title: EZH2_MinusTag_CLIP_Rep1


title: EZH2_PlusTag_CLIP_Rep2

status: Public on Feb 20 2024

type: SRA

city: Pasadena

first: Jimmy

middle: K

last: Guo

address: 1200 E California Blvd

country: USA

institute: Caltech

library_source: transcriptomic

data_processing:
Paired-end RNA sequencing reads were trimmed to remove adaptor sequences using Trim Galore! v0.6.2 and assessed with FastQC v0.11.8. Read pairs were then aligned to a combined genome reference containing the sequences of repetitive and structural RNAs (ribosomal RNAs, snRNAs, snoRNAs, 45S pre-rRNAs, tRNAs) using Bowtie2. The remaining reads were then aligned to a combined genome reference containing the mouse (mm10) and human (hg38) genomes using STAR aligner181. PCR duplicates were removed using the Picard MarkDuplicates function. For mixing experiments, only reads that mapped uniquely in the genome and unambiguously to the human or mouse genomes were kept for further analysis. For experiments done in a single species, the appropriate reference genome and alignments were used (mm10 for mouse and hg38 for human). These reads were then used for downstream analysis.  For computing enrichments, all human (hg38) and mouse (mm10) annotated genes (RefSeq, downloaded from UCSC GRCh38 and GRCm38, respectively) were used as a reference set except for the genes encoding the transfected proteins. We treated exonic regions and intronic regions of each annotated gene as separate reference genes for computing enrichment. For each reference gene, we enumerated 100 nucleotide windows that span across the gene; for each window, we calculated: (i) the number of reads overlapping the window in the protein elution sample (e.g., CLIP or CLAP) and (ii) the maximum of either the number of observed reads over the window or the median read count over all windows within the gene in the input sample. Because all windows overlapping a gene should have the same expression level in the input sample, this approach provides a conservative estimation of the input coverage because it prevents windows from being scored as enriched if the input values over a given window are artificially low due to stochastic fluctuation, while at the same time accounting for any non-random issues that lead to increases in read counts over a given window (i.e., alignment artifacts leading to non-random assignment or pileups).  To directly compare the number of reads within each window between sample and input, we normalized each window count by the total number of reads sequenced and the overall complexity within each sample. For example, if one sample was sequenced twice as deeply as another, then we would expect to observe – on average – twice as many reads over a given window for that sample. To account for the differences in the number of mapped (aligned) reads between samples, we scaled the total number of sequenced reads by the proportion of aligned reads within each sample.  For each window, we computed enrichment by dividing the normalized sample counts by the normalized input counts. Nominal p-values were calculated for each window using a binomial test where k (number of successes) is defined as the number of reads in the protein elution samples within the window, N (number of trials) is the sum of the number of reads in the protein elution and input samples, and p (probability of success) is the expected number of reads in the elution sample divided by the sum of the expected number of reads per window in elution and input samples. (The expected number of reads is defined as the total number of reads scaled by the proportion of aligned reads within each sample). For plotting and reporting purposes, we considered all regions with a nominal binomial p-value < 10-6 as significant. However, the overall results reported are robust to the precise p-value cutoff used.
Assembly: mm10, hg38
Supplementary files format and content: tab-delimited text file includes gene window enrichment calculations
Supplementary files format and content: bedgraph

molecule: total RNA

organism:
Homo sapiens
Mus musculus

source_name: HEK239T, PSM33

extract_protocol:
Cells were washed once with PBS and then crosslinked on ice using 0.25 J cm-2 (UV 2.5k) of UV at 254 nm in a Spectrolinker UV Crosslinker. Cells were then scraped from culture dishes, washed once with PBS, pelleted by centrifugation at 1500g for 4 min, and flash-frozen in liquid nitrogen for storage at -80 °C.
Cells were lysed in 1 mL lysis buffer (50mM Tris pH 7.5, 100mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1X Promega PIC). For CLIP samples, RNA was digested with Ambion RNase I (1:3,000 dilution) to achieve a size range of 100-500 nucleotides in length. We used a fixed RNase condition for all experiments to enable comparison between proteins. Lysates were precleared by mixing with Protein G beads (Dynabeads) for 30 minutes at 4°C. Target proteins were immunoprecipitated overnight at 4°C from 10 million cells with 5 μg of antibody coupled to 40 μL of Protein G beads in 100 μL lysis buffer. Antibodies were pre-coupled to beads for 1 hour at room temperature with mixing, followed by 3 washes with lysis buffer to remove unbound protein. After immunoprecipitation, beads were washed four times with high salt wash buffer (50 mM TrisHCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) and four times with wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20). RNA and protein were eluted by incubating in NLS elution buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 2% N-lauroylsacrosine, 2.5 mM TCEP) supplemented with 100 mM DTT at 50°C for 20 minutes. Samples were then run through an SDS-PAGE gel and transferred to a nitrocellulose membrane using the iBlot transfer system. The region 70 kDa above the molecular weight of the protein of interest was isolated and treated with Proteinase K (NEB), followed by purification with RNA Clean & Concentrate-5 (Zymo). Anti-V5 antibody (Bethyl, A190-120A) was used for all CLIP experiments.  For CLAP samples, cells were resuspended in 1 mL of cold lysis buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with 1X Protease Inhibitor Cocktail (Promega), 200 U of RiboLock (NEB), 20 U of TURBO DNase (Ambion), and 1X manganese/calcium mix (0.5mM CaCl2, 2.5 mM MnCl2). Samples were incubated on ice for 10 minutes and then at 37°C for 10 minutes at 1150 rpm shaking on a ThermoMixer (Eppendorf). Lysates were cleared by centrifugation at 15000g for 2 minutes and the supernatant was collected for capture to HaloLink Resin (Promega). For each CLAP capture, 200 μL of 25% HaloLink Resin (50 μL of HaloLink Resin total) was used per 10 million cells. Resin was washed three times with 2 mL of 1X TBS (50 mM Tris pH 7.5, 150 mM NaCl) and incubated in 1X Blocking Buffer (50 mM HEPES, pH 7.5, 10 μg/mL Random 9-mer, 100 μg/mL BSA) for 20 minutes at room temperature with continuous rotation. After the incubation, resin was washed three times with 1X TBS. Cleared lysate was mixed with 50 μL of HaloLink Resin and incubated at 4°C overnight with continuous rotation. The captured protein bound to resin was washed three times with lysis buffer at room temperature and then three times at 90°C for 3 minutes while shaking at 1200 rpm with each of the following buffers: 1X ProK/NLS buffer (50 mM HEPES, pH 7.5, 2% NLS, 10 mM EDTA, 0.1% NP-40, 10 mM DTT), high salt buffer (50 mM HEPES, pH 7.5, 10 mM EDTA, 0.1% NP-40, 1M NaCl), 8M urea buffer (50 mM HEPES, pH 7.5, 10 mM EDTA, 0.1% NP-40, 8 M Urea), and Tween buffer (50 mM HEPES, pH 7.5, 0.1% Tween 20, 10 mM EDTA). After the last wash, samples were centrifuged at 7500g for 30 seconds and supernatant was discarded. For elution, HaloLink Resin was resuspended in 100 μL of ProK/NLS buffer + 10 μL of Proteinase K (NEB) and incubated at 50°C for 20 minutes while shaking at 1200 rpm. Elutions were then transferred to microspin cups (Pierce, Thermo Fisher), centrifuged at 2000g for 30 seconds, and purified with RNA Clean and Concentrate-5 (Zymo).

