model
Read distribution was estimated using the Negative Binomial Generalized log-linear model implemented in the R/Bioconductor package DESeq2 v1.30.0
Differential gene expression was tested using the nbinomWaldTest function and p-values were corrected using the Benjamini-Hochberg (BH) approach
Assembly: GRCh38
Supplementary files format and content: text file with raw counts

source_name: K-562 B2M tdTomato

growth_protocol: B2MtdTomato K-562 cells were cultured in RPMI-1640 supplemented with 10% Fetal Bovine Serum, 1% L-glutamine, and 1% penicillin/streptomycin at 37 °C in a 5% CO2 humidified incubator

treatment_protocol: nucleofection of ETRs mRNA and gRNA

value: K-562 B2M tdTomato

value: lymphoblast cells

value: B2M tdTomato

value: B2M epi-silencing


title: Tripartite ETR targeting B2M with a full length gRNA treated K-562 B2M tdTomato cells, replicate 3

description: Library name: Tripartite_B4F4_20_R3


title: AKI, C6706∆toxR, replicate 2


title: brain.input_Ctr2

description: Library name: sample11


title: AKI, C6706∆toxR, replicate 3


title: HNRNPC_MUT_KD3

city: Cleveland

first: Soon Yeul

last: Yi

address: CWRU Wood Research Building W127A, 2109 Adelbert Rd,

department: Biochemistry

institute: Case Western Reserve University

description:
Library name: JL4774_HNRNPC_MUT_KD3
V5-HNRNPC-MUT-EndoKD-Rep3

data_processing:
Raw fastq files were used as an input for CLIPittyClip processing pipeline (PMID: 38658162)
Assembly: GRCh38.p14
Supplementary files format and content: tab-delimited text file containing peak coordinates

growth_protocol: HEK293 cell lines were grown and maintained at 37°C in DMEM media containing 5% FBS and 1% NEAA.

extract_protocol:
Cell pellets were lysed with PBS lysis buffer (1x PBS, 0.1% SDS, 0.5% NaDoc, 0.5% NP-40, 1 protease inhibitor tablet (Roche)) for 10 minutes on ice. Lysate was then treated with RQ1 DNase (Promega) for 5 minutes at 37°C with shaking at 1100RPM. The lysates were then briefly treated with RNase I at 1:100 or 1:1000 dilutions at the same condition as DNase treatment. Lysates were clarified by centrifuging on tabletop 4°C centrifuge for 10 minutes. For V5-tagged protein samples, V5 antibody (Addgene, cat. #218107-rAB) was used. For endogenous HNRNPC IP in competition CLIP samples, anti-HNRNPC antibody (Invitrogen, cat. #PA5-22280) was used. Immunoprecipitated samples were labeled with radioactivity (gamma-32P-ATP (Revvity)) and ran on SDS-PAGE in non-reducing conditions. The gels were transferred to a nitrocellulose membrane, and bands 10-30kDa above hnRNPC (32kDa) were excised. The excised membranes were treated with proteinase K (Roche) to release the protein-bound RNAs from the membrane.
CLIP footprints were reverse-transcribed using the BrdU incorporation and bead-capture strategy described previously (PMID: 24613350).

treatment_protocol: Expression of the exogenous V5-tagged HNRNPC/RBM25 was induced by treating the cells with 1ug/mL doxycycline for 48 hours. For competition CLIP conditions, wildtype or mutant RBM25 (harboring the HNRNPC RRM) was overexpressed and RNA-bound HNRNPC was immunoprecipitated. For knockdown conditions, siRNA targeting endogenous HNRNPC was transfected prior to doxycycline induction, such that doxycycline simultaneously induced expression of the exogenous V5-tagged HNRNPC construct and depletion of endogenous HNRNPC. Cellular media was replaced with ice-cold PBS for crosslinking using 254nm UV light, on ice, for a dose of 400mJ/cm^2 and a dose of 200mJ/cm^2. Samples were scraped into tubes, centrifuged and cell pellets stored at -80°C until use.

value: Human embryonic Kidney

value: Doxycycline


title: HNRNPC_MUT_KD2

description:
Library name: JL4774_HNRNPC_MUT_KD2
V5-HNRNPC-MUT-EndoKD-Rep2


title: brain.input_Ctr3

description: Library name: sample12

value: brain

value: water


title: Mock treated K-562 B2M tdTomato cells, replicate 3

description: Library name: UT_R3

value: Mock


title: HNRNPC_MUT_KD1

description:
Library name: JL4774_HNRNPC_MUT_KD1
V5-HNRNPC-MUT-EndoKD-Rep1


title: brain.ip_B12_1

description: Library name: sample13

value: vitamin VB12


title: HNRNPC_MUT_CTRL2

description:
Library name: JL4774_HNRNPC_MUT_CTRL2
V5-HNRNPC-MUT-CTRL-Rep2


title: Tripartite ETR targeting B2M with a 16nt gRNA treated K-562 B2M tdTomato cells, replicate2

description: Library name: Tripartite_B4F4_16_R2


title: brain.input_Ctr1

description: Library name: sample10


title: brain.ip_B12_2

description: Library name: sample14


title: HNRNPC_MUT_CTRL3

description:
Library name: JL4774_HNRNPC_MUT_CTRL3
V5-HNRNPC-MUT-CTRL-Rep3


title: brain.input_B6_3

description: Library name: sample9

value: vitamin VB6


title: AKI, C6706∆toxR, replicate 1

value: {delta}toxR

value: AKI growth


title: Tripartite ETR targeting B2M with a full length gRNA treated K-562 B2M tdTomato cells, replicate 2

