title: Untreated Xist-RMCE ESC clone 3 H3K27me3

value: Tri-Methyl-Histone H3 (Lys27) (C36B11) (H3K27me3) (Cell Signaling, cat #:9733)


title: gex_gp33_5

status: Public on Feb 21 2025

type: SRA

city: Freising

first: Gustavo

middle: P.

last: de Almeida

state: Bavaria

address: Liesel-Beckmann-Str. 1

department: Division of Animal Physiology and Immunology

country: Germany

institute: Technical University of Munich

description: scRNA-Seq + TCR (10x Genomics)

library_source: transcriptomic single cell

data_processing:
References refdata-cellranger-arc-mm10-2020-A-2.0.0.tar.gz and refdata-cellranger-vdj-GRCm38-alts-ensembl-7.0.0.tar.gz were retrieved from 10xgenomics.com and used to align the reads of the gene expression and TCR assay with cellranger multi (v7.1.0).
Assembly: GRCm38.p6 (release 98) - GENCODE Release M23
Supplementary files format and content: Annotated data matrix (anndata) object saved in h5ad-file format. The file contains the preprocessed, normalized and annotated count data of all samples.

molecule: polyA RNA

organism: Mus musculus

source_name: spleen

extract_protocol:
Spleens were harvested from infected mice and macerated through a 100µm Nytex filter, washed with DMEM and erythrocyte lysis was performed using a hypotonic ACK medium. Lysis was stopped by adding DMEM containing 10% FCS. Cells were counted using a Neubauer counting chamber  stained with flurorescently labelled antibodies against CD8a,  CD44 as well as a fixable live/dead dye and multimers loaded with either gp33 or np396 and conjugated to different flurochromes. Single, live reporter+ CD8 T cells reacting to either of the multimers were sorted and used for library generation.
Gene expression and T-cell receptor V(D)J libraries were prepared by using the Chromium Next GEM Single Cell 5’ Reagent Kit v2 (10X Genomics), Chromium Single Cell Mouse TCR Amplification Kit (10X Genomics) and Chromium Next GEM Chip K Single Cell Kit (10X Genomics) following the manufacturer’s protocol (CG000331 Rev E). Dual Index Kit TT Set A (10X Genomics) was used for multiplexing (i7 and i5 index read, 10bp). The samples were sequenced in a paired-end run (read 1: 26bp, read 2: 90bp) on a NovaSeq6000 platform using S1 v1.5 (100 cycles) sequencing kits (Illumina). Bcl2fastq software (v2.20.0.422) was used for demultiplexing and generating .fastq files.  The libraries were sequenced in a paired-end run (read 1: 26bp, read 2: 90bp) on a NovaSeq6000 platform using S1 v1.5 (100 cycles) sequencing kits (Illumina). Bcl2fastq software (v2.20.0.422) was used for demultiplexing and generating .fastq files.

treatment_protocol: TCF1 reporter mice were infected with LCMV Armstrong, splenocytes were harvested 4 weeks post infection.

tag: tissue

value: spleen

tag: cell line

value: CD8+ T cells

tag: genotype

tag: epitope

tag: phenotype

tag: time point

value: day 30 post infection

tag: treatment

value: LCMV Armstrong

tag: library type

value: GEX


title: 3-day 1000ng/mL dox-treated Airn-RMCE ESC clone 5 H3K27me3

city: Chapel Hill

first: Joseph

middle: Mauro

last: Calabrese

state: NC

address: 120 Mason Farm Road

country: USA

institute: UNC Chapel Hill

library_source: genomic

data_processing:
ChIP-Seq reads were aligned using bowtie2 with default parameters.
Aligned reads that had a mapping quality greater than or equal to 30 were extracted with samtools 28, and allele-specific read retention (i.e., reads that overlap at least one B6 or CAST SNP) was performed as in 30,31 using a custom Perl script (intersect_reads_snps16.pl).
Assembly: mm10
Supplementary files format and content: WIG files were created using a custom perl script. Total, non-allelic read counts are divided by total reads of the dataset in million (i.e. RPM).

molecule: genomic DNA

source_name: RMCE Embryonic Stem Cells

growth_protocol: B6/CAST F1-hybrid Rosa26-RMCE ESCs from 12 were grown on gelatin-coated plastic dishes in a humidified Thermo Fisher Forma Series II water-jacketed incubator at 37°C and under 5% CO2. Cells were grown in DMEM high glucose plus sodium pyruvate (Gibco 11995-065), 15% ESC-qualified fetal bovine serum (Gibco 26140-079), 0.1mM non-essential amino acids (Gibco 11140-050), 100U/mL penicillin-streptomycin (Gibco 15140-122), 2mM L-glutamine (Gibco 25030-081), 0.1mM β-mercaptoethanol (Sigma-Aldrich 63689), and 1:500 LIF conditioned media produced from Lif-1Cα (COS) cells (kind gift of N. Hathaway).

extract_protocol:
ChIP beads were resuspended in Elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) and placed on a 65°C heat block for 17 minutes with frequent vortexing. ChIP DNA was then reverse crosslinked in 0.5% SDS and 100mM NaCl overnight at 65°C, followed by a 1-h RNaseA (3L; Thermo Scientific EN0531) treatment at 37°C and a 2.5-h Proteinase K (10L; Invitrogen 25530015) treatment at 56°C. DNA was then extracted with 1 volume of phenol:chloroform:isoamyl alcohol (Sigma-Aldrich P3803) and precipitated with 2 volumes 100% ethanol, 1/10 volume 3M sodium-acetate pH 5.4, and 1/1000 volume linear acrylamide (Invitrogen AM9520) overnight at -20°C. Precipitated DNA was then extracted with a 30-minute max speed spin at 4°C, washed once with ice-cold 80% ethanol, and resuspended in TE.
ChIP-Seq libraries were prepared with NEBNext End Repair Module (NEB, cat #: E6050S), A-tailing by Klenow Fragment (3’--5’ exo-; NEB, cat #: M0212S), and TruSeq 6-bp index adaptor ligation by Quick ligase (NEB, cat #: M2200S), and NEBNext High-Fidelity 2X PCR Master Mix (NEB, cat #: M0541S). All DNA clean-up steps were performed with AMPure XP beads (Beckman Coulter, cat #: A63880). Single-end, 75-bp sequencing was performed using an Illumina NextSeq 500/550 High Output v2.5 kit (Illumina, cat #: 20024906) on a NextSeq 500 System.

treatment_protocol: Prior to crosslinking, RMCE cell lines (with rtTA) were treated with 0-1000ng/mL doxycycline (1000ng/mL if not otherwise specified) for 72 hours (or longer, 7 days). Adhered cells were crosslinked with 0.6% formaldehyde (Fisher Scientific BP531-500) in DMEM media with 5% FBS for 10min at room temperature, then quenched with 125mM glycine for 5min at room temperature. Crosslinked cells were then washed twice with ice-cold PBS and scraped with ice-cold PBS with 0.05% Tween (Fisher Scientific EW-88065-31) and PIC (Sigma Aldrich P8340). The cells were then spun at 1,200 x g at 4°C to remove PBS, followed by resuspension in ice-cold PBS with PIC and divided into 10-million cell aliquots.

value: RMCE Embryonic Stem Cells

value: 3 days with 1000ng/mL doxycycline

value: Airn-RMCEs

tag: chip antibody


title: DMS-MaPseq, spt4∆, dms treated, biol rep 3

city: Worcester

first: Leonard

last: Schärfen

state: MA

address: 368 Plantation Street

department: RNA Therapeutics Institute

institute: UMass Chan Medical School

library_source: transcriptomic

data_processing:
The modular CoSTseq data analysis pipeline, implemented including all custom sub modules in Snakemake, is available at https://github.com/NeugebauerLab/CoSTseq. Raw paired-end reads were quality filtered and trimmed with fastp and then aligned to the reference genome with STAR, without generating new splice junctions. Ribosomal RNA was separated based on alignment coordinates and both mRNA and rRNA were deduplicated separately with UMICollapse.
Assembly: sacCer3
Supplementary files format and content: _counts.pkl files contain count data that can be loaded using CoSTseq data processing tools or the "pickle" Python package. Each file includes four lists specifying number of mutations and coverage for each genomic position for both strands: mutation count forward strand, mutation count reverse strand, coverage forward strand, coverage reverse strand. Each list contains 18 numpy arrays: 16 for each chromosome, one for mitochondrial DNA, one for the KanMX cassette used to obtain KO strains.
Supplementary files format and content: _counts_rrna.pkl files contain count data for rrna only that can be loaded using CoSTseq data processing tools or the "pickle" Python package. Each file includes two numpy arrays specifying number of mutations and coverage for each rRNA position.
Library strategy: DMS-MaPseq

organism: Saccharomyces cerevisiae

source_name: BY4741 spt4::kanMX

growth_protocol: Liquid cultures from individual colonies of Saccharomycescerevisiae BY4741 were grown in liquid YPAD medium to an OD600 around 0.6 at 30ºC with 200rpm shaking. YKW100 was grown in YPA Gal/Raf and then shifted to YPAG or Gal/Raf.

extract_protocol:
Cells were pelleted at 3,500×g and washed with stop buffer without isoamyl alcohol, then resuspended in 600µl lysis buffer (45mM NaOAc, 6mM EDTA, pH 5.5) with 40µl 20% SDS added after resuspension. The mixture was then incubated at 65ºC for 30s with vigorous shaking, added to 650µl hot (65ºC) acid-phenol:chloroform:IAA 125:24:1, pH 4.5 and incubated for 2min at 65ºC with shaking. Samples were placed on ice for 5min, then spun at 20,000×g at room temperature. The aqueous layer was subsequently again extracted with acid-phenol, then with chloroform and RNA was precipitated with isopropanol.
CoSTseq: Approximately 80µg of input RNA were incubated with Dynabeads MyOne Streptavidin C1 (Invitrogen) according to the manufacturer’s recommendations. The supernatant of the pulldown was retained for mRNA isolation. Beads were washed twice with high salt buffer (2M NaCl, 50mM Tris⋅HCl pH 7.4, 0.5% Triton X-100), once with binding buffer (300mM NaCl, 10mM Tris⋅HCl pH 7.4, 0.1% Triton X-100), and once with low salt buffer (5mM Tris⋅HCl pH 7.4, 0.1% Triton X-100); 500µl each. Nascent RNA was extracted from beads twice using Trizol and chloroform and resuspended in 3.75µl H2O. Reverse transcription was performed in a 10µl volume with an optimized KCl based buffer made of 75mM KCl, 3mM MgCl2, 50mM Tris⋅HCl pH 8.3 and 0.5µl RNase inhibitor using the GsI2c-MBP fusion protein (commercially TGIRT-III), which was purified as described previously. RT reactions were pre-incubated with 100nM annealed DNA/RNA heteroduplex adapter with a single G overhang to capture the biotin-C at the 3’-end (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTG, rArArGrArUrCrGrGrArArGrArGrCrArCrArCrGrUrCrUrGrArArCrUrCrCrArGrUrCrArC/3SpC3/) for 30min at room temperature. Then 1.25µM dNTP were added and the reaction was incubated at 25°C for 10min, 42°C for 10min, 50°C for 10min, 55°C for 10min, 60°C for 30min, 65°C for 20min, and 75°C for 15min. 1µl of RNase H (NEB) was added and the sample incubated at 37ºC for 30min, following clean-up using MinElute PCR Purification kit (Qiagen). The 5’-end adapter (/5Phos/NNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT/3SpC3/) was first adenylated using MthRNALigase (NEB), purified using Oligo Clean & Concentrator (Zymo Research) and then ligated to cDNA using Thermostable5’AppDNA/RNALigase (NEB) according to the manufacturer’s instructions. Adapter ligated cDNA was again cleaned using spin column purification. PCR was carried out with KAPAHiFiHotStart polymerase (Roche) using Illumina TruSeq index primers (i5: AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACG, i7: CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTG) for 16-20 cycles. Final libraries were size selected in the 200-1,000 bp range by cutting from a polyacrylamide gel. DMS-MaPseq: RNA was precipitated from the supernatant of the biotin pulldown using ethanol. 50 µg were used as input for the Dynabeads Oligo(dT)25 mRNA purification kit (Invitrogen) and mRNA was purified according to the manufacturer’s instructions. RNA was then fragmented using 10 mM ZnCl2 at 94 ºC for 55 s and precipitated using isopropanol. End repair using T4 PNK was performed according to the manufacturer’s instructions. Fragmented mRNA was then size selected by differential silica binding with RNA Clean & Concentrator-5 spin columns (Zymo Research) using 33% ethanol. RT was carried out as described above, except the DNA strand contained a random overhang (N) instead of a G. Adapter ligation, PCR and sequencing were performed as described above. Mature rRNA DMS-MaPseq was carried out by fragmentation and library preparation without any prior enrichment steps.