tag: cell line

value: HEK239T, PSM33

tag: cell type

value: Human embryonic kidney cells, Mouse embryonic stem cells


title: PTBP1_PlusTag_CLAP_Rep1

data_processing:
Paired-end RNA sequencing reads were trimmed to remove adaptor sequences using Trim Galore! v0.6.2 and assessed with FastQC v0.11.8. Read pairs were then aligned to a combined genome reference containing the sequences of repetitive and structural RNAs (ribosomal RNAs, snRNAs, snoRNAs, 45S pre-rRNAs, tRNAs) using Bowtie2. The remaining reads were then aligned to a combined genome reference containing the mouse (mm10) and human (hg38) genomes using STAR aligner181. PCR duplicates were removed using the Picard MarkDuplicates function. For mixing experiments, only reads that mapped uniquely in the genome and unambiguously to the human or mouse genomes were kept for further analysis. For experiments done in a single species, the appropriate reference genome and alignments were used (mm10 for mouse and hg38 for human). These reads were then used for downstream analysis.  For computing enrichments, all human (hg38) and mouse (mm10) annotated genes (RefSeq, downloaded from UCSC GRCh38 and GRCm38, respectively) were used as a reference set except for the genes encoding the transfected proteins. We treated exonic regions and intronic regions of each annotated gene as separate reference genes for computing enrichment. For each reference gene, we enumerated 100 nucleotide windows that span across the gene; for each window, we calculated: (i) the number of reads overlapping the window in the protein elution sample (e.g., CLIP or CLAP) and (ii) the maximum of either the number of observed reads over the window or the median read count over all windows within the gene in the input sample. Because all windows overlapping a gene should have the same expression level in the input sample, this approach provides a conservative estimation of the input coverage because it prevents windows from being scored as enriched if the input values over a given window are artificially low due to stochastic fluctuation, while at the same time accounting for any non-random issues that lead to increases in read counts over a given window (i.e., alignment artifacts leading to non-random assignment or pileups).  To directly compare the number of reads within each window between sample and input, we normalized each window count by the total number of reads sequenced and the overall complexity within each sample. For example, if one sample was sequenced twice as deeply as another, then we would expect to observe – on average – twice as many reads over a given window for that sample. To account for the differences in the number of mapped (aligned) reads between samples, we scaled the total number of sequenced reads by the proportion of aligned reads within each sample.  For each window, we computed enrichment by dividing the normalized sample counts by the normalized input counts. Nominal p-values were calculated for each window using a binomial test where k (number of successes) is defined as the number of reads in the protein elution samples within the window, N (number of trials) is the sum of the number of reads in the protein elution and input samples, and p (probability of success) is the expected number of reads in the elution sample divided by the sum of the expected number of reads per window in elution and input samples. (The expected number of reads is defined as the total number of reads scaled by the proportion of aligned reads within each sample). For plotting and reporting purposes, we considered all regions with a nominal binomial p-value < 10-6 as significant. However, the overall results reported are robust to the precise p-value cutoff used.
Assembly: mm10, hg38
Supplementary files format and content: tab-delimited text file includes gene window enrichment calculations
Supplementary files format and content: bedgraph
Library strategy: CLAP-seq


title: EZH2_MinusTag_CLAP_Rep1


title: EED_PlusTag_Input


title: PTBP1_PlusTag_CLAP_Rep2


title: EZH2_MinusTag_Input_Rep1


title: EZH2_MinusTag_CLIP_Rep2


title: EZH2_MinusTag_CLAP_Rep2


title: EED_PlusTag_CLIP_Rep1


title: EED_PlusTag_CLAP_Rep2


title: EZH2_MinusTag_Input_Rep2


title: EED_MinusTag_Input


title: EED_PlusTag_CLIP_Rep2


title: EED_MinusTag_CLAP_Rep2


title: EED_MinusTag_CLIP_Rep1


title: EZH2_PlusTag_CLAP_Rep1


title: Brain 7 - diencephalon, scRNAseq

city: Berlin

first: Nina

last: Mitic

address: Hannoversche Strasse 28

department: Berlin Institute for Medical Systems Biology

country: Germany

institute: Max-Delbrück-Center for Molecular Medicine

description: scRNA-seq library of adult brain for transcriptomic profiling

library_source: transcriptomic single cell

data_processing:
The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect, the gene expression matrices were processed with the SoupX tool (Young & Behjati, 2020) for removing ambient RNA contamination.
A subset of libraries was also mapped with Velocyto (v0.17.17) (La Manno et al, 2018) to create count matrices for unspliced and spliced molecules.
The targeted lineage tracing libraries were aligned using bwa mem3 (v.0.7.12) to individual references of endogenous genes used in the lineage tracing experiment (actb1, actb2, cfl1, cirbpb, rpl39 and ube2e1). The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors.
For custom code and further downstream analysis, see: https://github.com/nimitic/radial-glia.
Assembly: GRCz11
Supplementary files format and content: Tab-separated barcode files and matrix files along with the tab-separated gene file (supplementary file) are the output of CellRanger mapping, further processed by the SoupX package for ambient RNA removal.
Supplementary files format and content: Loom files are the output of velocyto mapping (distinguishing spliced and unspliced mRNAs).
Supplementary files format and content: filtered_scars.csv files contain the output of a custom scar filtering pipeline.

molecule: polyA RNA

organism: Danio rerio

source_name: adult brain

extract_protocol:
Adult zebrafish brain tissue was extracted, optionally dissected to select a specific region of interest (telencephalon, diencephalon, mesencephalon or rhombencephalon) and then dissociated with a trypsin protocol. Briefly, the samples were incubated in 750 µl of HBSS-glucose solution and dissocated with 15 µl of 0.05% trypsin-EDTA (Gibco) for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA-EBSS-HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter, washed with cold HBSS and resuspended in 100-200 µl HBSS with 0.05% BSA, then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM-seq samples, a papain dissociation protocol (Worthington kit, according to manufecturer's instructions) was used followed by an adapted scSLAM-seq protocol for labeling nascent transcripts (see sample-specific entry).
The standard transcriptome libraries were constructed according to the manufacturer's protocol (10X Genomics). For targeted libraries of scars used for lineage tracing, the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target, specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit, so they could be sequenced on the same platform.

tag: tissue

value: adult brain

tag: tissue region

value: diencephalon

value: mixed (tissue dissociation)

tag: genotype


title: HN878, 12h unexposed, rep1

city: San Antonio

first: Anna

last: Allue Guardia

state: Texas

address: 8715 Military Dr W

institute: Texas Biomedical Research Institute

description: Sample 27

data_processing:
Raw sequencing reads were trimmed (phred score of 30) and aligned to the reference genome H37Rv (Genbank NC_000962.3) using STAR v2.5.3a with default values in the Partek Flow Genomic Analysis software.
Transcripts were normalized and quantified as Transcripts per Kilobase Million (TPM) using the Partek E/M (Expectation/Maximization) algorithm.
Differentially expressed genes (DEGs) were identified using the DESeq2 method with an FDR cut-off of 0.1 and a minimum fold change (FC) of 2 in the iDEP.94 software.
Assembly: H37Rv (Genbank NC_000962.3)
Supplementary files format and content: Raw counts

organism: Mycobacterium tuberculosis

source_name: Bacterial culture

growth_protocol: M.tb strains were cultured from frozen stocks for 14 days at 37°C in 7H11 agar plates (BD BBL), supplemented with oleic acid, albumin, dextrose and catalase (OADC).

extract_protocol:
Bacterial pellets were used for RNA extraction using the Quick-RNA Fungal/Bacterial Miniprep kit (Zymo Research). Briefly, the pellet was resuspended in Lysis buffer and transferred to a ZR BashingBead Lysis tube. A bead beating procedure was performed in a BioSpec Mini-Beadbeater-24 (6 cycles of 45 s at 3,800 rpm, with 3 min intervals on a cooler rack). RNA in the supernatant was isolated using Zymo-spin columns, with an in-column DNAse I treatment, and eluted in nuclease-free water. A second DNAse treatment was performed on the isolated RNA using the TURBO DNAse reagent (Thermo Fisher Scientific), and RNA was further purified using the RNA Clean & Concentrator kit (Zymo Research).
A total RNA stranded library was prepared using the Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research). Briefly, 500 ng of RNA were used as input to construct the library, which included a rRNA depletion step of 30 min followed by 14 cycles of PCR. 75bp paired-end (PE) sequencing was conducted using a NextSeq 500 Mid Output Illumina platform.

treatment_protocol: Single bacterial suspensions were obtained and exposed to human ALFs for 15 min or 12 ± 2 hours at 37°C. After exposure, ALF was washed away and bacterial pellet incubated in RNAProtect (Qiagen) for 10 min at room temperature (RT), centrifuged at 13,000 × g and stored at −80 °C until further use. For each of the strains, unexposed bacteria were used as controls.