description: Library name: Tripartite_B4F4_20_R2


title: Mock treated K-562 B2M tdTomato cells, replicate 2

description: Library name: UT_R2


title: HNRNPC_MUT_CTRL1

description:
Library name: JL4774_HNRNPC_MUT_CTRL1
V5-HNRNPC-MUT-CTRL-Rep1


title: brain.input_B6_2

description: Library name: sample8


title: brain.input_B6_1

description: Library name: sample7


title: AKI, C6706∆ompU, replicate 1

value: {delta}ompU


title: HNRNPC_WT_KD2

description:
Library name: JL4774_HNRNPC_WT_KD2
V5-HNRNPC-WT-EndoKD-Rep2


title: HNRNPC_WT_CTRL3

description:
Library name: JL4774_HNRNPC_WT_CTRL3
V5-HNRNPC-WT-CTRL-Rep3


title: Mock treated K-562 B2M tdTomato cells, replicate 1

description: Library name: UT_R1


title: HNRNPC_WT_KD3

description:
Library name: JL4774_HNRNPC_WT_KD3
V5-HNRNPC-WT-EndoKD-Rep3


title: AKI, C6706∆ompU, replicate 2

value: O1 biovar El Tor


title: HNRNPC_WT_KD1

description:
Library name: JL4774_HNRNPC_WT_KD1
V5-HNRNPC-WT-EndoKD-Rep1


title: AKI, C6706∆ompU-pOueS-C6706, replicate 1

value: Strain C6706


title: AKI, C6706∆ompU-pOueS-C6706, replicate 2


title: Tripartite ETR targeting B2M with a 16nt gRNA treated K-562 B2M tdTomato cells, replicate 1

description: Library name: Tripartite_B4F4_16_R1


title: brain.input_B2_2

description: Library name: sample5

value: vitamin VB2


title: Mesenchymal cells, late time-point MCs 2, scRNA-seq

city: Ann Arbor

first: Lu

last: Lu

address: 100 Washtenaw Ave.

department: Department of Computational Medicine and Bioinformatics

institute: University of Michigan, Ann Arbor

description:
10X Genomics
3705-NW-1_filtered_feature_bc_matrix.h5

data_processing:
Cell Ranger ARC pipeline (version 2.0.0) with default parameter settings was used to process the raw Chromium Single Cell Multiome ATAC + Gene Expression sequencing data. During alignment steps, reads were mapped to the human reference genome (GRCh38) provided by 10x Genomics. The filtered count matrices output by the pipeline were used for further analysis.
Assembly: GRCh38
Supplementary files format and content: Cell Ranger output files: filtered_feature_bc_matrix.h5

source_name: Mesenchymal cells

growth_protocol: MCs were plated on 10cm dishes and grown to full confluence.

extract_protocol:
For 10X Multiome RNA+ATAC-sequencing, nuclear isolation was performed according to demonstrated protocols from 10X Genomics (CG000366, Revision C). Briefly, culture media was aspirated and plates were washed two times with cold PBS. Cells were isolated using a cell scraper and 5mL of ice-cold PBS into a 15mL conical tube and spun at 500 x g at 4C. The supernatant was removed and the cells were resuspended using 1X lysis buffer containing 10mM Tris-HCl (Invitrogen), 10mM NaCl (Fisher Scientific), 3mM MgCl2 (Promega), 0.1% Tween-20 (Fisher Scientific), 0.1% Nonidet P40 Substitute (Sigma-Aldrich), 0.01% Digitonin (Thermo Fisher Scientific), 1% BSA (Sigma-Aldrich), 1mM DTT (Sigma-Aldrich), and 1U/uL RNasin Plus Ribonuclease Inhibitor (Promega) in Nuclease-Free Water (Invitrogen) and incubated on ice for 5 minutes. Cells were monitored until less than 10% of cells remained unlysed. The pellet was then washed two times with centrifugation at 500 x g for 5 minutes at 4C, each time removing the supernatant. Finally, 10,000 target nuclei were resuspended in a diluted nuclei resuspension buffer (10X Genomics), pooling 2-3 cell lines for each submission.
Single-cell multiome libraries were prepared using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (10X Genomics) according to manufacturer’s instructions. ATAC sequencing, library preparation, and 3’ gene expression was performed with 50,000 reads per cell depth. Sequencing was performed by the Advanced Genomics Core at the University of Michigan using a NovaSeq 6000 (Illumina) platform.
scRNA-seq + scATAC-seq

value: Mesenchymal cells

tag: library type

value: mRNA


title: Tripartite ETR targeting B2M with a full length gRNA treated K-562 B2M tdTomato cells, replicate 1

description: Library name: Tripartite_B4F4_20_R1

value: K-562 B2M tdTomato

value: lymphoblast cells

value: B2M tdTomato


title: brain.input_B2_3

description: Library name: sample6


title: AKI, WT C6706, replicate 2

description: AKI WT_2

value: WT


title: Mesenchymal cells, late time-point MCs 2, scATAC-seq

library_source: genomic single cell

value: DNA


title: brain.input_B2_1

description: Library name: sample4

value: brain


title: AKI, WT C6706, replicate 1

description: AKI WT_1


title: A549_IAV_mock_2

city: Taipei

first: Po-Chih

last: Wu

address: No.109, Ln. 130, Sec. 1, Academia Rd., Nangang Dist., Taipei City 115, Taiwan