treatment_protocol: 2.8ml culture were removed, spun down, and washed with cold PBS. The pellet was resuspended in 10ml cold 0.5% sarkosyl and incubated on ice for 20min. Cells were then pelleted at 400×g. Run-on reactions were performed in a 300µl volume with 25µM biotin-11-CTP (Jena Biosciences), 125µM each ATP, UTP and GTP, 0.5% sarkosyl, 20mM Tris⋅HCl pH 7.4, 200mM KCl, 5mM MgCl2 and 2mM dithiothreitol at 30ºC for 2min with mild shaking. 200µl prewarmed 2.5x structure probing buffer (750mM Bicine⋅KOH pH 8.0, 500mM CH3CO2K pH 8.0, 12.5mM MgCl2) and 25µl DMS were added and incubated for 4min at 30ºC with mild shaking. The reaction was stopped by the addition of 1ml ice cold stop buffer (20mM Tris⋅HCl pH 7.4, 200mM KCl, 4.29M 2-mercaptoethanol, 50% isoamyl alcohol).

tag: strain

value: BY4741 spt4::kanMX

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 spt4::kanMX

value: DMS


title: 84_24h_HDF78M_20Gy

city: Rochester

first: Fathima

last: Athar

state: New York

address: 500 Joseph C Wilson Blvd.

department: Biology

institute: University of Rochester

data_processing:
Fastp (0.23.4): Default parameters Salmon (v1.5.1): salmon quant --gcBias -l A
Assembly: GCA_003508835.1 (Eonycteris spelaea), EptFus1.0_HiC (DNA Zoo Consortium, Eptesicus fuscus), GCA_004027435.1 (Artibeus jamaicensis), Myoluc2.0_HiC (DNA Zoo Consortium, Myotis lucifugus), GCF_000001635.27 (Mus musculus), and GCA_000001405.29 (Homo sapiens)
Supplementary files format and content: Row count and matrix

organism: Homo sapiens

source_name: Fibroblast

extract_protocol:
Total RNA was extracted from irradiated or non-irradiated fibroblasts from four bat species, laboratory mice, wild-caught mice and humans using PureLinkTM RNA Mini Kit (Thermo Fisher Scientific) following manufacturer’s instructions.
NGS-TruSeq Stranded mRNA libraries were generated and sequenced with Illumina NovaSeq 6000 single-end 75 bp sequencing at the University of Rochester Genomics Research Center.

value: Fibroblast

tag: cell type

value: wildtype


title: gex_gp33_3

description: scRNA-Seq + TCR (10x Genomics)


title: 85_24h_HDF70F_Con


title: DMS-MaPseq, spt4∆, dms treated, biol rep 1


title: 3-day 1000ng/mL dox-treated Airn-RMCE ESC clone 2 H3K27me3


title: 83_24h_HDF78M_10Gy


title: CD8_TCF1+_PD1hi_mouse5

data_processing:
Fastq files were processed with an adapted version of the nf-core (36) pipeline for rnaseq, using nextflow (v22.04).
Trimgalore (v0.6.7), a wrapper around FastQC (v0.11.9) and Cutadapt (v3.4) performed quality and adapter trimming of the reads.
The trimmed reads were aligned to GRCm38 with STAR (v2.6.1d) and quantified with Salmon (v1.5.2).
Assembly: GRCm38
Supplementary files format and content: tab-delimited text file includes raw gene counts for each sample

molecule: total RNA

extract_protocol:
TotalRNA was isolated using the ReliaPrep RNA Cell Miniprep System (Promega). Quality and quantity of isolated RNA was analyzed with the Bioanalyzer RNA Pico Chip (Agilent).
cDNA synthesis and library preparation was performed with the SMART-Seq v4 PLUS Ultra Low Input RNA kit (Takara) following the manufacturer's protocol. Libraries were sequenced on a single-end run (1x100bp, dual-index) on a NovaSeq6000 (Illumina) using SP 100 v1.5 chemistry (Illumina).

treatment_protocol: Sorted cells were collected in DNA LoBind tubes (Eppendorf). Cells were centrifuged, media was removed and pellet was frozen until RNA extraction.

value: day 7 post infection


title: 82_24h_HDF78M_Con


title: gex_gp33_2

description: scRNA-Seq + TCR (10x Genomics)


title: DMS-MaPseq, wildtype, untreated, biol rep 1

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0

value: untreated


title: Untreated Airn-RMCE ESC clone 2 H3K27me3

value: Untreated

value: Tri-Methyl-Histone H3 (Lys27) (C36B11) (H3K27me3) (Cell Signaling, cat #:9733)


title: Untreated Airn-RMCE ESC clone 5 H3K27me3


title: DMS-MaPseq, wildtype, untreated, biol rep 2


title: 3-day 1000ng/mL dox-treated Empty-RMCE ESC clone 3 H3K27me3

value: Empty-RMCEs


title: 81_24h_HDF70M_20Gy


title: DMS-MaPseq, wildtype, dms treated, biol rep 3


title: CD8_TCF1+_PD1hi_mouse4

value: spleen

value: CD8+ T cells


title: 80_24h_HDF70M_10Gy


title: CD8_TCF1+_PD1lo_mouse4

value: LCMV Armstrong


title: DMS-MaPseq, spt4∆, dms treated, biol rep 2


title: 79_24h_HDF70M_Con


title: CD8_TCF1+_PD1hi_mouse3


title: Untreated Empty-RMCE ESC clone 3 H3K27me3

value: RMCE Embryonic Stem Cells


title: DMS-MaPseq, wildtype, dms treated, biol rep 1


title: Untreated Empty-RMCE ESC clone 1 H3K27me3


title: CD8_TCF1+_PD1lo_mouse5


title: CD8_TCF1+_PD1lo_mouse3


title: TSCs rep 3 RNA

description:
tsc_6-2_rep3_rna
tsc_pooled_rna_mm10_neg.wig.gz
tsc_pooled_rna_mm10_pos.wig.gz

data_processing:
Sequencing reads (fastq files) were mapped both to the GENCODE vM25 mouse genome (“basic CHR” from https://www.gencodegenes.org/mouse/release_M25.html) [for B6] and to a version of the same genome modified to incorporate CAST single-nucleotide polymorphisms (SNPs) [for CAST]. Mapping was performed with STAR (v2.7.9a), using two-pass mapping and the option “--outFilterMultimapNmax 1” to consider only uniquely mapping reads.
Using a custom Perl script (intersect_reads_snps16.pl) and a file containing a list of CAST SNPs (sangercast07_30_20), aligned reads in the B6 and CAST SAM files were parsed to identify reads clearly originating from either the B6 or CAST allele (i.e. reads that overlap at least 1 B6 or CAST SNP).
For genome browser viewing, strand-specific mm10-aligned reads were separated using samtools: samtools view -Sq 30 (negative) -F 0x10 or (positive) -f 0x10
Assembly: mm10
Supplementary files format and content: WIG files were created using a custom perl script. Per dox condition/genotype, a pooled WIG file was generated using reads from the two or more biological replicates/clones during the STAR alignment.

source_name: Trophoblast Stem Cells

growth_protocol: B6/CAST F1-hybrid Rosa26-RMCE ESCs were grown on gelatin-coated plastic dishes in a humidified Thermo Fisher Forma Series II water-jacketed incubator at 37°C and under 5% CO2. Cells were grown in DMEM high glucose plus sodium pyruvate (Gibco 11995-065), 15% ESC-qualified fetal bovine serum (Gibco 26140-079), 0.1mM non-essential amino acids (Gibco 11140-050), 100U/mL penicillin-streptomycin (Gibco 15140-122), 2mM L-glutamine (Gibco 25030-081), 0.1mM β-mercaptoethanol, and 1:500 LIF conditioned media produced from Lif-1Cα (COS) cells (kind gift of N. Hathaway). TSCs were cultured as in (Quinn et al. 2006). Briefly, TSCs were cultured on gelatin-coated, pre-plated irradiated mouse embryonic fibroblast (irMEF) feeder cells in TSC media (RPMI [Gibco 11875093], 20% qualified FBS [Gibco 26140079], 0.1mM penicillin-streptomycin [Gibco 15140122], 1mM sodium pyruvate [Gibco 11360070], 2mM L-glutamine [Gibco 25030081], 100uM beta-mercaptoethanol [Sigma-Aldrich 63689]) supplemented with 25ng/mL FGF4 (Gibco PHG0154) and 1ug/mL Heparin (Sigma-Aldrich H3149) just before use, at 37°C in a humidified incubator at 5% CO2. At passage, TSCs were trypsinized with 0.125% Trypsin-EDTA in PBS solution (Gibco 25200-072) for ~4 minutes at room temperature and gently dislodged from the plate with a sterile, cotton-plugged Pasteur pipette. To deplete irMEFs from TSCs prior to all harvests, TSCs were pre-plated for 45 minutes at 37°C, transferred to a fresh culture plate, and then cultured for three days in 70% irMEF-conditioned TSC media supplemented with growth factors as above.

extract_protocol:
RMCE ESCs or MEF feeder-free TSCs were washed twice with ice-cold PBS and harvested with 1 mL TRIzol (Thermo Fisher 15596026). Total RNA was prepared via standard TRIzol-chlorofom extraction, per manufacturer’s instructions, except for the addition of 4μL 5μg/μL linear acrylamide (Thermo Fisher AM9520) to promote precipitation in isopropanol. RNA pellets were fully resuspended in RNase-free water, RNA concentrations were measured via Qubit 2.0 fluorometer (RNA high sensitivity kit, Thermo Fisher Q32852), and integrity of RNA was confirmed by visualizing rRNA bands on an agarose gel. Working stocks of 100ng/μL total RNA were prepared, and all RNA samples were stored at -80°C.
Libraries for RNA sequencing were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche 08098140702) and KAPA Unique Dual-Indexed Adapters (Roche 08861919702) or TruSeq adapters, with each library’s input consisting of 9μL 100ng/μL total RNA (900 ng) mixed with 1μL of a 1:250 dilution of ERCC RNA spike-in controls (Thermo Fisher 4456740). RNA fragmentation was performed at 94°C for 6min. Libraries were pooled and sequenced on an Illumina NextSeq500 platform with a 75-cycle NextSeq 500/550 High Output Kit v2.5 (Illumina 20024906).

treatment_protocol: Prior to harvesting, RMCE cell lines (with rtTA) were treated with 0-1000ng/ml doxycycline (1000ng/ml if not otherwise specified) for 72 hours (or longer, 7 days).

value: Trophoblast Stem Cells

value: TSCs


title: 78_24h_HDF3_20Gy


title: 76_24h_HDF3_Con


title: CD8_TCF1+_PD1hi_mouse2


title: 77_24h_HDF3_10Gy


title: CoSTseq, stm1∆, dms treated, biol rep 3

library_source: other

data_processing:
The modular CoSTseq data analysis pipeline, implemented including all custom sub modules in Snakemake, is available at https://github.com/NeugebauerLab/CoSTseq. Raw paired-end reads were quality filtered and trimmed with fastp and then aligned to the reference genome with STAR, without generating new splice junctions. Ribosomal RNA was separated based on alignment coordinates and both mRNA and rRNA were deduplicated separately with UMICollapse.
Assembly: sacCer3
Supplementary files format and content: _counts.pkl files contain count data that can be loaded using CoSTseq data processing tools or the "pickle" Python package. Each file includes four lists specifying number of mutations and coverage for each genomic position for both strands: mutation count forward strand, mutation count reverse strand, coverage forward strand, coverage reverse strand. Each list contains 18 numpy arrays: 16 for each chromosome, one for mitochondrial DNA, one for the KanMX cassette used to obtain KO strains.
Supplementary files format and content: _counts_rrna.pkl files contain count data for rrna only that can be loaded using CoSTseq data processing tools or the "pickle" Python package. Each file includes two numpy arrays specifying number of mutations and coverage for each rRNA position.
Library strategy: CoSTseq

molecule: other

source_name: BY4741 stm1::kanMX

value: BY4741 stm1::kanMX

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 stm1::kanMX


title: CoSTseq, stm1∆, dms treated, biol rep 2

value: DMS


title: CoSTseq, stm1∆, dms treated, biol rep 1


title: CD8_TCF1+_PD1lo_mouse2


title: 3-day 1000ng/mL dox-treated Xist-RMCE clone 5 RNA

description:
xist-rmce_clone-5_3d_1000dox_rna
xist-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
xist-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz

value: 3 days with 1000ng/mL doxycycline

value: Xist-RMCEs


title: 3-day 1000ng/mL dox-treated Empty-RMCE ESC clone 1 H3K27me3


title: TSCs rep 1 RNA

description:
tsc_6-2_rep1_rna
tsc_pooled_rna_mm10_neg.wig.gz
tsc_pooled_rna_mm10_pos.wig.gz