value: Bacterial culture

tag: strain

tag: treatment

tag: duration


title: W-7642, 12h unexposed, rep1

description: Sample 31


title: Brain 9 - telencephalon, scRNAseq

value: telencephalon


title: W-7642, 12h unexposed, rep2

description: Sample 32


title: Brain 7 - telencephalon, scRNAseq


title: EED_MinusTag_CLIP_Rep2


title: W-7642, 12h ALF, rep2

description: Sample 30

value: ALF


title: W-7642, 12h ALF, rep1

description: Sample 29


title: HN878, 12h unexposed, rep2

description: Sample 28


title: Brain 6 - telencephalon, scRNAseq


title: EED_MinusTag_CLAP_Rep1


title: Brain 7 - rhombencephalon, scRNAseq

value: rhombencephalon


title: HN878, 12h ALF, rep1

description: Sample 25


title: H37Rv, 12h unexposed, rep1

description: Sample 23


title: KPC1199 cells, sgCtrL, rep2

city: Shanghai

first: Zhengyan

last: Zhang

address: State Key Laboratory of Systems Medicine for Cancer, Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Cancer Institute, Shanghai Jiao Tong University,

country: China

description: KPC1199_cells_gene.count.matrix.annot.xls

data_processing:
The raw paired end reads were trimmed and quality controlled by fastp with default parameters.
Then clean reads were separately aligned to  Mouse-mm39 with orientation mode using HISAT2  software
The mapped reads of each sample were assembled by StringTie  in a reference-based approach
differential expression analysis was performed using the DESeq2
Assembly: mm39
Supplementary files format and content: excel sheet includs count of each sample

organism: Mus musculus

source_name: Pancreatic tumor

growth_protocol: KPC1199 cells were maintained in a humidified incubator at 37 °C with 5% CO2 and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 IU m-1 of penicillin/streptomycin (P/S). All cell lines used were tested negative for Mycoplasma.

extract_protocol:
Total RNA was extracted from the tissue using TRIzol® Reagent according the manufacturer’s instructions. Then RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies).
RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions (Illumina, San Diego, CA)

treatment_protocol: sgCtrL or Sin3Bsg1 KPC1199 cells + or – IFNγ indicates that PDAC cells were treated with or without 10 ng/ml IFNγ for 24 hours.

value: Pancreatic tumor

value: Pancreatic tumor cell

value: WT

value: No


title: KPC1199 cells,Sin3Bsg1,rep1

institute: Ren Ji Hospital, School of Medicine, Shanghai Cancer Institute, Shanghai Jiao Tong University,

value: Sin3B Knockout


title: EED_PlusTag_CLAP_Rep1


title: KPC1199 cells,Sin3Bsg1,rep2


title: HN878, 12h ALF, rep2

description: Sample 26


title: H37Rv, 12h ALF, rep2

description: Sample 22


title: KPC1199 cells, sgCtrL, rep1


title: KPC1199 cells,Sin3Bsg1,+IFNg,rep3

description: KPC1199_cells+-IFNg_gene.count.matrix.annot.xls

value: with IFNg 10ng/ml for 24hr


title: KPC1199 cells,Sin3Bsg1,+IFNg,rep1


title: CDC1551, 12h unexposed, rep1

description: Sample 19


title: KPC1199 cells,Sin3Bsg1,+IFNg,rep2


title: CDC1551, 12h ALF, rep2

description: Sample 18


title: H37Rv, 12h ALF, rep1

description: Sample 21


title: KPC1199 cells,sgCtrL,+IFNg,rep3


title: CDC1551, 12h ALF, rep1

description: Sample 17


title: W-7642, 15min unexposed, rep2

description: Sample 16


title: KPC1199 cells,sgCtrL,+IFNg,rep2


title: KPC1199 cells,Sin3Bsg1,-IFNg,rep3

value: without IFNg


title: KPC1199 cells,sgCtrL,+IFNg,rep1


title: W-7642, 15min ALF, rep2

description: Sample 14


title: W-7642, 15min ALF, rep1

description: Sample 13


title: KPC1199 cells,Sin3Bsg1,-IFNg,rep2


title: H37Rv, 12h unexposed, rep2

description: Sample 24


title: KPC1199 cells,sgCtrL,-IFNg,rep3


title: KPC1199 cells,Sin3Bsg1,-IFNg,rep1


title: KPC1199 cells,sgCtrL,-IFNg,rep2


title: HN878, 15min unexposed, rep1

description: Sample 11


title: KPC1199_Sin3Bsg1_Sin3B,Cut&tag

library_source: genomic

data_processing:
Sequenced reads were trimmed for adaptor sequence and to discard reads shorter than 50bp, then mapped to mm10 whole genome using Bowtie2 v. 2.4.2
Reads Per Kilobase of exon per Megabase of library size were calculated using homer annotatePeaks.pl
Peaks were called using MACS2 version 2.2.7.1 with the parameters -B  -t  --broad -g mm --broad-cutoff 0.1
Assembly: mm10
Supplementary files format and content: bigWig
Library strategy: CUT&Tag

molecule: genomic DNA

extract_protocol:
Briefly, tumor cell were generated and collected .CUT&Tag assay was performed following the manual of hyperactive pG-Tn5/pA-Tn5 transposase for CUT&Tag kit (TD901, Vazyme).
DNA library were prepared according to manufacturer’s instructions of Trueprep index kit v2 (TD202, Vazyme)

value: no


title: HN878, 15min ALF, rep2

description: Sample 10


title: HN878, 15min ALF, rep1

description: Sample 9


title: CDC1551, 12h unexposed, rep2

description: Sample 20


title: KPC1199 cells,sgCtrL,-IFNg,rep1


title: H37Rv, 15min unexposed, rep2

description: Sample 8


title: H37Rv, 15min ALF, rep2

description: Sample 6


title: KPC1199_sgCtrL_Sin3B,Cut&tag


title: KPC1199_Sin3Bsg1_H3K27Ac,Cut&tag


title: H37Rv, 15min ALF, rep1

description: Sample 5


title: H37Rv, 15min unexposed, rep1

description: Sample 7


title: KPC1199_Sin3Bsg1+IFNg_H3K27Ac,Cut&tag


title: KPC1199_sgCtrL+IFNg_H3K27Ac,Cut&tag


title: CDC1551, 15min unexposed, rep2

description: Sample 4


title: W-7642, 15min unexposed, rep1

description: Sample 15


title: CDC1551, 15min unexposed, rep1

description: Sample 3


title: KPC1199_sgCtrL_H3K27Ac,Cut&tag


title: CDC1551, 15min ALF, rep1

description: Sample 1


title: CDC1551, 15min ALF, rep2

description: Sample 2


title: HN878, 15min unexposed, rep2

description: Sample 12


title: SOX2 knockdown bulk RNA-seq C.i1_clone1 +Dox biol_rep2

city: Planegg

first: Wolfgang

last: Enard

address: Großhadernerstr. 2

institute: Ludwig-Maximilians-Universität München

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i1_clone1_2
BC:  CTACGTTCTTAG

data_processing:
Fastq data file quality assessment was conducted using fastqc (Andrews, S. (2010). FastQC: A quality control tool for high throughput sequence data. Available at: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
polyA trimming was performed with cutadapt (Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. journal, 17(1), 10-12.).
Quality filtering, mapping and counting was done using the zUMIs pipeline (Parekh, S., Ziegenhain, C., Vieth, B., Enard, W., & Hellmann, I. (2018). zUMIs—A fast and flexible pipeline to process RNA sequencing data with UMIs. GigaScience, 7(6), giy059).
Assembly: hg38, gorGor6, macFas6, all extended by the KRAB-dCas9 encoding sequence that was integrated in the genomes of the cell lines
Supplementary files format and content: We provide a separate count table per species as rds files
Supplementary files format and content: The sample conditions are noted in the additional sample_annotation_data_submission file.

organism: Macaca fascicularis

source_name: KRAB-dCas9 iPSCs

growth_protocol: Cell were grown in SF03 +Dox (1 µg/mL) or -Dox for 4 days.

extract_protocol:
Cells that were cultured in 24-wells, were lysed in 200 µL Buffer RLT Plus supplemented with 1 % 2-Mercaptoethanol and then stored at -80 °C until processing.
Libraries were constructed following the published prime-seq protocol. (Janjic, A., Wange, L.E., Bagnoli, J.W. et al. Prime-seq, efficient and powerful bulk RNA sequencing. Genome Biol 23, 88 (2022). https://doi.org/10.1186/s13059-022-02660-8); https://www.protocols.io/view/prime-seqs9veh66
polyA-capture bulk RNA-seq

value: KRAB-dCas9 iPSCs

value: iPSCs

value: +Dox


title: AD-like skin, uninfected (4th replicate)

city: Zurich

first: Genome

last: Informatics

address: Winterthurerstrasse 190

department: Functional Genomics Center Zurich (FGCZ)

country: Switzerland

institute: ETH ZURICH / University of Zurich

data_processing:
Rna-seq reads were aligned with STAR-aligner using the genome build GENCODE Mus musculus GRCm39 Release M26
Gene expression values were computed with the function featureCounts from the R package Rsubread.
Assembly: GENCODE Mus musculus GRCm39 Release M26
Supplementary files format and content: tab-delimited .txt files include 2 columns: ensemble feature identifier and count number.