country: Taiwan

institute: TFDA

description: Library name: Mock-2

data_processing:
Raw sequencing reads in FASTQ format were first subjected to quality control, including trimming of low-quality bases using Trimmomatic.
The cleaned reads were then aligned to the reference genome using HISAT2, producing BAM files.
Gene-level read counts were generated using featureCounts, resulting in a counts matrix.
Genes were then ranked based on expression changes, and Gene Set Enrichment Analysis (GSEA) was performed to identify biological pathways significantly enriched in the data.
Assembly: GRCh38
Supplementary files format and content: A counts matrix CSV file for all samples

growth_protocol: A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS. Cultures were routinely tested and confirmed to be free of mycoplasma contamination. Cells were infected with H1N1 (WSN) influenza virus at an MOI of 5 for 1 hour. After virus adsorption, the inoculum was removed, and fresh medium with or without BAY 11-7082 was added. Cells were then incubated for an additional 8 hours at 37 °C under 5% CO₂ before RNA was harvested.

extract_protocol:
Cells were washed with PBS, and lysed in RLT buffer (Qiagen, Germany). Total RNAs were isolated with the RNeasy Mini Kit (Qiagen) and treated with DNase I (Qiagen) following the manufacturer's instructions. RNA samples were stored at -80°C and used for preparation of libraries for RNA sequencing.
RNA sequencing libraries were prepared using an Illumina Stranded mRNA Prep library preparation kit (Illumina, San Diego, CA, USA) in accordance with the manufacturer’s instructions.


title: brain.input_B12_3

description: Library name: sample3


title: brain.input_B12_2

description: Library name: sample2


title: Mesenchymal cells, late time-point MCs 1, scRNA-seq

description:
10X Genomics
3660-NW-1_filtered_feature_bc_matrix.h5


title: A549_IAV_mock_1

description: Library name: Mock-1


title: A549_IAV_infected_2

description: Library name: IAV-DMSO-2


title: HNRNPC_WT_CTRL1

description:
Library name: JL4774_HNRNPC_WT_CTRL1
V5-HNRNPC-WT-CTRL-Rep1

value: Human embryonic Kidney

value: Doxycycline


title: HNRNPC_inRBM25_MUT_3

description:
Library name: JL4460_HNRNPC_inRBM25_MUT_3
HNRNPC-in-RBM25-MUT-OE-Rep3


title: HNRNPC_WT_CTRL2

description:
Library name: JL4774_HNRNPC_WT_CTRL2
V5-HNRNPC-WT-CTRL-Rep2


title: A549_IAV_infected_1

description: Library name: IAV-DMSO-1


title: HNRNPC_inRBM25_MUT_2

description:
Library name: JL4460_HNRNPC_inRBM25_MUT_2
HNRNPC-in-RBM25-MUT-OE-Rep2


title: Mesenchymal cells, late time-point MCs 1, scATAC-seq


title: Mesenchymal cells, early time-point MCs 2, scRNA-seq

description:
10X Genomics
6745-PM-2_filtered_feature_bc_matrix.h5


title: G11

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using bowtie
Raw read counts were calculated to create a matrix
expression matrix of differentially expressed genes
expression matrix of differentially alternative splicing genes
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include alternative splicing values for differentially AS genes in  each sample

source_name: Adipocytes

extract_protocol:
Fat tissues frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Inguinal white adipose tissues

tag: processed

value: AAV expressing scrambled control were injected to the iWAT


title: brain.input_B12_1

description: Library name: sample1

value: vitamin VB12


title: A549_IAV_infected_BAY_2

description: Library name: IAV-BAY-7082-2


title: Mesenchymal cells, early time-point MCs 1, scRNA-seq

description:
10X Genomics
6475-PM-1_filtered_feature_bc_matrix.h5


title: HNRNPC_inRBM25_MUT_1

description:
Library name: JL4460_HNRNPC_inRBM25_MUT_1
HNRNPC-in-RBM25-MUT-OE-Rep1


title: A549_IAV_infected_BAY_1

description: Library name: IAV-BAY-7082-1


title: Mesenchymal cells, early time-point MCs 2, scATAC-seq


title: HNRNPC_inRBM25_WT_2

description:
Library name: JL4460_HNRNPC_inRBM25_WT_2
HNRNPC-in-RBM25-WT-OE-Rep2


title: Mesenchymal cells, early time-point MCs 1, scATAC-seq


title: G8


title: HNRNPC_inRBM25_WT_3

description:
Library name: JL4460_HNRNPC_inRBM25_WT_3
HNRNPC-in-RBM25-WT-OE-Rep3


title: G7


title: A4

value: AAV expressing mouse Hnrnpa2b1 CDS were injected to the iWAT


title: NDB_POD30

city: Tucson

first: Kellen

last: Chen

state: AZ

address: 1501 N Campbell Ave

department: Surgery

institute: University of Arizona

description:
Library name: NDB_POD30
10x Genomics

data_processing:
Base calls were converted to reads using the Cell Ranger (10X Genomics; version 3.1) implementation of mkfastq
Then aligned against the mm10 (mouse) genome using Cell Ranger’s count function with SC3Pv3 chemistry and 5,000 expected cells per sample
Cell barcodes representative of quality cells were delineated from barcodes of apoptotic cells or background RNA based on a threshold of having at least 200 unique transcripts profiled, less than 10,000 total transcripts, and less than 10% of their transcriptome of mitochondrial origin.
Unique molecular identifiers (UMIs) from each cell barcode were retained for all downstream analysis.
Raw UMI counts were normalized with a scale factor of 10,000 UMIs per cell and subsequently natural log transformed with a pseudocount of 1 using the R package Seurat (version 3.1.1).
Highly variable genes were identified, and cells were scaled by regression to the fraction of mitochondrial transcripts. Aggregated data was then evaluated using uniform manifold approximation and projection (UMAP) analysis
Assembly: mm10 (mouse)
Supplementary files format and content: Tab seperated values files and matrix files