title: 74_24h_HDF2_10Gy

value: Fibroblast

value: wildtype


title: DMS-MaPseq, wildtype, dms treated, biol rep 2

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0


title: CoSTseq, dbp7∆, dms treated, biol rep 2

source_name: BY4741 dbp7::kanMX

value: BY4741 dbp7::kanMX

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 dbp7::kanMX


title: CD8_TCF1+_PD1lo_mouse1

value: day 7 post infection


title: 73_24h_HDF2_Con


title: CoSTseq, dbp7∆, dms treated, biol rep 1


title: 3-day 1000ng/mL dox-treated Xist-RMCE clone 3 RNA

description:
xist-rmce_clone-3_3d_1000dox_rna
xist-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
xist-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz


title: Sham

city: Nanjing

first: junjun

last: xiong

address: No. 300 Guangzhou Road, Nanjing

department: Department of Orthopedics

country: China

institute: The First Affiliated Hospital of Nanjing Medical University

data_processing:
The comparison software Bowtie2 was used to compare the data to the reference genome, filtering out reads with low comparison quality values, and finally using the reads that were uniquely aligned to the genome (uniquely aligned reads) for subsequent analysis of the information.
After sequencing and analyzing the DNA products of ChIP, we searched for the binding sites of target proteins on a genome-wide basis, and performed peak calling on a genome-wide basis using the MACS2 analysis software, with a threshold value of q-value<0.05, and analyzed the location of the peaks and the sequence information of the peak regions to filter out the peak-related genes. We will analyze the peak location and sequence information of the peak region to screen out the peak-related genes. The peaks will be detected separately for each treatment group, and if there are multiple biological replicates in a treatment group, the peaks will be detected in a single sample first, and then merged with the peaks in the group to output a unified peak data.
Assembly: mm10
Supplementary files format and content: SCI.bw
Supplementary files format and content: Sham.bw

source_name: spinal cord

extract_protocol:
To isolate astrocytes, single-cell suspensions of spinal cords were prepared. Astrocytes were labeled with anti-ACSA-2-conjugated MicroBeads (130-096-054, Miltenyi Biotec) on ice and then separated using a MACS multistage separator according to the manufacturer’s instructions.
After obtaining the DNA samples, the samples were first tested for quality. After passing the quality test, library construction was carried out, the specific steps are as follows: 1. DNA fragment end repair, add A base at the 3' end, and connect the sequencing junction. 2. PCR amplification and library fragment size selection (generally 100-300bp, including the junction sequence). 3. After the library construction is completed, the library will be tested for quality. The library that passes the quality test will be used for on-line sequencing.

value: spinal cord

value: astrocyte

tag: antibody

value: Sham


title: 72_24h_HDF1_20Gy


title: TSCs rep 2 RNA

description:
tsc_6-2_rep2_rna
tsc_pooled_rna_mm10_neg.wig.gz
tsc_pooled_rna_mm10_pos.wig.gz

value: Untreated


title: CoSTseq, dbp3∆, dms treated, biol rep 3

source_name: BY4741 dbp3::kanMX

value: BY4741 dbp3::kanMX

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 dbp3::kanMX


title: SCI

value: SCI


title: 75_24h_HDF2_20Gy


title: 3-day 1000ng/mL dox-treated Xist-RMCE clone 1 RNA

description:
xist-rmce_clone-1_3d_1000dox_rna
xist-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
xist-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz

value: RMCE Embryonic Stem Cells


title: CD8_TCF1+_PD1hi_mouse1

value: spleen

value: CD8+ T cells

value: LCMV Armstrong


title: CoSTseq, dbp7∆, dms treated, biol rep 3

value: DMS


title: 71_12d_M32_10Gy

value: Fibroblast

value: wildtype


title: 71_24h_HDF1_10Gy


title: 72_12d_M32_20Gy


title: CoSTseq, dbp3∆, dms treated, biol rep 2


title: 3-day 1000ng/mL dox-treated Xist-RMCE clone 4 RNA

description:
xist-rmce_clone-4_3d_1000dox_rna
xist-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
xist-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz

value: 3 days with 1000ng/mL doxycycline

value: Xist-RMCEs


title: CoSTseq, dbp3∆, dms treated, biol rep 1


title: 70_12d_M32_Con_F


title: 100_12d_HDF3_20Gy


title: 7-day 1000ng/mL dox-treated Airn-RMCE clone 5, rep 2 RNA

description:
airn-rmce_clone-5_7d_1000dox_rep2_rna
airn-rmce_7d_1000dox_pooled_rna_mm10_neg.wig.gz
airn-rmce_7d_1000dox_pooled_rna_mm10_pos.wig.gz

value: 7 days with 1000ng/mL doxycycline

value: Airn-RMCEs


title: 3-day 10ng/mL dox-treated Xist-RMCE clone 3 RNA

description:
xist-rmce_clone-3_3d_10dox_rna
xist-rmce_3d_10dox_pooled_rna_mm10_neg.wig.gz
xist-rmce_3d_10dox_pooled_rna_mm10_pos.wig.gz

value: 3 days with 10ng/mL doxycycline


title: CoSTseq, spt4∆, dms treated, biol rep 3

value: BY4741 spt4::kanMX

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 spt4::kanMX


title: control rep 2

city: Memphis

first: Yong-Dong

last: Wang

state: TN

address: 262 Danny Thomas Pl, MS1135

institute: St. Jude Children's Research Hospital

description: 10x Genomics

data_processing:
Illumina 10X Genomics scRNAseq base-call files were converted to FASTQ, which aligned to the hg38 genome and transcriptome using the CellRanger v3.0.2 pipeline, for a gene–cell expression matrix.
The count matrices were processed for filtering and downstream analysis using Partek Flow software.
Assembly: hg38
Supplementary files format and content: tab-delimited values files and matrix files

source_name: cortical organoid

extract_protocol:
Sample was pooled together with three cortical organoids for RNA extraction. Dissociated cells were filtered through a 40 µm cell strainer, and centrifuged at 300G for 5 mins, papain removed and resuspended in 1% FBS in PBS.
Using the Chromium Single Cell 3’ Reagent Kits v3.1 (10X Genomics), 4000-7000 cells were captured per lane, single-cell libraries were sequenced on a NovaSeq S2 flow cell

value: cortical organoid

tag: group

value: control

value: 100 days


title: 69_M32_Con_P


title: CoSTseq, spt4∆, dms treated, biol rep 1


title: control rep 1

description: 10x Genomics


title: 68_12d_M29_20Gy


title: 7-day 1000ng/mL dox-treated Airn-RMCE clone 2, rep 2 RNA

description:
airn-rmce_clone-2_7d_1000dox_rep2_rna
airn-rmce_7d_1000dox_pooled_rna_mm10_neg.wig.gz
airn-rmce_7d_1000dox_pooled_rna_mm10_pos.wig.gz

value: RMCE Embryonic Stem Cells


title: proband (family 1) rep 2

description: 10x Genomics

value: proband


title: 3-day 10ng/mL dox-treated Xist-RMCE clone 5 RNA

description:
xist-rmce_clone-5_3d_10dox_rna
xist-rmce_3d_10dox_pooled_rna_mm10_neg.wig.gz
xist-rmce_3d_10dox_pooled_rna_mm10_pos.wig.gz


title: 7-day 1000ng/mL dox-treated Airn-RMCE clone 2, rep 1 RNA

description:
airn-rmce_clone-2_7d_1000dox_rep1_rna
airn-rmce_7d_1000dox_pooled_rna_mm10_neg.wig.gz
airn-rmce_7d_1000dox_pooled_rna_mm10_pos.wig.gz


title: 70_24h_HDF1_Con


title: CoSTseq, wildtype, untreated, biol rep 5

value: a his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0

value: untreated


title: 67_12d_M29_10Gy


title: proband (family 1) rep 1

description: 10x Genomics


title: 3-day 1000ng/mL dox-treated Airn-RMCE clone 5, rep 2 RNA

description:
airn-rmce_clone-5_3d_1000dox_rep2_rna
airn-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
airn-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz


title: Family_2_maternal_4

description: Family 2 maternal replicate 4

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence and low-quality sequence, then mapped to hg38 whole genome using CLC Genomic Workbench RNA seq tool. TPM for transcripts were reported.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include TPM values for all 17 samples

extract_protocol:
Sample was pooled together with three cortical organoids for RNA extraction using Quick-RNA Miniprep Kit (Zymo Research) according to the manufacturer’s instructions
For sequencing, biological replicates were prepared with the TruSeq stranded mRNA library prep kit (Illumina) and sequenced on the Illumina NovaSeq 6000 platform.


title: 66_12d_M29_Con_F


title: CoSTseq, wildtype, untreated, biol rep 4


title: 7-day 1000ng/mL dox-treated Airn-RMCE clone 5, rep 1 RNA

description:
airn-rmce_clone-5_7d_1000dox_rep1_rna
airn-rmce_7d_1000dox_pooled_rna_mm10_neg.wig.gz
airn-rmce_7d_1000dox_pooled_rna_mm10_pos.wig.gz


title: CoSTseq, spt4∆, dms treated, biol rep 2

value: DMS


title: 64_12d_M24_20Gy

value: Fibroblast


title: 3-day 1000ng/mL dox-treated Airn-RMCE clone 5, rep 1 RNA

description:
airn-rmce_clone-5_3d_1000dox_rep1_rna
airn-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
airn-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz


title: 65_M29_Con_P

value: wildtype


title: CoSTseq, wildtype, untreated, biol rep 1


title: CoSTseq, wildtype, untreated, biol rep 3


title: CoSTseq, wildtype, untreated, biol rep 2


title: CoSTseq, wildtype, dms treated, biol rep 9


title: 63_12d_M24_10Gy


title: Family_2_maternal_3

description: Family 2 maternal replicate 3


title: Family_2_maternal_1

description: Family 2 maternal replicate 1


title: 62_12d_M24_Con_F


title: Untreated Airn-RMCE clone 5 RNA

description:
airn-rmce_clone-5_no-dox_rna
airn-rmce_no-dox_pooled_rna_mm10_neg.wig.gz
airn-rmce_no-dox_pooled_rna_mm10_pos.wig.gz

value: Untreated


title: 3-day 1000ng/mL dox-treated Airn-RMCE clone 2, rep 1 RNA

description:
airn-rmce_clone-2_3d_1000dox_rep1_rna
airn-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
airn-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz

value: 3 days with 1000ng/mL doxycycline


title: Family_2_proband_4

description: Family 2 proband replicate 4


title: CoSTseq, wildtype, dms treated, biol rep 8


title: Family_2_proband_3

description: Family 2 proband replicate 3


title: CoSTseq, wildtype, dms treated, biol rep 7


title: 60_12d_M16_20Gy


title: Untreated Airn-RMCE clone 2 RNA

description:
airn-rmce_clone-2_no-dox_rna
airn-rmce_no-dox_pooled_rna_mm10_neg.wig.gz
airn-rmce_no-dox_pooled_rna_mm10_pos.wig.gz


title: 3-day 1000ng/mL dox-treated Empty-RMCE clone 4 RNA

description:
empty-rmce_clone-4_3d_1000dox_rna
empty-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
empty-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz

value: Empty-RMCEs


title: CoSTseq, wildtype, dms treated, biol rep 6


title: 3-day 1000ng/mL dox-treated Airn-RMCE clone 2, rep 2 RNA

description:
airn-rmce_clone-2_3d_1000dox_rep2_rna
airn-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
airn-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz

value: Airn-RMCEs


title: Family_2_proband_1

description: Family 2 proband replicate 1


title: 3-day 1000ng/mL dox-treated Empty-RMCE clone 3 RNA

description:
empty-rmce_clone-3_3d_1000dox_rna
empty-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
empty-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz


title: Family_2_maternal_2

description: Family 2 maternal replicate 2


title: 59_12d_M16_10Gy


title: CoSTseq, wildtype, dms treated, biol rep 5


title: 58_12d_M16_Con_F


title: 57_M16_Con_P


title: Family_1_paternal_3

description: Family 1 paternal replicate 3


title: 3-day 1000ng/mL dox-treated Empty-RMCE clone 2 RNA

description:
empty-rmce_clone-2_3d_1000dox_rna
empty-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
empty-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz


title: 3-day 1000ng/mL dox-treated Empty-RMCE clone 1 RNA

description:
empty-rmce_clone-1_3d_1000dox_rna
empty-rmce_3d_1000dox_pooled_rna_mm10_neg.wig.gz
empty-rmce_3d_1000dox_pooled_rna_mm10_pos.wig.gz


title: 57_24h_M32_20Gy


title: Family_1_paternal_2

description: Family 1 paternal replicate 2


title: CoSTseq, wildtype, dms treated, biol rep 4


title: CoSTseq, wildtype, dms treated, biol rep 3


title: Family_1_paternal_1

description: Family 1 paternal replicate 1


title: CoSTseq, wildtype, dms treated, biol rep 2


title: yki 8-day [8Y6]

city: Boston

first: Yanhui

last: Hu

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v6.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) using default setting.
The counts matrices were then analyzed in Seurat and batch correction was performed using the Harmony algorithm before creating a UMAP and performing clustering analysis.
Assembly: BDGP6.32
Supplementary files format and content: 2022-02-09_matrix_Ying_yki-tumor.csv
Supplementary files format and content: normalized count matrix
Supplementary files format and content: metadata_Ying_yki-tumor final.csv
Supplementary files format and content: cell annotation
Library strategy: snRNA-seq

organism: Drosophila melanogaster

source_name: Whole-body

growth_protocol: All flies were kept on standard cornmeal fly food supplemented with yeast and agar. Crosses were grown at 18°C to inactivate Gal4. Adult offspring flies were collected within 48 hours after emerging, kept at 18°C for another 24 hours and then incubated at 29°C for 5 or 8 days to induce transgene expression.