source_name: ear skin

extract_protocol:
Isolation of total RNA from murine ear skin was performed using TRI reagent (Sigma-Aldrich) according to the manufacturer’s protocol with the modification that the tissue was homogenized using a TissueLyzer (Qiagen) for 6 minutes at 25 Hz before extraction of the nucleic acids with chloroform.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: ear skin

value: C57BL/6 WT

value: MC903 (10 days), olive oil (7 days)


title: control skin, uninfected (3rd replicate)

value: EtOH (10 days), olive oil (7 days)


title: AD-like skin, uninfected (3rd replicate)


title: Brain 4 - telencephalon, scRNAseq

value: adult brain

value: telencephalon

value: mixed (tissue dissociation)


title: SOX2 knockdown bulk RNA-seq C.i1_clone1 +Dox biol_rep1

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i1_clone1_1
BC:  TCAGGTCGTGGA


title: AD-like skin, uninfected (2nd replicate)


title: control skin, uninfected (4th replicate)


title: SOX2 knockdown bulk RNA-seq C.i2_clone1 -Dox biol_rep4

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i2_clone1_8
BC:  AAGTGCACGCCA

value: -Dox


title: AD-like skin, uninfected (1st replicate)


title: control skin, uninfected (1st replicate)


title: SOX2 knockdown bulk RNA-seq C.i2_clone1 -Dox biol_rep2

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i2_clone1_6
BC:  AGTCCGGTGTGA


title: SOX2 knockdown bulk RNA-seq C.i2_clone1 -Dox biol_rep3

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i2_clone1_7
BC:  TCACGACGACAG


title: control skin, uninfected (2nd replicate)


title: control skin, M. pachydermatis associated for 7 days (4th replicate)

value: EtOH (10 days), M. pachydermatis (7 days)


title: SOX2 knockdown bulk RNA-seq C.i2_clone1 +Dox biol_rep4

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i2_clone1_4
BC:  ATCGCTGCTCAG


title: AD-like skin, M. pachydermatis associated for 7 days (4th replicate)

value: MC903 (10 days), M. pachydermatis (7 days)


title: control skin, M. pachydermatis associated for 4 days (4th replicate)

value: EtOH (10 days), M. pachydermatis (4 days)


title: AD-like skin, M. pachydermatis associated for 4 days (4th replicate)

value: MC903 (10 days), M. pachydermatis (4 days)


title: SOX2 knockdown bulk RNA-seq C.i2_clone1 -Dox biol_rep1

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i2_clone1_5
BC:  CATCAACTCGAA


title: AD-like skin, M. pachydermatis associated for 7 days (3rd replicate)


title: SOX2 knockdown bulk RNA-seq C.i2_clone1 +Dox biol_rep3

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i2_clone1_3
BC:  TGTTATGGCCAA


title: AD-like skin, M. pachydermatis associated for 4 days (3rd replicate)


title: SOX2 knockdown bulk RNA-seq C.i1_clone1 -Dox biol_rep4

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i1_clone1_8
BC:  GGATATTCCACA


title: SOX2 knockdown bulk RNA-seq C.i1_clone1 -Dox biol_rep3

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i1_clone1_7
BC:  TTGAATGTGCGA


title: SOX2 knockdown bulk RNA-seq C.i2_clone1 +Dox biol_rep1

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i2_clone1_1
BC:  GTAGGCGCATGA


title: control skin, M. pachydermatis associated for 4 days (3rd replicate)


title: AD-like skin, M. pachydermatis associated for 7 days (2nd replicate)


title: SOX2 knockdown bulk RNA-seq C.i1_clone1 -Dox biol_rep2

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i1_clone1_6
BC:  TCACCGCCTCAA


title: SA-3h-ΔΔ-replicate 2, scRNA-seq

city: Wuhan

first: Xiaodan

last: Yan

state: Hubei Province

address: Donghu Road, No. 115, Wuchang District,

department: Medical Research Institute

institute: Wuhan University

description:
RiboD-mSPLiT
0612-2
SA rRNA and mRNA reads.txt

data_processing:
The demultiplexing, barcodes processing, gene counting and aggregation of scRNA-seq data were made by scripts compiled in Python 2.7.15 as previously described with some modifications. [lattman, W. Jiang, P. Oikonomou, S. Tavazoie, Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing. Nature microbiology 5, 1192 (Oct, 2020).]. Downstream analysis of single cell data was performed using pipelines detailed in Seurat v4.3.0.
For corralation analysis of bulk RNA-Seq and scRNA-seq data, scRNA-seq data was combined with the UMI sums of all cells. Single-cell and bulk transcriptomes of E.coli were compared by computing the Pearson correlation of log2 transcripts reads of each gene between the two measurements.
For scRNA-seq data mRNA / total RNA ratio analysis, we removed the barcodes sequence contained in the front, and the interference of the last ten bases, and kept only 20 bases for mapping. Then we mapped the data from the different species to the corresponding reference genomes.
(Detailed codes were saved in "Detailed Computational Pipeline.docx" and "scripts" compressed package .)
Assembly: E.coli MG1655 K12
Supplementary files format and content: matrix files
Supplementary files format and content: xlsx file include raw counts for RNA-seq and UMI sums of scRNA-seq data
Supplementary files format and content: tab-delimited files includes raw counts for samples of the same species

organism: Staphylococcus aureus

source_name: bacterial cell

extract_protocol:
For RiboD-mSPLiT, all  cells were from pure culture in vitro. Cells were harvested by centrifuge;
For RiboD-mSPLiT, library was performed accarding to the Materials and Methods section;

value: S. aureus 25923

value: bacterial cell

value: WT

value: exponential growth phase


title: SOX2 knockdown bulk RNA-seq C.i1_clone1 -Dox biol_rep1

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i1_clone1_5
BC:  CTCCAACCTTAA

value: KRAB-dCas9 iPSCs

value: iPSCs


title: SA-3h, replicate 2, scRNA-seq

description:
mSPLiT
0119-F
SA rRNA and mRNA reads.txt


title: AD-like skin, M. pachydermatis associated for 4 days (2nd replicate)

value: ear skin

value: C57BL/6 WT


title: SOX2 knockdown bulk RNA-seq C.i1_clone1 +Dox biol_rep4

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i1_clone1_4
BC:  AGAACGACACAA

value: +Dox


title: EC-3h, replicate 2, scRNA-seq

description:
mSPLiT
0615-2
ECOLI rRNA and mRNA reads.txt

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


title: EC-3h, replicate 1, scRNA-seq

description:
mSPLiT
Result of correlation analysis between mSPLiT and RiboD-mSPLiT.xlsx
0615-1
ECOLI rRNA and mRNA reads.txt


title: control skin, M. pachydermatis associated for 4 days (2nd replicate)


title: control skin, M. pachydermatis associated for 7 days (2nd replicate)


title: SOX2 knockdown bulk RNA-seq C.i2_clone1 +Dox biol_rep2

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i2_clone1_2
BC:  TCACTGTACCGG


title: EC-3h-ΔΔ, replicate 1, scRNA-seq

description:
RiboD-mSPLiT
Result of correlation analysis between mSPLiT and RiboD-mSPLiT.xlsx
0427-3
ECOLI rRNA and mRNA reads.txt


title: SOX2 knockdown bulk RNA-seq C.i1_clone1 +Dox biol_rep3

description:
sample_annotation_data_submission.xlsx
Sox2_macaque.dgecounts.rds
SOX2_KD_bulkRNA-seq_C.i1_clone1_3
BC:  CTCTTCTACTAG


title: R5249

first: Peng

last: Jin

address: Ruijin Rd. II

department: Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics

institute: Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

data_processing:
Single-cell RNA-sequencing was performed on two primary AML patient BMs with the Chromium 10x Genomics 3’ gene expression kit version 3.1 according to the manufacturer’s instructions. Single-cell expression matrices were obtained from raw sequence data (FASTQ) with the 10x Genomics Cell Ranger Software Suite v4.0.0 (GRCh38 reference). Cells with a mitochondrial count ratio higher than 0.05 and <200 or >2500 expressed genes were removed from the dataset. To reduce patient-related bias, we performed data integration using the Seurat package, and a total of 12,853 cells were retained. Briefly, cells from different patients were integrated with the ‘‘FindIntegrationAnchors’’ and ‘‘IntegrateData’’ functions in Seurat package. Principal component analysis was performed using the top 2,000 highly variable genes and selected for downstream analysis. We utilized the single-cell genotyping tool, VarTrix (https://github.com/10XGenomics/vartrix), to perform cell-variant assignment. VarTrix is a software tool for extracting single cell variant information from 10x single cell gene expression datasets as well as 10x single cell DNA datasets. VarTrix itself does not perform variant calling, and it takes a set of previously defined variant calls and use that to identify those variants in the single cell data. According to the pipeline, we provided a pre-called variant in VCF format based on U2AF1S34F in the 21:43104346-43104346 locus, then VarTrix uses Smith-Waterman alignment to evaluate reads that map to this variant locus and assign single cells to the variant. VarTrix will produce genome matrices in the same Market Exchange format that CellRanger uses. This is a sparse matrix format that can be read by common packages, which containing information about each variant for each cell barcode.
Assembly: hg38
Supplementary files format and content: RDS files were generated using seurat package in Rstudio and values represent counts of genes (row) detected in cells (column). Txt files were meta information for cells in RDS files.