source_name: freshly isolated cells from wound tissue

growth_protocol: Freshly isolated murine cells from murine skin or wounds.

extract_protocol:
Tissue was enzymatically digested, cells were collected and washed. These cellular suspensions were then submitted for droplet-based microfluidic single cell RNA sequencing (scRNA-seq) using the 10x Chromium Single Cell platform (Single Cell 3’ v3, 10x Genomics, USA).
cDNA was fragmented followed by end repair and A-tailing at 65°C for 30min. cDNA were double-sided size selected using SpriSelect beats. Sequencing adaptors were ligated to the cDNA at 20°C for 15min. cDNA was amplified using a sample-specific index oligo as primer, followed by another round of double-sided size selection using SpriSelect beads.

treatment_protocol: Murine wounds were created using our excisional wounding protocol, in either leptin-receptor knockout, wildtype mice, or wildtype mice fed with a high-fat diet, then submitted for scRNA-seq analysis.

value: wildtype


title: HNRNPC_inRBM25_WT_1

description:
Library name: JL4460_HNRNPC_inRBM25_WT_1
HNRNPC-in-RBM25-WT-OE-Rep1


title: NDB_POD7

description:
Library name: NDB_POD7
10x Genomics

value: freshly isolated cells from wound tissue


title: Atherosclerotic plaques, thin-cap fibroatheroma (TCFA), rep5

first: Yuanyuan

last: Wei

address: No. 130, Dong'an Road

institute: Fudan University

data_processing:
The fastx_clipper software was used to remove splice sequences from the 3' end and low-quality fragments.
FastQC is used to evaluate the quality of the sequence after quality control.
Bowtie software was used to align the sequences to the reference genome, Rfam database, RepBase database, and miRBase database.
DESeq software was used for statistical differences.
Miranda is used for target prediction
Assembly: hg38
Supplementary files format and content: Raw counts of all miRNAs in the 13 samples

source_name: Carotid artery

extract_protocol:
Total RNAs were extracted using miRNeasy Mini Kit (Qiagen), followed by subsequent treatment with the RNase-Free DNase Set (Qiagen). 200 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using QIAseq miRNA Library Kit (Qiagen, 331502, Germany) following manufacturer's protocols.

value: Carotid artery

tag: condition

value: thin-cap fibroatheroma (TCFA)


title: RBM25_MUT_3

description:
Library name: JL4460_RBM25_MUT_3
V5-RBM25-MUT-Rep3


title: PNR_35_activated_cd4+_baseline

city: Bethesda

first: Michael

last: Sack

state: MD

address: 10 Center Dr

institute: NHLBI/NIH

description: Library name: SH8867_35

data_processing:
The RNA sequencing data was processed using default parameters with RNA-seek workflow (https://github.com/OpenOmics/RNA-seek)
preprocessing of the raw sequencing reads to remove low quality bases, adapters, and shorter reads using Cutadapt v1.18
alignment to human hg38 reference genome (GRCh38, Gencode Release 30) using STAR (v.2.7.6a) aligner in two-pass mapping mode
The gene expression counts estimated from number of reads mapping to each annotated gene using RSEM (v.1.3.3)
Assembly: hg38
Supplementary files format and content: tab-delimited file containing RSEM expected counts for each sample
Supplementary files format and content: differential expression results comparing NR vs baseline in activated CD4+ T cells
Supplementary files format and content: differential expression results comparing NR vs baseline in naive CD4+ T cells

source_name: PBMC

extract_protocol:
Total RNA from CD4+ T cells at baseline and after four weeks of Nicotinamide Riboside supplementation were extracted with the miRNeasy Micro Kit (Qiagen) and RNA quality and integrity was assessed by Qubit 3.0 fluorometer (Thermo Fisher Scientific) and Agilent Bioanalyzer (Agilent Technologies).
Libraries were prepared using TruSeq stranded Total RNA HT kit (Illumina) and sequenced in a HISeq 3000 (Illumina)

treatment_protocol: Study subjects were screened prior to signing consent and subjects were given a 4-week supplementation of Nicotinamide Riboside (250 mg bid)

value: PBMC

value: activated CD4+ T cells

tag: time

value: baseline


title: Atherosclerotic plaques, thin-cap fibroatheroma (TCFA), rep4


title: RBM25_WT_3

description:
Library name: JL4460_RBM25_WT_3
V5-RBM25-WT-Rep3


title: RBM25_WT_2

description:
Library name: JL4460_RBM25_WT_2
V5-RBM25-WT-Rep2


title: Atherosclerotic plaques, thin-cap fibroatheroma (TCFA), rep1


title: Atherosclerotic plaques, thin-cap fibroatheroma (TCFA), rep2


title: Atherosclerotic plaques, late fibroatheroma (LFA), rep4

value: late fibroatheroma (LFA)