extract_protocol:
Whole-body flies without heads were flash-frozen using liquid nitrogen and were homogenized in 1ml dounce in buffer of 250 mM sucrose, 10 mM Tris pH 8.0, 25 mM KCl, 5mM MgCl, 0.1% Triton-X, 0.5% RNasin plus (Promega, N2615), 1X protease inhibitor (Promega, G652A), 0.1 mM DTT, then filtered through 40 um cell strainer and 40 um Flowmi (BelArt, H13680-0040). Samples were centrifuged, washed, and resuspended in 1 X PBS with 0.5% BSA and 0.5% RNasin plus. The suspension was filtered again with 40 um Flowmi immediately before FACS sorting. Nuclei were stained with DRAQ7TM Dye (Invitrogen, D15106) and sorted using Sony SH800Z Cell Sorter at Systems Biology Flow Cytometry Facility at Harvard Medical School. After sorting, nuclei were collected and re-suspend at 700-800 cells/µl in 1 X PBS buffer with 0.5% BSA and 0.5% RNasin plus. snRNAseq was performed according to the 10X genomics protocol (Chromium Next GEM Single Cell 3’_v3.1_Rev_D)
Libraries were generated according to 10X genomics Next GEM Single Cell 3’ Library and Gel Bead Kit v3.1

treatment_protocol: Female flies were incubated for 5 or 8 days at 29°C

value: Whole-body

tag: Sex

value: Female

value: esg-GAL4, tub-GAL80TS > UAS-GFP, UAS-yki3SA


title: Family_1_maternal_3

description: Family 1 maternal replicate 3

value: cortical organoid


title: 56_24h_M32_10Gy


title: yki 8-day [8Y5]

description: 10x Genomics


title: 61_M24_Con_P


title: CoSTseq, wildtype, dms treated, biol rep 1


title: Family_2_proband_2

description: Family 2 proband replicate 2


title: 55_24h_M32_Con


title: Family_1_maternal_2

description: Family 1 maternal replicate 2


title: yki 8-day [8Y4]

description: 10x Genomics


title: HG001 (p)ppGpp0 ΔguaBA, replicate 3

city: Tübingen

first: Christiane

last: Wolz

address: Elfriede-Aulhorn-Straße 6

department: Interfaculty Institute for Medical Microbiology and Infection Medicine

institute: University of Tübingen

data_processing:
The sequencing was demultiplexed with bcl2fastq (v2.19.0.316) and quality checked with fastq (v0.20.1) and visualized with MultiQC (v1.7).
CLC Genomics Workbench 20.0.3
Sequences were trimmed for low quality sequence using CLC genomci workbench (qualitiy limit 0.05)
Trimmed sequences were mapped to reference genome (NZ_CP018205.1) using CLC genomic workbench (parameters: mismatch cost: 2; insertion cost: 3; deletion cost: 3; lenght fraction: 0.8; similarity fraction: 0.8)
Read count extraction and normalization were performed using CLC genomic benchwork
Assembly: reference genome of HG001 (NZ_CP018205.1)
Supplementary files format and content: tab-separated txt files (GE): RPKM values, TPM values, total and unique gene reads calculated from gene expression data

organism: Staphylococcus aureus

growth_protocol: Samples were grown aerobically in chemically-defined medium (CDM) at 37°C under agitation (200 rpm) for 24 hours

extract_protocol:
Bacteria were harvested by centrifugation. Bacterial pellets were immediately resuspended in TRIzol (Thermo Fisher Scientific) and mechanically lysed using zirconia/silica beads in a high speed homogenizer. RNA was further purified using the ExpressArt® RNA ready Add-on Kit for TRIzol extraction (AmpTec). DNase digest of the sample was directly performed on the column.
Per sample, a total amount of 100 ng RNA was subjected to rRNA depletion followed by cDNA library construction with the IlluminaTM Stranded Total RNA Prep Ligation with Ribo Zero Plus Kit according to the manufacturer’s instructions. Libraries were sequenced as single-read (100 bp read length) on a NextSeq platform (Illumina) using the NextSeqTM 500 Mid Output Kit v2.5.

treatment_protocol: not applicable, no treatment before cell harvest

value: (p)ppGpp0 [delta]guaBA

tag: time

tag: culture conditions

value: chemically-defined medium (CDM), aerobic growth at 37°C and 200 rpm in liquid culture


title: yki 8-day [8Y3]

description: 10x Genomics


title: 54_24h_M29_20Gy


title: Family_1_maternal_1

description: Family 1 maternal replicate 1

value: 100 days


title: HG001 (p)ppGpp0 ΔguaBA, replicate 2


title: wild type 8-day [8W2]

description: 10x Genomics

value: esg-GAL4, tub-GAL80TS > UAS-GFP


title: 52_24h_M29_Con


title: 53_24h_M29_10Gy


title: Family_1_proband_3

description: Family 1 proband replicate 3


title: HG001 (p)ppGpp0 ΔguaBA, replicate 1


title: wild type 8-day [8W1]

description: 10x Genomics


title: 51_24h_M24_20Gy


title: HG001 ΔguaBA, replicate 3

value: [delta]guaBA


title: yki 5-day [Y5]

description: 10x Genomics


title: HG001 ΔguaBA, replicate 2

description: Sample was excluded from differential gene expression (DGE) analysis since the strain was clearly not a ΔguaBA mutant strain. PCA analysis indicates that it is rather a (p)ppGpp0 mutant and therefore was possibly mixed up during RNAseq processing


title: pooled cells from 4 stages, mRNA-derived cDNA

city: Chengdu

first: Zhonghan

last: Li

state: Sichuan

address: 29 Wangjiang Road

department: College of Life Sciences

institute: Sichuan University

description:
poly A RNA
3' mRNA library: read1 file contains cell barcode and UMI; read2 file contains transcript

data_processing:
Pre-processing of sequencing results to generate count matrices (gene expression,and HTO barcode counts) was performed using the Singleron celeScope pipeline where HTO sequences were concatenated and treated as Custom library with default settings and aligning to the hg38.
Further processing was done with Seurat v4.2.0 for cell and gene filtering, hashtag identification and clustering.
Assembly: hg38
Supplementary files format and content: IPS1_umi_tag.tsv containing UMI counts of hashtags for each barcode
Supplementary files format and content: CeleScope output files: barcodes.tsv, genes.tsv, matrix.mtx

extract_protocol:
Cells at each stage were lifted from plates by enzymatic digestion followed by incubated with respective TotalSeq antibodies. Then euqal number of  labelled cells were mixed togther for capturing and library preparation
library preparation was performed by Singleron Biotechnologies Inc, Nanjing using GEXSCOPE® Single Cell RNA Library Kit under manufacturer's protocols.

value: induced pluripotent stem cell-derived cells

value: stage-specific differentiation

value: mRNA

tag: antibodies/tags


title: HG001 ΔguaBA, replicate 1


title: 49_24h_M24_Con

tag: tissue

value: Fibroblast

value: wildtype


title: Family_1_proband_1

description: Family 1 proband replicate 1


title: 50_24h_M24_10Gy


title: pooled cells from 4 stages, HTO-derived cDNA

description:
Hashtag-derived oligonucleotide (HTO)
Cell Hashing library: read1 file contains cell barcode and UMI; read2 file contains Hashtag Antibody Derived Tag reads

molecule: protein

tag: cell line

value: HTO

value: Anti-CD298 and β2Μ/TotalSeq™-A0251 anti-human Hashtag 1 for iPSC;  Anti-CD298 and β2Μ/TotalSeq™-A0252 anti-human Hashtag 2 for PS;  Anti-CD298 and β2Μ/TotalSeq™-A0253 anti-human Hashtag 3 for PSM;  Anti-CD298 and β2Μ/TotalSeq™-A0254 anti-human Hashtag 4 for Sox9+ SLCs


title: yki 5-day [Y4]

description: 10x Genomics

tag: genotype


title: 48_24h_M16_20Gy


title: HG001 (p)ppGpp0, replicate 3

value: (p)ppGpp0 ([delta]rel, [delta]relP, [delta]relQ)


title: OE-TCF-9

city: Guangzhou

first: Keling

last: Wu

address: #7 Jin-Sui Road, Zhujiang New Town

institute: zhongshan ophthalmic center

description: case 3

data_processing:
Illumina® (NEB, USA) was used for establishment of sequencing libraries
Sequencing of the library preparations were performed on an Illumina NovaSeq platform
Clean reads obtained from processed raw reads were aligned to the human reference genome (GRCh38/hg38) by TopHat v2.0.12.
The reads numbers was matched to each gene by HTSeq v0.6.1
Differentially expression analysis of downregulated genes between the two groups were performed with DESeq R package (1.18.0)
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: OE-TCF vs NC-OE-TCF

source_name: retinal progentior cells (RPCs)

extract_protocol:
RPCs treated with Ad-NC and Ad-hTCF7L2 for two days were dissolved in TRIzol reagent (Invitrogen, USA).
About 1 µg RNA per sample was taken as input material for sample preparations. NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) was used for establishment of sequencing libraries. Then, sequencing of the library preparations were performed on an Illumina NovaSeq platform and 150-bp paired-end reads were generated.

value: retinal progentior cells (RPCs)

value: two days


title: yki 5-day [Y3]

description: 10x Genomics

value: Whole-body

value: Female

value: esg-GAL4, tub-GAL80TS > UAS-GFP, UAS-yki3SA


title: 47_24h_M16_10Gy


title: OE-TCF-6

description: case 2


title: wild type 5-day [W2]

description: 10x Genomics


title: HG001 (p)ppGpp0, replicate 1

value: chemically-defined medium (CDM), aerobic growth at 37°C and 200 rpm in liquid culture


title: 46_24h_M16_Con


title: wild type 5-day [W1]

description: 10x Genomics


title: OE-TCF-3

description: case 1


title: Family_1_proband_2

description: Family 1 proband replicate 2

value: cortical organoid

value: 100 days


title: HG001 wildtype, replcate 3


title: T12_STIM2

city: Geneve

first: Alan Yue Yang

last: Teo

address: Chem. des Mines 9, Campus Biotech

department: SV

country: Switzerland

institute: EPFL

data_processing:
The reads were aligned using Cell Ranger v7.1.0. and the final data matrix is generated as per Cellranger pipeline.
Assembly: GRCm39.109
Supplementary files format and content: Data were analyzed using Seurat v4

molecule: nuclear RNA

extract_protocol:
For snRNA-seq experiment, single-nucleus dissociation of the mouse lumbar spinal cord was performed according to our established procedures. Following euthanasia by isoflurane inhalation and cervical dislocation, the lower-thoracic and the lumbosacral spinal cord segments were immediately dissected and frozen on dry ice. Spinal cords were doused in 500 µL sucrose buffer (0.32 M sucrose, 10 mM HEPES [pH 8.0], 5 mM CaCl2, 3 mM Mg acetate, 0.1 mM EDTA, 1 mM DTT) and 0.1% Triton X-100 with the Kontes Dounce Tissue Grinder. 2 mL of sucrose buffer was then added and filtered through a 40 µm cell strainer. The lysate was centrifuged at 3200 g for 10 min at 4°C. The supernatant was then decanted, and 3 mL of sucrose buffer was added to the pellet for 1 min. We homogenized the pellet using an Ultra-Turrax and 12.5 mL of density buffer (1 M sucrose, 10 mM HEPES [pH 8.0], 3 mM Mg acetate, 1 mM DTT) was added below the nuclei layer. The tube was centrifuged at 3200 g at 4°C and supernatant poured off. Nuclei on the bottom half of the tube wall were collected with 100 µL PBS with 0.04% BSA and 0.2 U/µL RNase inhibitor. Finally, nuclei were resuspended through a 30 µm strainer, and adjusted to 1000 nuclei/µL.
snRNA-seq library preparation was carried out using the 10x Genomics Chromium Single Cell Kit Version 3. The nuclei suspension was added to the Chromium RT mix to achieve loading numbers of 2000-5000. For downstream cDNA synthesis (13 PCR cycles), library preparation and sequencing, the manufacturer’s instructions were followed.