organism: Homo sapiens

source_name: Bone marrow

extract_protocol:
Bone marrow mononuclear cells from AML patients were isolated after Ficoll–Isopaque density gradient centrifugation.
Single-cell RNA-sequencing was performed on two primary AML patient BMs with the Chromium 10x Genomics 3’ gene expression kit version 3.1 according to the manufacturer’s instructions.

tag: disease state

value: AML

value: Bone marrow

value: Bone marrow mononuclear cells


title: R5307


title: control skin, M. pachydermatis associated for 7 days (3rd replicate)


title: AD-like skin, M. pachydermatis associated for 7 days (1st replicate)


title: EC-24h-static-ΔΔ, replicate 1, scRNA-seq

description:
RiboD-mSPLiT
1214-7

value: 24 h static biofilm


title: modelAD_F

city: Irvine

first: Narges

last: Rezaie

state: California

address: 2218 Biological Sciences III

institute: University of California, Irvine

description: Mouse microglial single-cell in hippocampus and cortex, hippocampus nuclei, and cortex nuclei were collected for Split-seq and dispersed into 48 wells of a 96 well plate containing unique barcodes per well (16 wells of microglial cells, 16 wells of hippocampus nuclei, and 16 wells of cortex nuclei). Library preparation was performed following manufacturer’s instructions for the Single Cell Whole Transcriptome Kit, where the cells are mixed for the next 2 sets of barcoding reactions followed by sublibrary generation.

data_processing:
Illumina Split-seq: Reads were demultiplexed into 6 sublibraries using bcl2fastq and the provided adapter sequences.
Illumina Split-seq: Parse Biosciences' analysis software was run on the modelAD_A sublibrary: split-pipe --mode all  --chemistry v1 --genome_dir genomes/mm10 --fq1 5X_12kA_R1.fastq.gz --fq2 5X_12kA_R2.fastq.gz --output_dir analysis_12A --sample <replace with SAMPLE.txt>
Illumina Split-seq: Parse Biosciences' analysis software was run on the modelAD_B sublibrary: split-pipe --mode all  --chemistry v1 --genome_dir genomes/mm10 --fq1 5X_12kB_R1.fastq.gz --fq2 5X_12kB_R2.fastq.gz --output_dir analysis_12B --sample <replace with SAMPLE.txt>
Illumina Split-seq: Parse Biosciences' analysis software was run on the modelAD_C sublibrary: split-pipe --mode all  --chemistry v1 --genome_dir genomes/mm10 --fq1 5X_12kC_R1.fastq.gz --fq2 5X_12kC_R2.fastq.gz --output_dir analysis_12C --sample <replace with SAMPLE.txt>
Illumina Split-seq: Parse Biosciences' analysis software was run on the modelAD_D sublibrary: split-pipe --mode all  --chemistry v1 --genome_dir genomes/mm10 --fq1 5X_12kD_R1.fastq.gz --fq2 5X_12kD_R2.fastq.gz --output_dir analysis_12D --sample <replace with SAMPLE.txt>
Illumina Split-seq: Parse Biosciences' analysis software was run on the modelAD_E sublibrary: split-pipe --mode all  --chemistry v1 --genome_dir genomes/mm10 --fq1 5X_12kE_R1.fastq.gz --fq2 5X_12kE_R2.fastq.gz --output_dir analysis_12E --sample <replace with SAMPLE.txt>
Illumina Split-seq: Parse Biosciences' analysis software was run on the modelAD_F sublibrary: split-pipe --mode all  --chemistry v1 --genome_dir genomes/mm10 --fq1 5X_12kF_R1.fastq.gz --fq2 5X_12kF_R2.fastq.gz --output_dir analysis_12F --sample <replace with SAMPLE.txt>
Assembly: mm10
Supplementary files format and content: For Illumina Split-seq, all split-pipe (Parse Biosciences analysis software) outputs are intergrated and preprocessed, then stored in sparse cell-by-gene expression matrices in counts (ModelAD_count.mtx) where rows are cells and genes are columns along with corresponding cell metadata (cell_information.csv) and a list of expressed genes (gene_information.csv)

source_name: Cortex and hippocampus

extract_protocol:
Left cortex and left hippocampus tissues from 8 month old mice were snap frozen in liquid nitrogen and were prepared for single-nucleus isolation and fixation. The right half were pooled for microglia isolation
Illumina Split-seq: Parse Biosciences’ Single Cell Whole Transcriptome Kit (also called Split-seq).

tag: age

value: 8 month

value: Cortex and hippocampus


title: AD-like skin, M. pachydermatis associated for 4 days (1st replicate)


title: modelAD_E


title: control skin, M. pachydermatis associated for 7 days (1st replicate)

value: EtOH (10 days), M. pachydermatis (7 days)


title: 3h, RNA-seq

description: RNA-seq

data_processing:
RNA-Seq clean data was mapped to the MG1655 k12 genome using the BWA aligner software (version 0.7.17-r1188, https://github.com/lh3/bwa.git).
Then we converted the sam files to bam files using samtool.
The bam files were counted by featureCounts (v2.0.1, https://github.com/topics/featurecounts) to generate expression results. (Detailed codes were saved in "Detailed Computational Pipeline.docx".)
Assembly: E.coli MG1655 K12
Supplementary files format and content: tab-delimited files includes raw counts for RNA-seq sample
Supplementary files format and content: xlsx file include raw counts for RNA-seq and UMI sums of scRNA-seq data

extract_protocol:
RNA was harvested using Bacteria RNA Extraction Kit (Vazyme). 1μg of total RNA was used for construction of sequencing libraries;
RNA libraries for RNA-seq were prepared using Total RNA-seq (H/M/R) Library Prep Kit following manufacturer's protocols;


title: SA-3h-ΔΔ-replicate 1, scRNA-seq

description:
RiboD-mSPLiT
0612-1
SA rRNA and mRNA reads.txt


title: modelAD_D


title: Chip, IgG, replicate 2

description: ChIP-seq

data_processing:
Clean data was mapped to the MG1655 k12 genome using the BWA aligner software (version 0.7.17-r1188, https://github.com/lh3/bwa.git) ,Then we converted the sam files to bam files using samtool.
Then bam files were then analysed using macs2(https://github.com/taoliu/MACS/) and ChIPseeker.
We used samtool (v1.9) and bedtools (v2.29.2) to extract the corresponding fasta file from "narrow.peak" format files.
MEME(https://meme-suite.org/meme/) was used to find binding motif enriched in the sequencing data. (Detailed codes were saved in "Detailed Computational Pipeline.docx".)
Assembly: E.coli MG1655 K12
Supplementary files format and content: narrowPeak (except for Input sample)

extract_protocol:
Cells were fixed with 1% formaldehyde for 30 min. After permeabilization, DNase digestion and cell lysis, HipH protein binded DNA fragments were collected by co-IP.
Library was performed accarding to the Materials and Methods section.


title: SA-3h, replicate 1, scRNA-seq

description:
mSPLiT
0119-E
SA rRNA and mRNA reads.txt


title: control skin, M. pachydermatis associated for 4 days (1st replicate)

value: EtOH (10 days), M. pachydermatis (4 days)


title: modelAD_C


title: D2 Foxp1cKO + Foxp1AAV #3 (mCherry)

city: Dallas

first: Genevieve

last: Konopka

address: 5323 Harry Hines Blvd.