title: PNR_35_activated_cd4+_NR

description: Library name: SH8867_36

value: NR


title: PNR_34_activated_cd4+_NR

description: Library name: SH8867_34


title: PDB_POD7

description:
Library name: PDB_POD7
10x Genomics

tag: diet

value: High-fat diet


title: PDB_POD2

description:
Library name: PDB_POD2
10x Genomics

tag: tissue


title: A2


title: A1


title: RBM25_WT_1

description:
Library name: JL4460_RBM25_WT_1
V5-RBM25-WT-Rep1

tag: cell line


title: RBM25_MUT_1

description:
Library name: JL4460_RBM25_MUT_1
V5-RBM25-MUT-Rep1

tag: cell type


title: NDB_POD0

description:
Library name: NDB_POD0
10x Genomics

source_name: freshly isolated cells from skin tissue

value: freshly isolated cells from skin tissue

tag: genotype


title: Atherosclerotic plaques, thin-cap fibroatheroma (TCFA), rep3


title: NDB_POD2

description:
Library name: NDB_POD2
10x Genomics


title: PNR_34_activated_cd4+_baseline

description: Library name: SH8867_33


title: PNR_19_activated_cd4+_NR

description: Library name: SH8867_28


title: Atherosclerotic plaques, late fibroatheroma (LFA), rep1


title: Atherosclerotic plaques, late fibroatheroma (LFA), rep2


title: CSF, LUAD LM, 6

first: Qingjian

last: Ou

address: Siping Rd

department: Medical

institute: Tongji University

description:
Library name: CSFLUAD6
10× genomics

data_processing:
Raw sequencing data were processed with the Cell Ranger software (10X Genomics) for demultiplexing and alignment to a reference genome (GRCH38).
Assembly: hg38
Supplementary files format and content: Raw counts

source_name: CSF

extract_protocol:
Samples were taken by the suction blister method.Subsequently tissues were enzymatically digested, using trypsin for 45 minutes at 37°C. The resulting single cell suspension was then passed through 100µm and 40µm nylon meshes and through a dead cell removal column before further processing.
Single-cell RNA-seq was performed using the 10X Genomics Chromium Single Cell Controller with the Chromium Single Cell 3′ V3 Kit following the manufacturer's instructions.

value: CSF


title: CSF, LUAD LM, 5

description:
Library name: CSFLUAD5
10× genomics


title: PDB_POD0

description:
Library name: PDB_POD0
10x Genomics


title: CSF, LUAD LM, 4

description:
Library name: CSFLUAD4
10× genomics


title: GDB_POD30

description:
Library name: GDB_POD30
10x Genomics

value: B6.BKS(D)-Leprdb/J db/db


title: PNR_14_activated_cd4+_baseline

description: Library name: SH8867_25


title: Atherosclerotic plaques, early fibroatheroma (EFA), rep4

value: early fibroatheroma (EFA)


title: PNR_4_activated_cd4+_NR

description: Library name: SH8867_24


title: Immortalized periodontal ligament cells compressive force replicate 2

city: Seoul

first: Hanjin

last: Bae

address: 28 Yeongeon-dong

institute: Seoul National University Hospital

description:
Library name: CS_2
Immortalized human periodontal ligament cells subjected to compressive force for 24 h.

data_processing:
Sequencing reads were aligned to the human reference genome (GRCh38) using Bowtie2. Read counts were generated using bedtools. Normalization was performed using edgeR with TMM normalization, and expression values are reported as log2-transformed normalized counts.
Assembly: Homo sapiens reference genome GRCh38 (hg38)
Supplementary files format and content: The processed data file contains a matrix of gene symbols and log2-transformed normalized expression values for all samples (Ctrl_1, Ctrl_2, CS_1, CS_2).

source_name: immortalized human periodontal ligament cells

growth_protocol: An immortalized human periodontal ligament cell line derived from cementifying fibroma cells was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, non-essential amino acids, GlutaMAX, 2-mercaptoethanol, and penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO₂.

extract_protocol:
Total RNA was extracted using the Direct-zol RNA Microprep Kit according to the manufacturer’s instructions. RNA quality was assessed using a NanoDrop 2000 spectrophotometer.
RNA libraries were prepared using the QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen), a poly(A)-based method, with 500 ng of total RNA per sample, following the manufacturer’s protocol.

treatment_protocol: Cells were subjected to compressive force for 24 hours. Control samples were maintained under identical conditions without compression.

tag: treatment

value: compressive force


title: PNR_14_activated_cd4+_NR

description: Library name: SH8867_26


title: Atherosclerotic plaques, early fibroatheroma (EFA), rep3


title: GDB_POD0

description:
Library name: GDB_POD0
10x Genomics


title: GDB_POD7

description:
Library name: GDB_POD7
10x Genomics


title: GDB_POD2

description:
Library name: GDB_POD2
10x Genomics


title: Atherosclerotic plaques, early fibroatheroma (EFA), rep2


title: Immortalized periodontal ligament cells compressive force replicate 1

description:
Library name: CS_1
Immortalized human periodontal ligament cells subjected to compressive force for 24 h.

value: immortalized human periodontal ligament cells


title: PNR_4_activated_cd4+_baseline

description: Library name: SH8867_23


title: PNR_1_activated_cd4+_NR

description: Library name: SH8867_20


title: Atherosclerotic plaques, early fibroatheroma (EFA), rep1


title: Immortalized periodontal ligament cells control replicate 2

description:
Library name: Ctrl_2
Immortalized human periodontal ligament cells cultured without compressive force for 24 h.