value: spinal cord

value: Nuclei

value: Spinal cord injury, subjected to epidural electrical stimulation


title: 18_RLNGL_RNA-seq

city: Dunsany

first: Dayle

last: Johnston

state: Ireland

address: AGRIC

department: Animal and Bioscience Research Department

country: Ireland

institute: Teagasc

data_processing:
The 3' end of the sequence reads were trimmed for Illumina adapters, low quality reads (quality score <  20), ambiguous nucleotides, and poly-G artefacts resulting from the NextSeq’s two-colour chemistry, using CutAdapt (version 1.18).
The cleaned sequence reads were evaluated for quality using FastQC.
Reads were aligned to the ARS-UCD1.2 bovine reference genome and read counts were generated by converting aligned reads into counts per gene using the Spliced Transcripts Alignment to a Reference (STAR) aligner (version 2.6.1b)
Genome_build: ARS-UCD1.2
Supplementary_files_format_and_content: TXT file of raw counts table of sequenced reads

organism: Bos taurus

source_name: BoHV1 challenged_cranial lung lobe_lesioned

extract_protocol:
Total RNA was extracted using the Qiagen RNeasy Plus Universal Mini Kit (Qiagen LTD, Manchester, UK), according to the manufacturer’s instructions including Appendix C of the manufacturer’s protocol.
RNA-Seq library preparation was performed using the TruSeq stranded mRNA Kit (Illumina, San Diego, California, USA).

tag: breed

value: Holstein-Friesian

value: BoHV1 challenged

value: cranial lung lobe

tag: tissue condition

value: lesioned

tag: shed

value: C


title: yki 5-day [Y6]

description: 10x Genomics


title: NC-OE-TCF-8

description: control 3

tag: treatment

value: Ad-NC


title: T12_STIM1


title: 17_RLNGL_RNA-seq


title: HG001 wildtype, replcate 2

value: wildtype


title: NC-OE-TCF-5

description: control 2

value: retinal progentior cells (RPCs)

value: two days


title: HG001 wildtype, replcate 1


title: 16_RLNGL_RNA-seq


title: T12_SCI4

value: Spinal cord injury


title: NC-OE-TCF-2

description: control 1


title: 15_RLNGL_RNA-seq


title: T12_SCI3


title: HG001 (p)ppGpp0, replicate 2

value: (p)ppGpp0 ([delta]rel, [delta]relP, [delta]relQ)


title: 4T07 cell,ChIP Cebpb

city: Shanghai

first: Yang

last: Dong

state: 上海

address: Shanghai

institute: Tongji University

description: Library name: Sample 2

data_processing:
Raw data (raw reads) of fastq format were firstly processed using fastp (version 0.19.11, Chen et al., 2018) software. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using BWA (v 0.7.12) and clean reads were aligned to the reference genome using BWA mem (v 0.7.12).
For a specific ChIP-seq binding site, individual reads mapping to the plus or minus strand and present significant enrich. For single-end sequencing, the fragment size was estimated with MACS2 (Zhang et al., 2008) predicted method with default parameters. MACS2 scan all the genome with a specific window size and calculate the reads enrichment level. A particular number of windows were used as samples to build enrichment model and to predict fragment size. The following peak calling analysis was based on this predicted fragment size.
After mapping reads to the reference genome, we used the MACS2 (version 2.1.0) peak calling software to identify regions of IP enrichment over background. A q-value threshold of 0.05 was used for all data sets. After peak calling, the distribution of chromosome distribution, peak width, fold enrichment, significant level and peak summit number per peak were all displayed.
Assembly: mm10
Supplementary files format and content: bigWig

growth_protocol: 4T07 cells were maintained in DMEM, high glucose supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
Cells were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin was sonicated for 15 minutes.
The purified DNA was used for ChIP-seq library preparation. The library was constructed by Novogene Corporation (Beijing, China). Subsequently, pair-end sequencing of sample was performed on Illumina platform (Illumina, CA, USA). Library quality was assessed on the Agilent Bioanalyzer 2100 system.

value: triple-negative breast cancer (TNBC) cell line

value: WT


title: T12_AD3

value: Spinal cord injury, subjected to autonomic dysreflexia


title: 13_RLNGL_RNA-seq


title: 4T07 cell,ChIP Input

description: Library name: Sample 1


title: AA_H_5

city: Hefei

first: Jingni

last: Zhang

address: No. 350 Shushanhu Road

institute: Anhui Institute of Optics and Fine Mechanics, Hefei Institutes of Physical Science, Chinese Academy of Sciences

description:
Library name: Sample 17
fpkm_AA_H_5

data_processing:
Sequencing was performed on the DNBSEQ platform with a read length of PE150.
The raw data obtained from sequencing was filtered using SOAPnuke.
HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts) is software used for aligning RNA-seq reads to a reference genome.
The clean data was aligned to the reference gene set using Bowtie2 (v2.3.4.3) . Gene expressionquantification was performed using RSEM (v1.3.1) software, and the expression clustering heatmap of genes across different samples was generated using pheatmap (v1.0.8) . Differential gene detection was carried out using DESeq2 (v1.4.5) (or DEGseq or PoissonDis ).
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on differentially expressed genes using the hypergeometric test implemented in the R package phyper.
Assembly: Homo_sapiens_9606.NCBI.GCF_000001405.39_GRCh38.p13.v2201
Supplementary files format and content: processed data file.xls

source_name: lung; bronchus

growth_protocol: BEAS-2B cells were maintained in BEGM in humidified atmosphere with CO2 at 37°C.

extract_protocol:
Total RNA was extracted from tissues using TRIzol (Invitrogen, Carlsbad, California, USA) according to manual instruction.
Total RNA was processed using mRNA enrichment. The mRNA was enriched using magnetic beads with Oligo(dT) to capture mRNA with a polyA tail. The obtained RNA was fragmented using a breaking buffer, and random N6 primers were used for reverse transcription.Subsequently, double-stranded cDNA was synthesized. The ends of the synthesized double-stranded DNA were blunted and 5' end-phosphorylated. A single-base overhang "A" was added to the 3' end, and a bubble-shaped adapter with a protruding "T" at the 3' end was ligated. The ligated products were PCR-amplified using specific primers. The PCR products were denatured to form single-stranded DNA, and single-stranded circular DNA libraries were generated using a bridged primer. The constructed libraries underwent quality control, and after passing, they were subjected to sequencing.

treatment_protocol: BEAS-2B cells were fed with BEGM with  acetaldehyde (0, 10, and 16 mM) for 24 h.

value: lung; bronchus

value: normal lung epithelial cells

value: exposed to 16 mM acetaldehyde for 24 hours


title: AA_H_3

description:
Library name: Sample 15
fpkm_AA_H_3


title: 11_RLNGL_RNA-seq

value: Holstein-Friesian

value: B


title: AA_H_2

description:
Library name: Sample 14
fpkm_AA_H_2


title: 4T07 cell,OE-Cebpb rep3

description: Library name: Sample 6

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Assembly: mm10
Supplementary files format and content: tab-delimted text file includes raw counts for each sample
Supplementary files format and content: tab-delimted text file includes RPKM counts for each sample

extract_protocol:
RNA was harvested using TRIzol (Invitrogen). 1.0 μg of total RNA was used for the construction of sequencing libraries.
Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample.

treatment_protocol: 4T07 cells were trasnfected with either pLenti-CMV-MCS or pLenti-CMV-Cebpb by lentivirus. Stable tracfected cells were selected by puromycin.

value: Cebpb overexpression


title: T12_AD2

value: spinal cord

value: Nuclei


title: T12_AD1


title: 7_RLNGL_RNA-seq

value: BoHV1 challenged

value: cranial lung lobe

value: lesioned


title: 4T07 cell,OE-Cebpb rep2

description: Library name: Sample 5


title: L6_SCI3


title: AA_H_1

description:
Library name: Sample 13
fpkm_AA_H_1


title: 4T07 cell,OE-Cebpb rep1

description: Library name: Sample 4


title: 6_RLNGL_RNA-seq

source_name: control_cranial lung lobe_lesioned

value: control

value: A


title: AA_L_6

description:
Library name: Sample 12
fpkm_AA_L_6

value: exposed to 10 mM acetaldehyde for 24 hours


title: 3_RLNGL_RNA-seq


title: AA_L_5

description:
Library name: Sample 11
fpkm_AA_L_5


title: 4T07 cell,OE-Control rep3

description: Library name: Sample 3

value: triple-negative breast cancer (TNBC) cell line


title: 2_RLNGL_RNA-seq


title: 4T07 cell,OE-Control rep2

description: Library name: Sample 2

value: WT


title: AA_L_4

description:
Library name: Sample 10
fpkm_AA_L_4


title: 1_RLNGL_RNA-seq


title: L6_AD3

value: Spinal cord injury, subjected to autonomic dysreflexia


title: L6_SCI1

value: Spinal cord injury


title: 4T07 cell,OE-Control rep1

description: Library name: Sample 1


title: L6_AD2


title: AA_L_3

description:
Library name: Sample 9
fpkm_AA_L_3

value: lung; bronchus

value: normal lung epithelial cells


title: AA_H_4

description:
Library name: Sample 16
fpkm_AA_H_4

value: exposed to 16 mM acetaldehyde for 24 hours


title: AA_L_1

description:
Library name: Sample 7
fpkm_AA_L_1


title: L6_AD1


title: Eonycteris spelaea5

description: Library name: Bat162Skin

data_processing:
Salmon, DESeq2
Assembly: Eonycteris spelaea: GCA_003508835.1; Eptesicus fuscus: HLeptFus2; Artibeus jamaicensis: GCA_004027435.1;
Supplementary files format and content: matrix;raw read counts

organism: Eonycteris spelaea

source_name: Skin

extract_protocol:
Trizol RNA extraction
rRNA depletion using Illumina TruSeq stranded total RNA RiboZero Gold kit

value: Skin

value: wildtype


title: AA_L_2

description:
Library name: Sample 8
fpkm_AA_L_2


title: 17_RLNGH_RNA-seq

source_name: BoHV1 challenged_cranial lung lobe_healthy

value: Holstein-Friesian

value: healthy

value: C


title: 16_RLNGH_RNA-seq

value: BoHV1 challenged

value: cranial lung lobe


title: Hb_7

city: Norwich

first: Alice

last: Godden

state: Not applicable

address: School of Biological Sciences, University of East Anglia, Norwich Research Park

country: United Kingdom

institute: University of East Anglia

data_processing:
Reads were trimmed, aligned with PatMan and then count matrices assembled before DESeq2 analyses
Assembly: GRCz10
Supplementary files format and content: piRNA_raw_counts.csv, raw counts of a subset of 24 out of the 40 samples that were analysed. These 24 were chosen due to noise observed in PCA clustering.
Supplementary files format and content: mirna_raw_counts.csv, raw counts for 39/40 samples as one sample failed to progress through our pipeline to the same high-quality as the other 39, so this sample was omitted from analyses.

organism: Danio rerio

source_name: sperm

extract_protocol:
Total RNA extraction
New England BioLabs kit NEBNext®Multiplex Small RNA Library Prep Set for Illumina® Set 1 and 2 (NEB #E7300 and NEB #E7580)
small RNA library prep for Illumina

value: sperm


title: RNA from WT E. coli (grown at 45 °C), biological replicates 1 and 2 combined

city: Martinsried

first: Kirsten

last: Jung

address: Großhaderner Str. 2-4

institute: LMU

data_processing:
Basecalling with Guppy v6.5.7 using default parameters.
Alignment to modified E. coli reference genome with duplicate tRNA/rRNA gene sequences removed (supplementary file "deduplicated_Ecoli_genome.fasta") with Minimap2 v2.26 using default parameters.
Read filtering with SAMtools v1.17 using default parameters.
Read quantification with Salmon using a gff3 annotation file ("Escherichia_coli_str_k_12_substr_mg1655_gca_000005845.ASM584v2.58.gff3") and modified E. coli reference transcriptome with duplicate tRNA/rRNA gene sequences removed (supplementary file "deduplicated_Ecoli_transcriptional_unit_coordinates.txt") for gene-level quantification.
Modification detection in aligned reads with ELIGOS2 using default parameters.
Aligned read indexing and preprocessing with Nanopolish v0.14 using default parameters.
Modification detection in indexed/preprocessed reads with Nanocompore using a minimum read depth of 20.
Modification detection in indexed/preprocessed reads with CHEUI using default parameters.
Assembly: Modified version of GCA_000005845 ASM584v2 with duplicate tRNA/rRNA gene sequences removed (supplementary file "deduplicated_Ecoli_genome.fasta").
Supplementary files format and content: Files ending in "ELIGOS_results.txt": tab-delimited text files output by ELIGOS2 including putative modification sites as genomic loci.
Supplementary files format and content: Files ending in "cheui_differential_m5C_sites.txt" and "cheui_differential_m6A_sites.txt": tab-delimited text files output by CHEUI-diff including putative modification sites (m5C or m6A) as relative positions within each RNA.
Supplementary files format and content: Files ending in "site.level.predictions.txt": tab-delimited text files output by CHEUI-solo including putative modification sites (5-methylcytosine [m5C] or N6-methyladenosine [m6A]) as relative positions within each RNA.
Supplementary files format and content: Files ending in "Nanocompore_results.tsv": tab-separated values files output by Nanocompore including putative modification sites as relative positions within each RNA.
Supplementary files format and content: Files ending in "Salmon_gene_counts.txt": tab-delimited text files output by Salmon including gene-level read counts.