department: Neuroscience

institute: University of Texas Southwestern Medical Center

data_processing:
cellranger mkfastq
cellranger count
cellbender
doubletfinder
harmony
seurat
Assembly: mm10 (GRCm38p6)
Supplementary files format and content: matrix.mtx.gz' has raw counts for all samples
Supplementary files format and content: barcodes.tsv' has list of barcodes for all samples matching with raw counts
Supplementary files format and content: features.tsv' has list of genes matching to raw counts
Supplementary files format and content: meta.tsv' has all meta information including assigned celltypes

source_name: Dorsal Striatum

extract_protocol:
We sacrificed P18 mice by rapid decapitation and quickly dissected the brain and transferred it into ice-cold nACSF that contained (mM): 125 NaCl, 25 NaHCO3, 10 Dextrose, 3 KCl, 1.25 NaH2PO4, 2 CaCl2.2H2O, 2 MgCl2 with 7.3-7.4 pH and 300-305 mOsm osmolality. We placed the brain onto an ice-cold 1.0 mm stainless steel brain matrix (Stoelting, Cat#51386) and obtained three 1.0 mm thick striatal sections which were then transferred into a petri-dish containing cold nACSF. Using forceps and under a dissection microscope, striatum was dissected out from these slices, flash frozen in liquid N2, and stored at -80°C. The striatal tissue was collected from P18 mice of required genotypes at different time points and stored. Protocol for isolating nuclei was adapted and modified from our previously published work6. Briefly, tissue was homogenized 20 times with pestle A, followed by 20 times with pestle B, in a glass Dounce tissue grinder (Sigma, Cat#D8938) in 2 ml ice-cold EZ Nuclei Lysis Buffer (Sigma, Cat #NUC-101). Homogenized samples were transferred into 15 ml tube with an additional 2 ml ice-cold EZ Nuclei Lysis Buffer, incubated on ice for 3 min and centrifuged at 500 X g for 5 min at 4°C. The supernatant was discarded, and nuclei pellet was resuspended in 3 ml ice-cold Nuclei Suspension Buffer (NSB; 1X PBS, 0.01% Ultrapure BSA, and 0.1% RNase inhibitor) and again centrifuged at 500 X g for 5 min at 4°C. The supernatant was then discarded, and nuclei pellet was resuspended in 300-400 µl NSB, and filtered through 40 µm size Flowmi cell strainer (Bel-Art Products, Cat# H13680-0040). An aliquot of the filtered nuclei suspension was mixed with Trypan Blue and transferred into a disposable cell counting chamber nuclei to visualize and count the healthy nuclei under a light microscope to determine desired nuclei concentration (targeting 700-1,200 nuclei/µl).
Libraries from a total of 9 mice (3 mice/genotype) were prepared using the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 (10X Genomics, Cat#1000121) targeting 10,000 nuclei per sample as per the manufacturer’s instructions. All mice were injected either with ControlAAV or Foxp1AAV in their striatum at P1 age where only a fraction of neurons was infected that were identified based on the presence of AAV transcripts. However, the proportion of AAV transcripts is exceptionally small in the pool of total transcripts that are captured by snRNA-seq experiment and therefore we used a modified approach 12 where we amplified the viral transcript specific sequences and prepared separate libraries. Briefly, we used 5% of the total cleaned up cDNA (amplified from the GEMs that were generated from each sample for the targeted 10,000 nuclei) and amplified the viral transcripts with reverse primer 10X_Enrich_R – 5’CTACACGACGCTCTTCCG3’ and forward primers 10X_WPRE_F1 – 5’ACGAGTCGGATCTCCCTTT3’ (for ControlAAV infected mice) and 10X_mCherry_F1 – 5’GCATGGACGAGCTGTACA3’ (for Foxp1AAV infected mice) for 25 cycles using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB, Cat# M0531S). We used different forward primers for ControlAAV and Foxp1AAV infected mice since ControlAAV vector has a WPRE sequence at the 3’end whereas we had to remove WPRE sequence from the AAVFoxp1 vector to accommodate Foxp1 and mCherry into the vector and thus have the mCherry sequence at 3’end of Foxp1AAV vector. The amplified PCR product was run on an agarose gel, where a ~250 bp product was excised from the gel and eluted using QIAquick Gel Extraction Kit (Qiagen, Cat#28706) in 20 µl elution buffer (NEB). 5% of this first round amplified product was used to perform a second round of amplification using the reverse primer, i. e. 10X_WPRE_F1 and forward primers 10X_WPRE_F2 – 5’GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACGAGTCGGATCTCCCTTT3’ (for AAVControl infected mice) and 10X_mCherry_F2 –5’GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTACAAGTAGACCCAGCTT3’ (for AAVFoxp1 infected mice) for 20 cycles using the same Phusion polymerase. Once again, this amplified PCR product was loaded on an agarose gel where similar size product (~250 bp) was excised and eluted in 30 µl elution buffer. The eluted PCR products were once again cleaned up using SPRIselect Reagent and subjected to sample-index PCR amplification according to the manufacturer’s instructions (10X Genomics) to generate cDNA libraries. Finally, both (total and AAV_transcript) libraries were subjected to quality control using 4200 TapeStation System (Agilent, Cat# G2991BA) and quantified using Qubit 2.0 Fluorometer (Invitrogen). For each sample, 5 nM of the total libraries and 0.5 nM of the AAV transcript libraries (10% of the total library) were pooled and sequenced on a NovaSeq 6000 (Illumina) platform on S4 flow cell.

value: Dorsal Striatum

value: D2 Foxp1cKO


title: Chip, IgG, replicate 1

description: ChIP-seq

value: bacterial cell

value: WT


title: EC-3h-ΔΔ, replicate 2, scRNA-seq

description:
RiboD-mSPLiT
0427-4
ECOLI rRNA and mRNA reads.txt

value: exponential growth phase


title: H3K27ac

city: Boulder

first: Edward

last: Chuong

state: CO

address: 3415 Colorado Ave.

institute: CU - Boulder

data_processing:
Real-Time Analysis v3.4.4 was used for basecalling.
Adapters and low quality reads were trimmed using BBDuk v38.05 using options ‘ktrim=r k=23 mink=11 hdist=1 tpe tbo qtrim=r trimq=10’. Trimmed reads were aligned to the hg38 assembly using BWA-MEM v0.7.15, and only uniquely mapping reads with a minimum MAPQ of 10 were retained. Fragments aligning to the mitochondrial genome were removed. Peak calling was performed using complete and size subsetted alignment files with MACS2 v2.1.1 with paired-end options ‘--format BAMPE --pvalue 0.01 --SPMR -B --call-summits’. Bigwig files were prepared from the MACS2 normalized bedgraph files using bedGraphToBigWig v4.
Assembly: hg38
Supplementary files format and content: signal tracks corresponding to read coverage per 1 million fragments in UCSC bigwig format

growth_protocol: HT1080 cells were routinely grown at 37C in 5% CO2 on coated plastic (VWR #10861-680, typically 3,000,000-5,000,000 cells in 10cm dishes) in DMEM + GlutaMAX (Gibco #10565018) supplemented with 1X penicillin-streptomycin (Gibco #15140122), 10% fetal bovine serum (Gibco #10437010), and 8 ug/mL Blasticidin S HCl (Gibco #A1113903; for dCas9-HT1080 selection).

extract_protocol:
CUT&RUN pulldowns were generated using a protocol from Meers et al. 2019 (PMID: 31232687, https://dx.doi.org/10.17504/protocols.io.zcpf2vn). All buffers were prepared according to the “High Ca2+/Low Salt” section of the protocol using 0.05% digitonin. 500,000 viable cells were used for each pulldown. The following antibodies were used: rabbit anti-mouse IgG (1:100, Abcam #ab46540) and rabbit anti-H3K27ac (1:100, Abcam #ab4759). pAG-MNase was added to each sample following primary antibody incubation at a final concentration of 700 ng/mL. Chromatin digestion, release, and extraction was carried out according to Meers et al. 2019 (PMID: 31232687, https://dx.doi.org/10.17504/protocols.io.zcpf2vn). Pulldown success was determined by Qubit dsDNA High Sensitivity and TapeStation 4200 HSD5000 before proceeding with library preparation.
Libraries were generated using a modified protocol for use with the KAPA HyperPrep Kit. Briefly, the full volume of each pulldown (~30 uL) was diluted to 50 uL in nuclease-free water, and libraries were generated following the manufacturer’s protocol with the following modifications. Freshly diluted 0.200 uM single-index adapters were added to each library at a low concentration (9 nM) to minimize adapter dimer formation. Adapter-ligated libraries underwent a double-sided 0.8X/1.0X cleanup with KAPA Pure Beads. Purified, adapter-ligated libraries were amplified using the following PCR cycling conditions: 45 s at 98°C, 14x(15 s at 98°C, 10 s at 60°C), 60 s at 72°C. Amplified libraries underwent 2 double-sided 1.0X cleanups with KAPA Pure Beads. The final libraries were quantified using Qubit dsDNA High Sensitivity and TapeStation 4200 HSD5000. Libraries were pooled and sequenced on an Illumina NovaSeq 6000 (University of Colorado Genomics Core) as 150bp paired-end reads.