value: control


title: CSN, P3, rep1

city: White Plains

first: Julia

last: Kaiser

state: NY

address: 784 Mamaroneck Avenue

institute: Burke Neurological Institute

description:
2106-P3SCc
10x Genomics

data_processing:
Raw read files were processed and aligned to the mouse reference, mm10 (GENCODE vM23/Ensembl 98) using 10x Genomics Cell Ranger 6.0.1 count and aggr (Zheng et al., 2017). Using established R packages and custom-written code, cells with low read counts (empty droplets) or high numbers of mitochondrial gene products (±3x than median absolute deviation) were removed. Size-factor normalized logcounts were obtained (A. T. L. Lun, Bach, et al., 2016; McCarthy et al., 2017) and batch-corrected (Haghverdi et al., 2018). After integration of three sample types (P1 SCPN, P3 SCPN, P3 CSN) using the top 2000 most variable genes with min. mean normalized expression of 0.001 (A. Lun, 2019), dimensionality reduction (UMAP) was done on the batch-corrected log counts. The dataset was annotated with singleR  (Aran et al., 2019; A. T. L. Lun et al., 2020) using previously published single-cell data of the P0 mouse cortex to subset our dataset to Layer V neurons (Loo et al., 2019).
Assembly: mm10
Supplementary files format and content: Feature - barcode matrices in *.h5 format

source_name: CSN

extract_protocol:
tissue of retrogradely labeled mice was collected at P1 (SCPN group only) or P3 (SCPN or CSN group). All procedures were done rapidly and using cold solutions to minimize time between euthanasia and cell collection. Briefly, brains were dissected and sliced into 800 µm thick coronal sections which were subsequently processed under a fluorescent dissection scope (Nikon SMZ18) to extract cortex containing labeled cells. A single cell suspension of the samples was obtained by enzymatic (15min, 37°C) and mechanical dissociation (gentle trituration using rounded glass Pasteur pipettes of decreasing diameter (600um, 300um)) followed by filtration through a 40µm mesh (Biologix, 15-1040-1). SCPN were FACS-purified from this single cell suspension using a WOLF flow sorter (Nanocellect Biomedical) with standard settings (threshold for cell size adapted to 30.000). To decrease contamination and increase sorting efficacy, cells were initially sorted in a high concentration of ∼6 × 106 cells/ml followed by a second sort of the first round of sorted cells (lower concentration, ∼2 × 105 cells/ml). This allowed for enrichment of CTB 555 labeled cells in a timely manner. We collected ∼15.000 cells in 5ml per sort. Cells were enriched for downstream processing by centrifugation for 10min at 80g and processed directly for single-cell RNA-Seq.
Libraries were prepared using the Chromium Next GEM Single Cell 3’ Library Kit v3.1 and the Chromium i7 Multiplex barcodes.

value: CSN


title: SCPN, P3, rep2

description:
2106-P3Ped
10x Genomics

source_name: SCPN

value: SCPN


title: PNR_35_naive_cd4+_NR

description: Library name: SH8867_18

value: naive CD4+ T cells


data_processing:
Skewer software was used to remove splice sequences from the 3' end and low-quality fragments.
STAR software was used to align the sequences to the reference genome.
DEseq2 software was used for statistical differences.
StringTie software was used for transcript assembly.
rMATS software was used for alternative splicing events statistics.
Assembly: hg38
Supplementary files format and content: Raw counts of all genes (including mRNAs and lncRNAs) in the 13 samples
Supplementary files format and content: Raw counts of all transcripts (including mRNAs and lncRNAs) in the 13 samples

extract_protocol:
Total RNAs were extracted using miRNeasy Mini Kit (Qiagen), followed by subsequent treatment with the RNase-Free DNase Set (Qiagen). 500 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using VAHTSTM Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme, NR603, China) following manufacturer's protocols.

value: Carotid artery

value: thin-cap fibroatheroma (TCFA)


title: SCPN, P1, rep2

description:
2106-P1Ped
10x Genomics


title: CSF, LUAD LM, 3

description: 10× genomics

value: lung adenocarcinoma leptomeningeal metastases


title: Immortalized periodontal ligament cells control replicate 1

description:
Library name: Ctrl_1
Immortalized human periodontal ligament cells cultured without compressive force for 24 h.


title: SCPN, P3, rep1

description:
2012-P3Ped
10x Genomics


title: PNR_35_naive_cd4+_baseline

description: Library name: SH8867_17

value: PBMC

value: baseline


title: PNR_34_naive_cd4+_NR

description: Library name: SH8867_16

value: NR


title: SCPN, P1, rep1

description:
2012-P1Ped
10x Genomics


title: PNR_1_activated_cd4+_baseline

description: Library name: SH8867_19

value: activated CD4+ T cells


title: CSF, LUAD LM, 1

description: 10× genomics


title: CSF, LUAD LM, 2

description: 10× genomics


title: PNR_34_naive_cd4+_baseline

description: Library name: SH8867_15


title: B. subtilis library,  Δrho background, after chloramphenicol selection

city: Cambridge

first: K Julia

last: Dierksheide

state: MA

address: 31 Ames St.