organism: Escherichia coli

source_name: whole cells

growth_protocol: Bacteria were grown in LB at 37 °C with 200 rpm shaking.

extract_protocol:
RNA was isolated using the PCI protocol (Sambrook & Russell, 2006) with some modifications (Petrov et al., 2022).
Nanopore MinION sequencing libraries were prepared with tRNA/rRNA-depleted polyadenylated RNA following the manufacturer’s protocol (ONT #SQK-RNA002).

treatment_protocol: After growth at 37 °C to an OD600 of 0.5, 45 °C samples were heat-shocked with the addition of 90 °C LB, then grown at 45 °C.

value: Growth at 45 °C; tRNA/rRNA-depletion and polyadenylation


title: Eonycteris spelaea4

description: Library name: Bat161Skin


title: L6_SCI2

value: spinal cord

value: Nuclei


title: 18_RLNGH_RNA-seq


title: Control_6

description:
Library name: Sample 6
fpkm_Control_6

value: WT

value: Control


title: Ha_8


title: Eonycteris spelaea3

description: Library name: Bat106Skin


title: 15_RLNGH_RNA-seq


title: Hb_6


title: Control_5

description:
Library name: Sample 5
fpkm_Control_5


title: 14_RLNGH_RNA-seq


title: 13_RLNGH_RNA-seq


title: Control_4

description:
Library name: Sample 4
fpkm_Control_4


title: Ha_7


title: Lb_10


title: 12_RLNGH_RNA-seq

value: B


title: Eonycteris spelaea1

description: Library name: Bat108Skin


title: Control_3

description:
Library name: Sample 3
fpkm_Control_3

value: lung; bronchus


title: 56_12d_Art3_20Gy

data_processing:
Fastp (0.23.4): Default parameters Salmon (v1.5.1): salmon quant --gcBias -l A
Assembly: GCA_003508835.1 (Eonycteris spelaea), EptFus1.0_HiC (DNA Zoo Consortium, Eptesicus fuscus), GCA_004027435.1 (Artibeus jamaicensis), Myoluc2.0_HiC (DNA Zoo Consortium, Myotis lucifugus), GCF_000001635.27 (Mus musculus), and GCA_000001405.29 (Homo sapiens)
Supplementary files format and content: Row count and matrix

organism: Artibeus jamaicensis

extract_protocol:
Total RNA was extracted from irradiated or non-irradiated fibroblasts from four bat species, laboratory mice, wild-caught mice and humans using PureLinkTM RNA Mini Kit (Thermo Fisher Scientific) following manufacturer’s instructions.
NGS-TruSeq Stranded mRNA libraries were generated and sequenced with Illumina NovaSeq 6000 single-end 75 bp sequencing at the University of Rochester Genomics Research Center.

value: Fibroblast


title: Control_2

description:
Library name: Sample 2
fpkm_Control_2

value: normal lung epithelial cells


title: Lb_9


title: 11_RLNGH_RNA-seq


title: 10_RLNGH_RNA-seq


title: 54_12d_Art3_Con_F


title: 88_12d_WM19_20Gy

organism: Mus spretus


title: Control_1

description:
Library name: Sample 1
fpkm_Control_1


title: Lb_7


title: 55_12d_Art3_10Gy


title: La_7


title: 9_RLNGH_RNA-seq


title: 53_Art3_Con_P


title: 87_12d_WM19_10Gy


title: 8_RLNGH_RNA-seq


title: Eonycteris spelaea2

description: Library name: Bat160Skin

value: Skin


title: 52_12d_Art2_20Gy

value: wildtype


title: 86_12d_WM19_Con_F


title: La_5


title: 7_RLNGH_RNA-seq

value: Holstein-Friesian


title: 51_12d_Art2_10Gy


title: 6_RLNGH_RNA-seq

source_name: control_cranial lung lobe_healthy

value: control

value: healthy

value: A


title: Hb_2


title: 49_Art2_Con_P

source_name: Fibroblast


title: 5_RLNGH_RNA-seq

value: cranial lung lobe


title: 83_12d_WM15_10Gy


title: Lb_6

value: sperm


title: 48_12d_Art1_20Gy


title: Ha_5


title: La_6


title: 47_12d_Art1_10Gy


title: Ha_6


title: 81_WM15_Con_P


title: 82_12d_WM15_Con_F


title: 3_RLNGH_RNA-seq


title: 2_RLNGH_RNA-seq


title: La_4


title: 46_12d_Art1_Con_F


title: 80_12d_WM14_20Gy


title: 79_12d_WM14_10Gy


title: 1_RLNGH_RNA-seq


title: 45_Art1_Con_P


title: 50_12d_Art2_Con_F


title: 85_WM19_Con_P


title: Hb_1


title: BLN, BoHV1, S18

source_name: bronchial lymph node

value: BoHV1 challenged

value: C


title: 84_12d_WM15_20Gy


title: Hb_4


title: 45_24h_Art3_20Gy


title: 4_RLNGH_RNA-seq


title: 76_12d_WM11_20Gy


title: 44_24h_Art3_10Gy


title: BLN, BoHV1, S16


title: Lb_5


title: 43_24h_Art3_Con


title: 75_12d_WM11_10Gy


title: 42_24h_Art2_20Gy


title: 74_12d_WM11_Con_F


title: BLN, BoHV1, S14


title: Ha_4


title: BLN, BoHV1, S15


title: 41_24h_Art2_10Gy


title: La_1


title: Ha_3


title: 73_WM11_Con_P


title: Hb_5


title: 39_24h_Art1_20Gy


title: BLN, BoHV1, S17


title: 68_24h_WM19_10Gy


title: BLN, BoHV1, S13


title: 78_12d_WM14_Con_F


title: Lb_3


title: 40_24h_Art2_Con


title: 69_24h_WM19_20Gy


title: 66_24h_WM15_20Gy


title: Lb_4


title: 67_24h_WM19_Con


title: 38_24h_Art1_10Gy


title: BLN, BoHV1, S12


title: BLN, BoHV1, S11


title: 37_24h_Art1_Con


title: BLN, BoHV1, S9


title: Hb_3


title: 65_24h_WM15_10Gy


title: BLN, BoHV1, S8


title: Slide_2_Pt-5_LiM-2

type: RNA

city: New York

first: Anirban

last: Maitra

state: NY

address: Smilow 910, 522 1st Ave

department: Perlmutter Cancer Center

institute: New York University Grossman School of Medicine

description:
Slide 2
CosMx_SCT_data.csv.gz
Meta.data_CoxMx.csv.gz
Run5961_PDAC_Slide2_exprMat_file.csv.gz
Run5961_PDAC_Slide2_fov_positions_file.csv.gz
Run5961_PDAC_Slide2_metadata_file.csv.gz
Run5961_PDAC_Slide2-polygons.csv.gz
Run5961_PDAC_Slide2_tx_file.csv.gz
Run5961_PDAC_Slide2_CellLabels.tar.gz
Run5961_PDAC_Slide2_CellOverlay.tar.gz
Run5961_PDAC_Slide2_CompartmentLabels.tar.gz

hyb_protocol: Hybridization was performed at 37°C overnight (16-18 h) in a hybridization chamber to prevent evaporation. Following overnight hybridization, incubation frame covers were removed, and samples were washed twice with 50% formamide (VWR) in 2x SSC at 37°C for 25 min, rinsed twice with 2X SSC for 2 min at RT, then blocked with 200 μL Blocking Buffer (NanoString) containing DAPI at 1:40 dilution for 15 min at RT. Samples were then incubated with a fluorophore-conjugated antibody cocktail against CD298/B2M, PanCK, CD45, and CD68 proteins at RT for 1 hr. After antibody staining, the samples were washed three times with 1x PBS for 5 min each at RT, then flow cells were attached to the sample slide using the Flow Cell Assembly Tool and Kit (NanoString).

scan_protocol: Target RNA readout on SMI instrument was performed following published protocols (He et al, 2022). Briefly, assembled flow cells were loaded onto the SMI instrument and samples were washed with Reporter Wash Buffer to rinse the samples and remove any air bubbles from the flow cell. Once loaded and washed, the entire flow cell was scanned, and field of views (FOVs) were placed on each slide for RNA readout. Prior to assay initiation, a 5-channel Z-stack image (4 antibodies and DAPI) was collected for each FOV. These images were used to perform single cell segmentation. Assay cycling was then initiated. The first RNA readout cycle was initiated by flowing 100 µL of Reporter Pool 1 into the flow cell and incubating for 15 min. After incubation, 1 mL of Reporter Wash Buffer was flowed across the flow cell to wash out any unbound reporter probes. The Reporter Wash Buffer was then replaced with Imaging Buffer. Eight Z-stack images (0.8 μm step size) of each FOV were acquired and then fluorophores on the reporter probes were UV-cleaved and washed out with Strip Wash Buffer. This fluidic and imaging procedure was then repeated for the remaining 15 reporter pools, and the 16 cycles of reporter hybridization-imaging was repeated 7 additional times to increase RNA detection sensitivity.

data_processing:
Raw image processing and feature extraction were performed using an in-house SMI data processing pipeline (He et al, 2022) which includes registration, feature detection, and localization. First, 3D rigid image registration was made using fiducials embedded in the samples matched with the fixed image reference established at the beginning of SMI run to correct for any shift. Next, RNA image analysis algorithm was used to identify reporter signature locations in X, Y, and Z axes along with an assigned confidence. The reporter signature locations and the associated features were collated into a single list. Lastly, the XYZ location information of individual target transcripts were extracted and recorded in a table by a secondary analysis algorithm, as described in He et al, 2022.
We then utilized Seurat45 (v.5.0) to analyze the single-cell level spatial transcriptomic data, following the standard single-cell RNA-seq data processing procedures. Specially, SCTransform function was applied to obtain the normalized transcript-by-cell count matrix. The normalized expression matrix (a panel of 1000 detected genes) was selected for subsequent PCA, SNN, graph construction, and cell clustering.
CSV file with normalized data("data" slot) from SCT

source_name: Liver metastasis

label_protocol: RNA ISH probes (Human Universal Cell Characterization Panel, 1000-plex) were denatured at 95°C for 2 min and then placed on ice before preparing the ISH probe mix (1nM ISH probes, 1x Buffer R, 0.2 U/μL SUPERaseIn™ in DEPC H2O). 150 μL ISH probe mix was pipetted into the incubation frame of each slide, then incubation frame covers were applied to help move the hybridization solution across the tissue.

extract_protocol: To prepare tissue samples for the CosMx™ SMI platform (NanoString Technologies Inc, Seattle, WA), slides with fresh frozen PDAC tissue sections were removed from -80°C freezer and immediately transferred to pre-cooled (4°C) 10% Neutral Buffered Formalin (NBF, EMS Diasum) and allowed to incubate for 2 h at 4°C. Slides were then transferred to room temperature (RT) 1x phosphate-buffered saline (1x PBS) and washed three times for 2 min each, ensuring a fresh coplin jar was used each time. After the final wash, slides were moved to a slide holder and baked vertically at 60°C for 30 min. Following baking, slides underwent heat-induced epitope retrieval (HIER) and proteinase digestion procedures to expose target RNAs and epitopes using the Manual Slide Preparation for RNA Assays User Manual (specifics can be found in NanoString manual MAN-10184-02). HIER was carried out at 100°C for 15min in Leica ER1 buffer and proteinase digestion used a cocktail of Proteinase K (5 μg/ml; ThermoFisher) and Protease A (NanoString). The samples were incubated with the proteinase digestion cocktail at RT for 30 min. The samples were then rinsed with fresh 1x PBS and incubated with 1.5:1000 diluted fiducials (Bangs Laboratory) in 2x saline sodium citrate, 0.001% Tween 20 (2x SSCT) solution for 5 min at RT. Excess fiducials were removed by rinsing with 1x PBS, followed by fixation with 10% NBF for 1 min at RT. Fixed samples were rinsed with Tris-glycine buffer (0.1M glycine, 0.1M Tris-base in DEPC H2O) and 1X PBS twice each for 5 min before being blocked using 100 mM N-succinimidyl acetate (NHS-acetate, ThermoFisher) in NHS-acetate buffer (0.1M NaP, 0.1% Tween pH 8 in DEPC H2O). Blocking lasted 15 min at RT. Prepared samples were rinsed twice with 2x saline sodium citrate (SSC) for 5 min each and then Adhesive Incubation Frames (NanoString) were placed on the slides to cover the samples.