treatment_protocol: cells were untreated


title: SP140 KD IFNB rep4

description:
raw_counts_genes_wt_wtB_kd_kdB.txt
normalized_counts_genes_wt_wtB_kd_kdB.txt

data_processing:
Real-Time Analysis v3.4.4 was used for basecalling.
Adapters and low quality reads were trimmed using BBDuk v38.05 with arguments 'ktrim=r k=23 mink=11 hdist=1 qtrim=r trimq=10 tpe tbo' and mapped to hg38 GENCODE v34 using salmon v0.13.1 with options ‘--libType A --validateMappings --rangeFactorizationBins 4 --gcBias’.
Differentially expressed genes were called using DESeq2 v1.38.3 with design '~genotype+treatment+genotype:treatment'. For the CRISPRi differential expression analysis, normalized count data is derived from DESeq2 comparisons between: 1) SP140-KD (4 replicates) vs GFP-gRNA control HT1080 samples (2 replicates); 2) IFNB treated SP140-KD (4 replicates) vs IFNB treated GFP-gRNA control HT1080 samples (2 replicates); and 3) IFNB treated GFP-gRNA control HT1080 samples (2 replicates) vs GFP-gRNA control HT1080 samples (2 replicates). Genes with zero counts across all samples were removed.
Assembly: Hg38
Supplementary files format and content: tab-delimited text files including DESeq2 raw counts
Supplementary files format and content: tab-delimited text files including DESeq2 normalized counts

growth_protocol: HT1080 cells were routinely grown at 37C in 5% CO2 on coated plastic (VWR #10861-680, typically 3,000,000-5,000,000 cells in 10cm dishes) in DMEM + GlutaMAX (Gibco #10565018) supplemented with 1X penicillin-streptomycin (Gibco #15140122), 10% fetal bovine serum (Gibco #10437010), 8 ug/mL Blasticidin S HCl (Gibco #A1113903; for dCas9-HT1080 selection), and 1 ug/mL Puromycin (Gibco #A1113803; for gRNA-HT1080 selection).

extract_protocol:
RNA from HT1080 was harvested using Zymo Quick-RNA Miniprep Plus Kit (Zymo #R1057).
Libraries were generated with 500 ng RNA using KAPA mRNA HyperPrep Kit (KAPA #KK8581) according to the manufacturer's instructions. Briefly, single-indexed adapters (KAPA #KK8700) or unique dual-indexed adapters (KAPA #KK8727) were added to each sample at a final concentration of 78 nM. Excess adapter was removed using sequential 0.63X and 0.70X KAPA Pure Bead (KAPA #KK8002) cleanups. Purified, adapter-ligated samples were amplified for 11 cycles according to the manufacturer's instructions and purified using a 1.0X KAPA Pure Bead cleanup.

treatment_protocol: Cells were either untreated or treated with 10 U/mL IFNB for 4h.

value: IFNB


title: SP140 KD IFNB rep3


title: modelAD_B


title: EC-24h-static-ΔΔ, replicate 2, scRNA-seq

description:
RiboD-mSPLiT
1214-8


title: IgG

value: IgG


title: Chip, HipH-flag-CDG, IP, replicate 2

description: ChIP-seq

tag: antibody

value: Anti-DDDDK-tag mAb (Monoclonal Antibody; MBL, M185-3L, 016)


title: modelAD_A


title: SP140 KD IFNB rep2


title: D2 Foxp1cKO + Foxp1AAV #1 (mCherry)


title: Chip, HipH-flag-CDG, IP, replicate 1

description: ChIP-seq


title: SP140 KD IFNB rep1


title: SPEN_GagnonCLAP_Rep2

value: Mouse embryonic stem cells


title: Chip, HipH-flag, IP, replicate 2

description: ChIP-seq


title: D2 Foxp1cKO + ControlAAV #3 (WPRE)


title: SPEN_GagnonCLAP_Rep1


title: SP140 KD untreated rep4

value: untreated


title: Chip, HipH-flag, IP, replicate 1

description: ChIP-seq


title: D2 Foxp1cKO + ControlAAV #2 (WPRE)


title: PSM33_Gagnon_Input


title: SP140 KD untreated rep2


title: YAM6_xrn1_Gs_2

city: Paris

first: Maxime

last: Wery

address: 26 rue d'Ulm

country: France

institute: Institut Curie

data_processing:
Reads were trimmed using Trim Galore
Reads were mapped on the S. cerevisiae reference genome (R64-2-1, including the 2-micron plasmid) and ERCC spike-in RNA sequences, using HISAT2 V2.2.0, with default parameters and maximum intron size of 5000.
Gene count was obtained with featureCounts v2.0.0.
Gene count was normalized using estimatesizefactorsformatrix() function from DESeq2 package (R software), using ERCC counts.
Normalized densities were computed for each sample as: normalized gene count / gene length
Assembly: R64-2-1
Supplementary files format and content: tab delimited file, containing normalized tag/nt for each sample

organism: Saccharomyces cerevisiae

source_name: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 xrn1Δ::kanMX4

growth_protocol: WT, xrn1-delta and upf1-delta cells were grown to mid-log phase in CSM with glucose as carbon source, and then shifted for 16 minutes in CSM where glucose has been replaced by glycerol and ethanol (glucose starvation). In parallel, control cells were maintained for the same time in glucose-containing CSM.

extract_protocol:
Total RNA was extracted using standard hot phenol procedure. Extracted RNA was ethanol-precipitated, resuspended in nuclease-free H2O (Ambion) and quantified using a NanoDrop 2000c spectrophotometer and/or a Qubit fluorometer with the Qubit RNA HS Assay kit (Life Technologies).
For each sample, 1 µg of total RNA was mixed with 2 µl of diluted ERCC RNA spike-in mix (1:100 dilution in nuclease-free H2O; Invitrogen). Ribosomal (r)RNAs were depleted using the Ribominus Eukaryote v2 kit (Ambion). Libraries were prepared using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina) and the IDT for Illumina – TruSeq RNA UD indexes (Illumina).

treatment_protocol: For the WT cells, CHX (100 μg/ml final concentration) or an equal volume of DMSO (Mock) was then added to each sample.

value: MATa his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 xrn1{delta}::kanMX4

value: Glucose starvation


title: D2 Foxp1CTL + ControlAAV #3 (WPRE)

value: D2 Foxp1CTL


title: mLND5-2

city: Pittsburgh

first: Amanda

middle: Catherine

last: Poholek

state: Pennsylvania

address: 4401 Penn Ave., Rangos Research Center 8129

department: Pediatrics

institute: University of Pittsburgh

description: fresh frozen Spatial transcriptome sequencing

data_processing:
For images of Seeker tile, serial sections were cut onto glass slides followed by staining and imaging as described.
Demultiplexed FastQ files were initially processed by the Curio Seeker bioinformatics pipeline
Assembly: mm10
Supplementary files format and content: MoleculesPerMatchedBead.mtx: Expression table Sparse Matrix (column=BeadBarcodes, row=Genes)
Supplementary files format and content: MatchedBeadLocation.csv: Spatial coordinates of Beadbarcodes
Supplementary files format and content: genes.tsv: Expression table genes
Supplementary files format and content: barcodes.tsv :Expression table BeadBarcode
Supplementary files format and content: image.tif: image of serial section

source_name: Mediastinal lymph node

extract_protocol:
Tile was removed and placed in a 1.5ml Eppendorf LoBind tube with Hybridization Reaction Mix. Reverse transcription was performed to generate cDNA followed by tissue clearing and Curio Seeker Bead resuspension.
Second stand synthesis was done followed by cDNA amplification, which after purifiying was subjected to tagmentation (Nextera XT) and library generation

treatment_protocol: naïve 1DERYFP CD4+ T cells (CD45.1+) were transferred into CD45.2+ recipients and were immunized with 25 µg of HDM intranasally daily for 3 days. Mediastinal lymph nodes (mLN) were isolated on day 4 and day 6, snap frozen and embedded in chilled optimal cutting temperature (OCT) compound (Tissue-Tek) on dry ice, and stored at -80 °C. Samples were cryosectioned (10 µm) at -20 °C on a cryostat (Leica) and mounted directly onto a 3 x 3 mm Curio Seeker Spatial Mapping Slide (Curio Biosciences)

value: Mediastinal lymph node

tag: time

value: Day 5

value: House dust mite intranasal


title: D2 Foxp1cKO + ControlAAV #1 (WPRE)


title: SP140 KD untreated rep1


title: D2 Foxp1cKO + Foxp1AAV #2 (mCherry)


title: IFNB rep2


title: D2 Foxp1CTL + ControlAAV #1 (WPRE)


title: D2 Foxp1CTL + ControlAAV #2 (WPRE)


title: YAM6_xrn1_1

value: Control


title: mLND3-2

value: Day 3


title: mLND5-1


title: IFNB rep1


title: YAM202_upf1_Gs_2

source_name: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 upf1Δ::kanMX4

value: MATa his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 upf1{delta}::kanMX4