department: Biology

institute: Massachusetts Institute of Technology

description: Library name: rho_postselection

data_processing:
First, the paired-end sequencing reads were aligned to the B. subtilis (NC_000964.3) and E. coli (NC_000913.3) genome using the bowtie package (version 1.2.3). 36 nt of each read was used for alignment, and alignments with more than 1 mismatch or an insert size of more than 5,000 bp were discarded (bowtie arguments: -trim3 10 -v 1 -X 5000). Any two reads with the same aligned start and end position and UMI (UMIs are present in the first 4nt of each index read) were collapsed to a single read. The number of unique reads mapping to each genomic fragment was calculated and normalized to the total number of reads in the sample. Reads mapping to the E. coli genome were combined and used for normalization to total reads, but were not included in further analysis.
Assembly: B. subtilis (NC_000964.3) and E. coli (NC_000913.3)
Supplementary files format and content: Indicates UMI collapsed counts for each fragment in each sample. Fragments are characterized by the columns "fragment_start", "fragment_end", "fragment_strand", which indicate the start position of the aligned read 1, the end position of the aligned read 2, and the strand of the genome the fragment is derived from (+ or - for plus and minus strand). The fragment_id combines all of this information to create a unique identifier for each fragment. The UMI collapsed reads for each sample are in the corresponding columns. The total reads mapping to the E. coli genome in each sample are in the final column under fragment_id "1_4641652_Eco_."

organism: Bacillus subtilis subsp. subtilis str. 168

source_name: Reporter library integrated at amyE, kanR gene disrupting rho

extract_protocol:
Sequencing libraries were prepared to quantify the frequency of each genomic fragment variant in the four experimental conditions. gDNA was extracted from the cell pellets using the Wizard Genomic DNA Purification kit (Promega) following manufacturer’s instructions.
To attach UMIs, a 2 cycle PCR was performed using 1.6 μg of gDNA per 100 μL PCR reaction (scaled as needed for library complexity) with primers oCEK071/oCEK083. This PCR reaction was then cleaned up with a DNA Clean & Concentrator-5 column, followed by a one-sided selection with a Select-a-Size DNA Clean & Concentrator MagBead Kit (Zymo Research) using a 260-bp cutoff, followed by a second clean up with the DNA Clean & Concentrator-5 column. This size-selected reaction was then amplified in a second PCR with primers oJT895/oCEK087. The second PCR reaction was concentrated using a DNA Clean & Concentrator-5 column, then gel-purified using an 8% TBE gel. These libraries were sequenced with 36-bp paired-end reads on a NextSeq500 (Illumina).

treatment_protocol: To perform chloramphenicol selection on the wild-type and Δrho B. subtilis libraries to enrich for genomic fragments with transcription termination activity, the libraries were grown in LB for ~10 doublings then back-diluted into selective media containing chloramphenicol. To begin the initial outgrowth, a single glycerol stock for each library was thawed and resuspended in 200 mL warm LB. These starter cultures were grown at 37°C with vigorous shaking to OD600 0.2. The wild-type and Δrho cultures were then back-diluted to OD600 0.003 and 0.015 in 200 mL warm LB, respectively.  Once the cultures reached OD600 0.2, they were harvested (pre-selection harvest). To begin the chloramphenicol selection, each culture was then back-diluted to OD600 0.002 in 100 mL of LB with 5 μg/mL chloramphenicol. The cultures were then grown with vigorous shaking at 37°C for 2.5 h and then harvested (post-selection harvest). To harvest cells, 11–14 mL of culture was pelleted at 4°C and then frozen in liquid nitrogen. Cell pellets were stored at −80°C until gDNA extraction.


title: PNR_19_naive_cd4+_NR

description: Library name: SH8867_10


title: Atherosclerotic plaques, late fibroatheroma (LFA), rep3

value: late fibroatheroma (LFA)


title: B. subtilis library, Δrho background, before chloramphenicol selection

description: Library name: rho_preselection

value: Reporter library integrated at amyE, kanR gene disrupting rho


title: Mouse, Lung,  Saline_1, scATAC

city: Chengdu

first: Ruijie

last: Hou

address: Renmin South Road

institute: State Key Laboratory of Biotherapy

description: Library name: Saline_1

data_processing:
The cellranger pipeline (version 3.1.0, 10X Genomics) was used to process raw sequencing output.
Assembly: mm10
Supplementary files format and content: Cell Ranger software output files (barcodes.tsv, features.tsv, matrix.mtx)

source_name: lung

extract_protocol:
The homogenized lung tissues were suspended in DMEM medium (C11995500BT, GIBCO) containing 1 mg/mL type I collagenase (C8140，Solarbio), 1 mg/mL type IV collagenase (C8160，Solarbio), 1 mg/mL Dispase II (D6430，Solarbio), and 0.2 mg/mL DNAase (D8071, Solarbio) at 37°C for 30 min. The digested lung tissues were filtered through a 70 μm filter membrane (BS-70-XBS, biosharp), and the filtrate was centrifuged and treated with red blood cell lysis buffer (R1010, Solarbio) for 10 min, centrifuged again under the same conditions, the pellet was washed once with sterile PBS (C10010500BT, GIBCO), and centrifuged again under the same conditions. Cell pellet was resuspended again with MACS mixture (2 mM EDTA, 0.1% BSA, 1% penicillin/streptomycin/antimitotic in DPBS) to obtain lung single cell suspension.
Single-cell ATAC sequencing (scATAC-seq) was performed using the 10X Chromium 3′ v3 kit (10X Genomics, Pleasanton, CA) according to the manufacturer's instructions.