value: Liver metastasis

tag: age

value: F

value: No treatment


title: La_3

value: WT


title: Slide 2_Pt-13_Pri

source_name: Primary PDAC

value: Primary PDAC

value: FOLFIRINOX, Capecitabine, gemcitabine/abraxane/ denosumab


title: Slide_2_Pt-5_LiM-1


title: BLN, BoHV1, S7

value: B


title: Slide_2_Pt-5_Pri


title: Ha_1


title: BLN,control, S6


title: 64_24h_WM15_Con

value: Fibroblast


title: BLN,control, S5


title: BLN, BoHV1, S10


title: 62_24h_WM14_10Gy

value: wildtype


title: La_2


title: 63_24h_WM14_20Gy


title: Lb_2


title: Hb_10


title: Slide_1_Pt-8_LiM-2

description:
Slide 1
CosMx_SCT_data.csv.gz
Meta.data_CoxMx.csv.gz
Run5961_PDAC_Slide1_exprMat_file.csv.gz
Run5961_PDAC_Slide1_fov_positions_file.csv.gz
Run5961_PDAC_Slide1_metadata_file.csv.gz
Run5961_PDAC_Slide1-polygons.csv.gz
Run5961_PDAC_Slide1_tx_file.csv.gz
Run5961_PDAC_Slide1_CellLabels.tar.gz
Run5961_PDAC_Slide1_CellOverlay.tar.gz
Run5961_PDAC_Slide1_CompartmentLabels.tar.gz

value: M


title: Lb_1


title: Slide_1_Pt-8_LiM-1


title: BLN,control, S4

value: control

value: Holstein-Friesian


title: Slide_1_Pt-8_Pri


title: BLN,control, S3

value: A


title: 60_24h_WM11_20Gy


title: La_8


title: BLN,control, S2


title: 28_12d_Efus8_20Gy

organism: Eptesicus fuscus


title: Ha_9


title: BLN,control, S1


title: Artibeus jamaicensis2

description: Library name: ArtSkin2

value: Skin


title: 61_24h_WM14_Con


title: Lb_8

value: sperm


title: human iPSC-derived Sox9- cells , biol rep 3

description:
column name is "Sox9neg biol rep 3" in processed data
gs_hSCL.csv

data_processing:
Quanlity filtering using fastp (version 0.20.0) with default parameters.
Trimmed sequence reads were mapped to hg19 using HISAT2 software (version 2.2.1)
Read counts for each gene were based on sense-strand data obtained using the featureCounts (version 2.0.1) software from the Subread package
Assembly: hg19
Supplementary files format and content: comma-separated values files including read counts for each Sample

extract_protocol:
RNA was harvested using TRI Reagent (Sigma).
RNA libraries for RNA-seq were prepared using NEBNext Ultra RNA Library Prep Kit following manufacturer's protocols.

tag: protocol

value: Both the human iPSC-derived Sox9+ sclerotome-like cells and human iPSC-derived Sox9- cells were obtained by differentiating the day-0 UC01 iPSCs (Sox9-IRES-tdTomato) to day-6 cells stepwise and sorting out the Sox9+ and Sox9- cells , respectively. All differentiation was conducted in chemically defined medium with insulin (CDMi) supplemented with indicated proteins and chemical molecules. To prepare CDMi, 50% IMDM (Gibco, Cat# 31980-097) and 50% F12 (Gibco, Cat# 31765-092) were mixed with 1% v/v concentrated lipids (Gibco, Cat# 11905-031), 450 μM monothioglycerol (Sigma, Cat# M6145), 0.7 μg/mL insulin (Solarbio, Cat# I8830), 15 μg/ml transferrin (Sigma, Cat# T0665), 1% v/v penicillin/streptomycin (Gibco, Cat# T0665), and 1 mg/mL polyvinyl alcohol (Sigma, Cat# P8136-250G). The Polyvinyl alcohol was first solved at 25 mg/ml in ultrapure water (UP H2O) by heating to 85 ℃ and then cooled on ice before addition to the medium. To start differentiation, day-0 UC01 iPSCs (Sox9-IRES-tdTomato) were gently washed with the wash medium (WM) containing 3 mg/ml bovine serum albumin (BSA) in DMEM/F12 and induced to primitive streak (PS) by CDMi containing Activin A (30 ng/ml, Solarbio, Cat#P00101), CHIR99021 (7 μM, MCE, Cat#HY-10182) and bFGF (20 ng/ml, Peprotech, Cat#100-18B) for 24 hours; the next day, day-1 PS was washed with WM and differentiated to presomitic mesoderm (PSM) by CDMi containing SB431542 (20 μM, MCE, Cat#HY-10431), CHIR99021 (3 μΜ), LDN193189 (125 nM, MCE, Cat#HY-12071A), bFGF (40 ng/ml) for another 24 hours; subsequently, the day-2 PSM was with WM and differentiated to somite (SM) in CDMi containing XAV939 (0.5 μM) and PD0325901 (1 μM, MCE, Cat#HY-10254) for 24 hours; the following day, the SM was washed with WM, and was induced to day-6 cells by CDMi containing SAG (200 nM) and LDN193189 (600nM) for 72 hours with medium changed every day.

value: induced pluripotent stem cell-derived cells

value: sclerotome differentiation


title: Hb_9


title: Artibeus jamaicensis1

description: Library name: ArtSkin1


title: human iPSC-derived Sox9- cells , biol rep 2

description:
column name is "Sox9neg biol rep 2" in processed data
gs_hSCL.csv


title: 25_Efus8_Con_P


title: 43_12d_ES162_10Gy


title: human iPSC-derived Sox9+ sclerotome-like cells , biol rep 3

description:
column name is "Sox9posi biol rep 3" in processed data
gs_hSCL.csv


title: La_10


title: human iPSC-derived Sox9- cells , biol rep 1

description:
column name is "Sox9neg biol rep 1" in processed data
gs_hSCL.csv


title: 24_24h_Efus_20Gy


title: Hb_8


title: 44_12d_ES162_20Gy


title: La_9


title: 42_12d_ES162_Con_F


title: Ha_10


title: 23_24h_Efus_10Gy


title: human iPSC-derived Sox9+ sclerotome-like cells , biol rep 2

description:
column name is "Sox9posi biol rep 2" in processed data
gs_hSCL.csv


title: 24_12d_Efus6_20Gy


title: 58_24h_WM11_Con


title: Artibeus jamaicensis3

description: Library name: ArtSkin3


title: 27_12d_Efus8_10Gy


title: Ha_2


title: 41_ES162_Con_P


title: 23_12d_Efus6_10Gy


title: 40_12d_ES161_20Gy


title: 26_12d_Efus8_Con_F


title: undifferentiated human iPSCs, biol rep 3

description:
column name is "hiPS biol rep 3" in processed data
gs_hiPSC.csv

value: Cells were routinely maintained and propagated in Pluripotency Growth Master 1 (PGM1, Cellapy, Cat# CA1007500) medium on matrigel-coated plates. The matrigel working solution was prepared in DMEM/F12 (Gibco, Cat# C11330500) at 1 mg/12 ml, and the coating was conducted at 37 ℃ for at least 1 hour. Cell splitting was carried out at 1:5 with 0.5 mM EDTA (ThermoFisher, Cat# AM9260G) in PBS when the confluency reached 80%.

value: induced pluripotent stem cells

value: culture with Pluripotency Growth Master 1


title: 22_24h_Efus_Con


title: 39_12d_ES161_10Gy


title: undifferentiated human iPSCs, biol rep 2

description:
column name is "hiPS biol rep 2" in processed data
gs_hiPSC.csv

value: WT


title: 38_12d_ES161_Con_F


title: 36_24h_ES162_20Gy


title: 21_24h_Efus8_20Gy


title: 37_ES161_Con_P


title: FBS, rep 5

city: Dallas

first: Kara

last: Poole

state: TX

address: 5323 Harry Hines Blvd

department: Ophthalmology

institute: UT Southwestern Medical Center

description: Library name: SerumP9

data_processing:
Standard bioinformatics analyses were conducted by Novogene Co.
Assembly: OryCun2
Supplementary files format and content: raw counts for each sample
Supplementary files format and content: FPKM values for each sample

organism: Oryctolagus cuniculus

source_name: keratocyte

growth_protocol: Primary keratocytes were maintained in defined serum-free media in a humidified incubator maintained at 5% CO2 and 37C.

extract_protocol:
Total RNA was extracted using the BioRad Aurum Total RNA Mini Kit according to the manufacturer's instructions, quantified by NanoDrop, and stored at -80°C for later shipping to Novogene for bulk RNA-seq.
Preparation of cDNA libraries was conducted by Novogene Co.

treatment_protocol:
First passage keratocytes were plated on collagen coated glass substrates in SF media (day 0).
Cells were cultured for a total of 6 days, with an initial media swap on day 1 (after the cells were able to attach for 24 hours), and a second media swap on day 3.
Media changes for our SF (keratocyte) condition were SF on day 1 and day 3.
Media changes for our FBS (fibroblast) condition were SF on day 1 and DMEM containing 10% FBS on day 3.
Media changes for our TGF-β1 (myofibroblast) condition were SF media supplemented with 5ng/mL TGF-β1 on day 1 and day 3.

value: keratocyte

value: 10% Fetal Bovine Serum


title: undifferentiated human iPSCs, biol rep 1

description:
column name is "hiPS biol rep 1" in processed data
gs_hiPSC.csv


title: FBS, rep 6

description: Library name: SerumP10


title: FBS, rep 4

description: Library name: SerumP8


title: FBS, rep 3

description: Library name: SerumP7


title: human iPSC-derived Sox9+ sclerotome-like cells , biol rep 1

description:
column name is "Sox9posi biol rep 1" in processed data
gs_hSCL.csv


title: 20_12d_Efus5_20Gy

value: Fibroblast


title: PBS treated rep5

city: Wuhan

first: Minghe

state: Hubei

address: Donghu Road

institute: WuHan University

data_processing:
Paired-end reads were harvested from sequencer, and were quality controlled by Q30.
After 3’ adaptor-trimming and low quality reads removing by cutadapt software (v4.6), the high quality clean reads were aligned to the reference genome with STAR software (v2.7.11b)
Raw read counts were extracted from the second column of the STAR output file 'ReadsPerGene.out.tab', which contained the unstranded RNA-seq read counts.
Assembly: Mus_musculus.GRCm39
Supplementary files format and content: Tab-delimited text file includes raw counts for each Sample

source_name: Hepa1-6 orthotopic tumor

extract_protocol:
Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA), and subjected to ribosomal RNA (rRNA) removal using GenSeq® rRNA Removal Kit (GenSeq, Inc., Shanghai, China).
The rRNA-depleted samples were used for library construction with GenSeq® Directional RNA Library Prep Kit (GenSeq, Inc., Shanghai, China) by following manufacturer’s recommendations.

value: Hepa1-6 orthotopic tumor

value: PBS


title: FBS, rep 8

description: Library name: SerumP12


title: 35_24h_ES162_10Gy

value: wildtype


title: FBS, rep 2

description: Library name: SerumP6


title: PBS treated rep4


title: 36_12d_ES160_20Gy


title: PBS treated rep2


title: 35_12d_ES160_10Gy


title: PBS treated rep3


title: FBS, rep 7

description: Library name: SerumP11


title: 19_24h_Efus8_Con


title: 22_12d_Efus6_Con_F


title: 19_12d_Efus5_10Gy


title: PBS treated rep1


title: 34_24h_ES162_Con


title: FBS, rep 1

description: Library name: SerumP5


title: 21_Efus6_Con_P


title: TGF-β1, rep 8

description: Library name: TGFB12

value: 5ng/mL TGF-β1


title: 20_24h_Efus8_10Gy


title: CAR-MEV treated rep5

value: CAR-MEV


title: 34_12d_ES160_Con_F


title: 18__24h_Efus6_20Gy


title: CAR-MEV treated rep4


title: TGF-β1, rep 7

description: Library name: TGFB11


title: 33_ES160_Con_P


title: CAR-MEV treated rep3


title: 17_Efus5_Con_P


title: 33_24h_ES161_20Gy


title: 17__24h_Efus6_10Gy


title: TGF-β1, rep 4

description: Library name: TGFB8


title: CAR-MEV treated rep2


title: 32_24h_ES161_10Gy


title: CAR-MEV treated rep1


title: TGF-β1, rep 3

description: Library name: TGFB7


title: 32_12d_ES130_20Gy


title: 16_24h_Efus6_Con


title: 15_24h_Efus5_20Gy


title: 31_24h_ES161_Con


title: VCaP ERG shRNA#2

city: Bloomington

first: Stephanie

last: Metcalf

state: IN

address: 1001 E Third St

institute: Indiana University Bloomington

description: Library name: ERG shRNA#2

data_processing:
Alignment of reads was performed using Bowtie2 for the reference genome hg19
Blacklist region was removed via bedtools
Duplicates were removed via Picard-2.14.0
MACS2 was used to call peaks with a p-value of 0.001
Assembly: Assembly: hg19
Supplementary files format and content: Supplementary files format and content: xlsx, broad peak

source_name: VCaP

growth_protocol: Cells were grown in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin

extract_protocol:
Cells were fixed with 1% v/v formaldehyde for 15 minutes and quenched with 2M Glycine for 5 minutes. Cells were then lysed and sonicated using a Diagenode Biorupter Pico.
ChIP-Seq libraries were generated by pooling at least three ChIPs per condition and sonicating to approximately 150bps. 10ng of IP was used as starting material and fragment end repair was done using Klenow DNA polymerase, T4 DNA ploymerase, and T4 DNA Ligase followed by QIAQuick PCR Purification. TruSeq adaptors were ligated via T4 DNA Ligase after which libraries were amplified through 18 cycles and products were size selected on a 2% agarose gel and AMPure XP bead cleanup.

value: VCaP


title: Part2

city: Durham

first: Seth

last: Zimmerman

address: 308 Research Dr.

institute: Duke University

description:
Library name: 2
Data for second two cell lines

data_processing:
Sequencing reads were de-multiplexed using fastx_barcode_splitter (v0.0.13) to identify inline barcodes and aligned to the reference library using bowtie (v1.0.0), allowing for 0 mismatches.  Sample sgRNA read counts were first normalized to total reads/sample.  sgRNA fold-change values were then calculated by comparing in vitro and in vivo sample sgRNA counts to their paired initial sample sgRNA count.  Gene target enrichment scores were then calculated by averaging the 5 sgRNA fold-change values within a sample.  Average sample enrichment fold-changes distributions were normalized by first zeroing each sample on the average enrichment of non-targeting control sgRNAs and then calculating a z-score.  To compare in vitro enrichment to in vivo enrichment, replicate sample z-scores were averaged and compared by T-test.  Resultant p-values were adjusted by calculating false discovery rates.
Supplementary files format and content: xlsx file contains raw sgRNA counts per sample
Library strategy: CRISPR Screen

source_name: Cell Line

growth_protocol: Lentivirus production-HEK293T cells were cultured to 70-80 % confluency and transfected with lentiviral packaging system at the ratio of 1:1:2 for psPAX2:pVSVg:lentiCRISPR v2 Blast using Fugene6 (Promega) transfection reagent.  After 48 to 72 hours, media was harvested from cells, centrifuged at 400 RCF for 3 minutes and filtered using a 45 µm pore filter (VWR) to remove cell debris.  Virus was either used immediately or snap frozen for future use.  Infection - For each replicate, 1.2 x 107 cells were infected at an MOI of 0.3 to ensure that most cells were infected by a single lentiviral particle.  Each infection was performed across 6 wells of a 6-well plate in 1 ml of the appropriate media containing virus at a predetermined concentration to reach the targeted MOI in the presence of 8 µg/ml polybrene (MilliporeSigma) and spun at 800 x g for 1 hour.  After 6 hours of culture at 37 °C, cells were washed in 1x Phosphate Buffered Saline (PBS; Sodium Chloride, 0.137 M; Potassium Chloride, 0.0027 M; Sodium Phosphate Dibasic, 0.01 M; Potassium Phosphate Monobasic, 0.0018 M), and the media was replaced.  18 hours later, each infection was pooled into a 15 cm culture dish and selected with 5 µg/ml blasticidin (Thermo Fisher Scientific), except for PANC-1 cells, which were selected with 20 µg/ml blasticidin.  Cells were continuously passaged and selected for 1 week after initial infection at which point 1 x 106 cells were pelleted and snap frozen as the initial sample.  1 x 106 cells were continuously passaged for 2 weeks for in vitro samples.  5 x 106 cells were transplanted into 1 male and 1 female Fox Chase SCID/Beige mouse (details below) (Charles River).   In vitro samples were pelleted and snap frozen after 2 weeks of growth. Xenograft transplant - For each transplant, 5 x 106 cells were resuspended in 200 µl sterile PBS and kept on ice until injection (less than 2 hours).  Mice were anesthetized in a bell jar with diffuse isoflurane, their injection site sterilized with iodine, and subcutaneously injected with 200 µl cell suspension in the right flank using a 30 G needle (BD).  Mice were monitored hourly for 24 hours, then afterward, three times a week for tumor growth and morbidity and moribundity.  Mice were humanely euthanized by CO2 asphyxiation followed by decapitation upon a tumor size of ~1 cm3 or upon signs of moribundity, and tumors removed and snap frozen during necropsy.

extract_protocol:
Cell samples were lysed in 1 ml DNAzol by vigorous pipetting according to the manufacture’s protocol (Molecular Research Center inc.).  DNA was precipitated in 0.5 ml 200 proof ethanol (VWR).  30 to 50 mg of tumor samples were lysed in 5 ml DNAzol by a motorized tissue homogenizer (Kinematica AG PT1200), centrifuged at 3,200 g for 15 minutes at 4 °C, and transferred to a new tube.  gDNA was then precipitated by 2.5 ml 200 proof ethanol.  Precipitated DNA was pelleted by centrifugation at 20,000 g, washed twice in 75% ethanol.  gDNA was solubilized in 0.5 ml or 1.0 ml of water for cell and tumor samples, respectively
To reduce PCR bias, amplification of sgRNA sequences from each sample gDNA was performed in triplicate and pooled at each step.  First round of PCR amplification was performed using 50 to 100 µg gDNA, Hotstart Taq (Takara), and 1 µM CRISPR F1 and CRISPR R1 primers (IDT). CRISPR F1 - AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG CRISPR R1 - TCTACTATTCTTTCCCCTGCACTGTtgtgggcgatgtgcgctctg Thermal cycles consisted of 95 °C for 5 minutes, followed by 18 cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 30 seconds, and a final cycle of 72 °C for 5 minutes.  Amplicons from the same samples were pooled and a second round of PCR amplification was performed to add sample barcode and Illumina adaptor sequences using 20 µl of PCR1 and 0.5 µM  CRISPR R2 and barcoded forward primers. See Supplemental Data 2 for barcode information. CRISPR R2 - CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtctactattctttcccctgcactgt Thermal cycles consisted of 95 °C for 5 minutes, followed by 20 cycles of 95 °C for 15 seconds, 58 °C for 30 seconds, 72 °C for 30 seconds, and a final cycle of 72 °C for 5 minutes.  Amplicons from the second round of PCR were pooled at equal concentrations, were purified using Ampure XP Beads according to the manufacture’s protocol (Beckman Coulter).  Purified amplicons were subjected to Illumina NexSeq 500 75bp single end sequencing.

value: Cell Line

value: MIAPACA, PANC1

value: PDAC

value: PtdIns Targeted CRISPR Library


title: 14_24h_Efus5_10Gy

city: Rochester

first: Fathima

last: Athar

address: 500 Joseph C Wilson Blvd.

institute: University of Rochester


title: TGF-β1, rep 1

description: Library name: TGFB5


title: 31_12d_ES130_10Gy


title: 13_24h_Efus5_Con


title: Part1

description:
Library name: 1
Data for first 3 cell lines

value: BXPC3, CFPAC1, HPAFII


title: 18_12d_Efus5_Con_F


title: TGF-β1, rep 6

description: Library name: TGFB10


title: 30_24h_ES160_20Gy


title: COV362 cells, chemo-resistant, CRISPR screen, PRMT5 high, rep4

city: Chicago

first: Harun

last: Ozturk

state: Illinois

address: 303. E. Superior, Lurie cancer center

institute: Northwestern University

description: Library name: Sample8

data_processing:
*library strategy: targeted PCR; CRISPR screen
Data were quality checked using FASTQC.
Reads were aligned to a fasta file containing sgRNAs sequences included in the library using the CB2 package.
Data were normalized and analyzed using the CB2 package.
Assembly: hg38
Supplementary files format and content: CSV files containing sgRNA and gene statistics.
Supplementary files format and content: The fasta file containing sgRNA sequences the library.

source_name: ovarian

growth_protocol: COV362 cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum, 1% Pen-Strep, and 1X GlutaMAX. The cells were maintained in a humidified incubator at 37 °C.

extract_protocol:
Cells were lysed for 24 hours at 55 °C in the presence of Proteinase K and RNase A. The next day, DNA was extracted using phenol-chloroform extraction.
Libraries were constructed using the custom oligos.

value: ovarian

value: high-grade sereous ovarian cancer cell line


title: 30_12d_ES130_Con_F


title: Serum Free, rep 8

description: Library name: SFree12

value: serum free


title: ∆rlmF∆rlmJ E. coli RNA biological replicates 1 and 2 combined

description:
WT_vs_rlm_ELIGOS_results.txt
WT_vs_rlm_cheui_differential_m5C_sites.txt
WT_vs_rlm_cheui_differential_m6A_sites.txt
rlm.cheui.m5C.site.level.predictions.txt
rlm.cheui.m6A.site.level.predictions.txt
WT_vs_rlm_Nanocompore_results.tsv
rlm_Salmon_gene_counts.txt

organism: Escherichia coli str. K-12 substr. MG1655

extract_protocol:
RNA was isolated using the PCI protocol (Sambrook & Russell, 2006) with some modifications (Petrov et al., 2022).
Nanopore MinION sequencing libraries were prepared with tRNA/rRNA-depleted polyadenylated RNA following the manufacturer’s protocol (ONT #SQK-RNA002).
Direct RNA sequencing

value: tRNA/rRNA-depletion and polyadenylation


title: Serum Free, rep 7

description: Library name: SFree11

value: keratocyte


title: TGF-β1, rep 5

description: Library name: TGFB9


title: COV362 cells, chemo-resistant, CRISPR screen, PRMT5 high, rep3

description: Library name: Sample7

value: WT


title: In vitro transcribed WT E. coli RNA replicates 1 and 2 combined

description:
WT_vs_IVT_ELIGOS_results.txt
WT_vs_IVT_cheui_differential_m5C_sites.txt
WT_vs_IVT_cheui_differential_m6A_sites.txt
IVT.cheui.m5C.site.level.predictions.txt
IVT.cheui.m6A.site.level.predictions.txt
WT_vs_IVT_Nanocompore_results.tsv
IVT_Salmon_gene_counts.txt

source_name: in vitro transcribed RNA


title: 29_ES130_Con_P

value: Fibroblast


title: 29_24h_ES160_10Gy


title: RNA from WT E. coli (grown at 37 °C), biological replicates 1 and 2 combined

description:
WT_vs_IVT_ELIGOS_results.txt
WT_vs_rlm_ELIGOS_results.txt
WT_vs_IVT_cheui_differential_m5C_sites.txt
WT_vs_rlm_cheui_differential_m5C_sites.txt
WT_vs_IVT_cheui_differential_m6A_sites.txt
WT_vs_rlm_cheui_differential_m6A_sites.txt
WT.cheui.m5C.site.level.predictions.txt
WT.cheui.m6A.site.level.predictions.txt
WT_vs_IVT_Nanocompore_results.tsv
WT_vs_rlm_Nanocompore_results.tsv
WT_Salmon_gene_counts.txt

value: Growth at 37 °C; tRNA/rRNA-depletion and polyadenylation


title: COV362 cells, chemo-resistant, CRISPR screen, PRMT5 high, rep2

description: Library name: Sample6


title: Eptesicus fuscus4

description: Library name: EFusSkin8

value: Skin

value: wildtype


title: TGF-β1, rep 2

description: Library name: TGFB6

value: 5ng/mL TGF-β1


title: COV362 cells, chemo-resistant, CRISPR screen, PRMT5 high, rep1

description: Library name: Sample5


title: Serum Free, rep 6

description: Library name: SFree10


title: Serum Free, rep 5

description: Library name: SFree9


title: COV362 cells, chemo-resistant, CRISPR screen, PRMT5 low, rep4

description: Library name: Sample4


title: 28_24h_ES160_Con


title: Serum Free, rep 4

description: Library name: SFree8


title: Eptesicus fuscus3

description: Library name: EFusSkin7


title: Serum Free, rep 3

description: Library name: SFree7


title: Eptesicus fuscus2

description: Library name: EFusSkin6


title: COV362 cells, chemo-resistant, CRISPR screen, PRMT5 low, rep3

description: Library name: Sample3


title: 27_24h_ES130_20Gy


title: Eptesicus fuscus1

description: Library name: EFusSkin5


title: 26_24h_ES130_10Gy


title: Serum Free, rep 2

description: Library name: SFree6


title: Serum Free, rep 1

description: Library name: SFree5


title: COV362 cells, chemo-resistant, CRISPR screen, PRMT5 low, rep1

description: Library name: Sample1


title: 25_24h_ES130_Con


title: COV362 cells, chemo-resistant, CRISPR screen, PRMT5 low, rep2

description: Library name: Sample2