value: lung


title: Mouse, Lung,  Bleomycin_1, scATAC

description: Library name: Bleomycin_1


title: B. subtilis library, bicyclomycin-benzoate, after chloramphenicol selection

description:
Library name: BCM_plus_postselection
Instrument: Singular Genomics G4

data_processing:
First, the paired-end sequencing reads were aligned to the B. subtilis (NC_000964.3) and E. coli (NC_000913.3) genome using the bowtie package (version 1.2.3)57. 40 nt of each read was used for alignment, and alignments with more than 1 mismatch or an insert size of more than 5,000 bp were discarded (bowtie arguments: -trim3 10 -v 1 -X 5000). Any two reads with the same aligned start and end position and UMI were collapsed to a single read. The number of unique reads mapping to each genomic fragment was calculated and normalized to the total number of reads in the sample. Reads mapping to the E. coli genome were combined and used for normalization to total reads in the enrichment calculations, but were not included in further analysis.
Assembly: B. subtilis (NC_000964.3) and E. coli (NC_000913.3)
Supplementary files format and content: Indicates UMI collapsed counts for each fragment in each sample. Fragments are characterized by the columns "fragment_start", "fragment_end", "fragment_strand", which indicate the start position of the aligned read 1, the end position of the aligned read 2, and the strand of the genome the fragment is derived from (+ or - for plus and minus strand). The fragment_id combines all of this information to create a unique identifier for each fragment. The UMI collapsed reads for each sample are in the corresponding columns. The total reads mapping to the E. coli genome in each sample are in the final column under fragment_id "1_4641652_Eco_."

source_name: Reporter library integrated at amyE

extract_protocol:
Sequencing libraries were prepared to quantify the frequency of each genomic fragment variant in the four experimental conditions. gDNA was extracted from the cell pellets using the Wizard Genomic DNA Purification kit (Promega) following manufacturer’s instructions. To reduce RNA contamination, the gDNA was then cleaned up with the genomic DNA clean and concentrator-10 (Zymo Research).
Libraries were generated using a two-step Q5 PCR protocol. To attach UMIs, a 2 cycle PCR was performed using 2 μg of gDNA per 150 μL PCR reaction (scaled as needed for library complexity) with primers oJD180/oJD182. This PCR reaction was cleaned up with two rounds of magnetic bead clean-up at a 1:1 ratio of beads to sample (PCRClean DX bead, Aline Biosciences). Half (pre-selection libraries) or one quarter (post-selection libraries) of this cleaned-up reaction was then amplified in a second PCR with primers oJD181/oJD183. The second PCR reaction was gel-purified using an 8% TBE gel. These libraries were sequenced with 50 bp paired-end reads on a G4 sequencer (Singular Genomics).

treatment_protocol: To perform chloramphenicol selection on the B. subtilis library to enrich for genomic fragments with transcription termination activity, the library was grown in LB for 5 generations, split into +BCM and DMSO control (−BCM) conditions for pre-treatment, and then back-diluted into selective media containing chloramphenicol and BCM or chloramphenicol and DMSO. To begin the initial outgrowth, a single glycerol stock was thawed, resuspended in warm LB, and back-diluted to OD600 0.006 in 50 mL warm LB. This starter culture was grown at 37°C with vigorous shaking to OD600 0.2. A second back-dilution to OD600 0.006 in 25 mL was then performed to pre-treat the cultures with bicyclomycin-benzoate (BCM) or a DMSO control. For this pre-treatment, cells were grown either in LB with 120 ng/mL BCM (+BCM treatment) or 2.5% DMSO (−BCM treatment). The pre-treatment cultures were then grown to OD600 0.2, at which point both cultures were harvested (pre-selection harvest). At the end of the +BCM pre-treatment, a small amount (3%) of residual chloramphenicol acetyltransferase is expected to remain in cells with inserts driving Rho termination, which may explain the slight increase in +BCM enrichment for these fragments relative to the non-terminated fragments (Fig. 1d). BCM concentration and pre-treatment duration were optimized using the kinB Rho termination site as a positive control. To begin the chloramphenicol selection, the BCM pre-treated culture (+BCM) was back-diluted to OD600 0.013 in 25 mL of LB with 5 μg/mL chloramphenicol and 120 ng/mL BCM, and the untreated culture (−BCM) was back-diluted to OD600 0.0075 in 25 mL of LB with 5 μg/mL chloramphenicol and 2.5% DMSO. The cultures were then grown with vigorous shaking at 37°C for 2.75 h before the post-selection harvest (OD600 0.24–0.27). To harvest cells, 11–14 mL of culture was pelleted at 4°C and then frozen in liquid nitrogen. Cell pellets were stored at −80°C until gDNA extraction. In total, cell pellets for four conditions were collected: +BCM culture before and after chloramphenicol selection; −BCM culture before and after chloramphenicol selection.

value: Reporter library integrated at amyE

value: bicyclomycin-benzoate


title: PNR_14_naive_cd4+_NR

description: Library name: SH8867_8


title: B. subtilis library, wild-type background, after chloramphenicol selection

description: Library name: wt_postselection


title: PNR_14_naive_cd4+_baseline

description: Library name: SH8867_7


title: PNR_4_naive_cd4+_NR

description: Library name: SH8867_6


title: PNR_19_naive_cd4+_baseline

description: Library name: SH8867_9