title: D2 Foxp1cKO + Foxp1AAV #2

value: Dorsal Striatum

value: D2 Foxp1cKO


title: YAM202_upf1_Gs_1


title: Untreated rep1


title: SP140 KD untreated rep3


title: D2 Foxp1cKO + Foxp1AAV #3


title: D2 Foxp1cKO + Foxp1AAV #1


title: LND5-3

data_processing:
Sequencing results were initially processed by spaceranger (10X Genomics) to align sequencing data with the image.
genes were filtered for more than 900 zeros threshold which resulted in matrix of 1932 genes by 3779 regions.
Assembly: mm10
Supplementary files format and content: barcodes.tsv.gz: list of spatial barcodes
Supplementary files format and content: features.tsv.gz: list of gene IDs
Supplementary files format and content: matrix.mtx.gz: gene expression count data in Matrix Market Exchange Format
Supplementary files format and content: aligned_fiducials.jpg.gz: aligned fiducials of the tissue image
Supplementary files format and content: detected_tissue_image.jpg.gz: image of tissue and spots
Supplementary files format and content: scalefactors_json.json: scalefactors in json format
Supplementary files format and content: tissue_hires_image.png.gz: hi-res image of tissue
Supplementary files format and content: tissue_lowres_image.png.gz: low-res image of tissue
Supplementary files format and content: tissue_positions_list.csv.gz: list of spatial barcodes and the coordinates specifying spots
Library strategy: spatial transcriptomics

source_name: mediastinal lymph node

extract_protocol:
The slides were sealed in individual 50 ml Falcon tube at -80 °C until further processing according to the manufacturer’s protocol (10x Genomics). The Visium Spatial Tissue Optimization Slide & Reagent kit (10x Genomics) was used to determine optimal permeabilization timing of 18 min. Immunofluorescence staining was done using “Methanol Fixation, Immunofluorescence Staining & Imaging for Visium Spatial Protocols (CG000312)”. Slides were stained with anti-B220 eFluor 450 (RA3-6B2, eBioscience), anti-CD4 Alexa Fluor 488 (N418, Biolegend), anti-CD45.1 Biotin (A20, Biolegend) followed by secondary detection with streptavidin Alexa Fluor-555 and anti-CD11c Alexa Fluor-647 (N418, Biolegend). Images were acquired using EVOS™ M7000 Imaging System (AMF7000) under the Visium assay mode. Tissue sections were then permeabilized, and mRNA molecules within cells captured by poly (dT) sequence on the slide surface, followed by on slide reverse transcription to generate cDNA.
cDNA is amplified and further processed into sequencing libraries according to the manufacturer’s protocol (10X Genomics).
Libraries were sequenced on an Illumina Nextseq2000 at 50,000 read pairs per spot covered by tissue.

treatment_protocol: naïve 1DERYFP CD4+ T cells (CD45.1+) were transferred into CD45.2+ recipients and were immunized with 25 µg of HDM intranasally daily for 3 days. Mediastinal lymph nodes (mLN) were isolated on day 4 and day 6, snap frozen and embedded in chilled optimal cutting temperature (OCT) compound (Tissue-Tek) on dry ice, and stored at -80 °C. Samples were cryosectioned (10 µm) at -20 °C on a cryostat (Leica) and mounted directly onto the 6.5 x 6.5 mm capture areas of a single Visium Spatial Gene Expression slide (10X Genomics).

value: mediastinal lymph node

value: House Dust Mite intranasal


title: YAM202_upf1_2


title: LND5-1


title: D2 Foxp1cKO + ControlAAV #3


title: LND3-3


title: YAM202_upf1_1


title: YAM6_xrn1_Gs_1


title: LG0812 Alec 17d rep 2

city: San Francisco

first: Wei

last: Wu

state: CA

address: 600 16th street

department: Department of Medicine

institute: University of California, San Franciso

data_processing:
RNA-Seq reads were mapped to the hg19 reference genome using STAR (Spliced Transcripts Align to a Reference, v2.4.2a).
The expression level of transcript per million (TPM) reads were quantified using RNA-Seq by Expectation-Maximization algorithm (RSEM v1.2.29).
The quantified gene expressions of 26,334 transcripts (including coding genes and non-coding genes) were processed in R studio.
Differentially expressed genes between tumor and normal samples were identified using the EdgeR algorithm.
Gene set enrichment analysis was done using GSEA 4.0.1 software.
Assembly: hg19
Supplementary files format and content: Quantified gene expression in TPM for each experimental condition

source_name: Lung cancer patient-derived xenograft

extract_protocol:
RNA was extracted from snap-frozen tissue or cell pellets. For tissue samples, tissue was minced using a liquid nitrogen-cooled mortar and pestle before RNA extraction. RNA isolation was performed using the RNeasy Mini kit (Qiagen) including an on-column DNase I digestion. RNA quality was assessed by automated electrophoresis using the RNA 6000 Pico Kit and an Agilent 2100 BioAnalyzer (Agilent Technologies, Inc.). RNA was quantified using the Qubit RNA HS Assay Kit and a Qubit 2.0 fluorometer (Thermo Fisher Scientific).
Library preparation and paired-end 150bp (PE150, Illumina) RNA sequencing was performed by Novogene (Novogene Corporation, Sacramento, USA).

value: Lung cancer patient-derived xenograft

value: ALK fusion-positive

value: Alectinib 6 mg/kg 17d


title: YAM6_xrn1_2


title: LND3-1


title: TET2_CLAP_Rep2

data_processing:
Paired-end RNA sequencing reads were trimmed to remove adaptor sequences using Trim Galore! v0.6.2 and assessed with FastQC v0.11.8. Read pairs were then aligned to a combined genome reference containing the sequences of repetitive and structural RNAs (ribosomal RNAs, snRNAs, snoRNAs, 45S pre-rRNAs, tRNAs) using Bowtie2. The remaining reads were then aligned to a combined genome reference containing the mouse (mm10) and human (hg38) genomes using STAR aligner181. PCR duplicates were removed using the Picard MarkDuplicates function. For mixing experiments, only reads that mapped uniquely in the genome and unambiguously to the human or mouse genomes were kept for further analysis. For experiments done in a single species, the appropriate reference genome and alignments were used (mm10 for mouse and hg38 for human). These reads were then used for downstream analysis.  For computing enrichments, all human (hg38) and mouse (mm10) annotated genes (RefSeq, downloaded from UCSC GRCh38 and GRCm38, respectively) were used as a reference set except for the genes encoding the transfected proteins. We treated exonic regions and intronic regions of each annotated gene as separate reference genes for computing enrichment. For each reference gene, we enumerated 100 nucleotide windows that span across the gene; for each window, we calculated: (i) the number of reads overlapping the window in the protein elution sample (e.g., CLIP or CLAP) and (ii) the maximum of either the number of observed reads over the window or the median read count over all windows within the gene in the input sample. Because all windows overlapping a gene should have the same expression level in the input sample, this approach provides a conservative estimation of the input coverage because it prevents windows from being scored as enriched if the input values over a given window are artificially low due to stochastic fluctuation, while at the same time accounting for any non-random issues that lead to increases in read counts over a given window (i.e., alignment artifacts leading to non-random assignment or pileups).  To directly compare the number of reads within each window between sample and input, we normalized each window count by the total number of reads sequenced and the overall complexity within each sample. For example, if one sample was sequenced twice as deeply as another, then we would expect to observe – on average – twice as many reads over a given window for that sample. To account for the differences in the number of mapped (aligned) reads between samples, we scaled the total number of sequenced reads by the proportion of aligned reads within each sample.  For each window, we computed enrichment by dividing the normalized sample counts by the normalized input counts. Nominal p-values were calculated for each window using a binomial test where k (number of successes) is defined as the number of reads in the protein elution samples within the window, N (number of trials) is the sum of the number of reads in the protein elution and input samples, and p (probability of success) is the expected number of reads in the elution sample divided by the sum of the expected number of reads per window in elution and input samples. (The expected number of reads is defined as the total number of reads scaled by the proportion of aligned reads within each sample). For plotting and reporting purposes, we considered all regions with a nominal binomial p-value < 10-6 as significant. However, the overall results reported are robust to the precise p-value cutoff used.
Assembly: mm10, hg38
Supplementary files format and content: tab-delimited text file includes gene window enrichment calculations
Supplementary files format and content: bedgraph
Library strategy: CLAP-seq

extract_protocol:
Cells were washed once with PBS and then crosslinked on ice using 0.25 J cm-2 (UV 2.5k) of UV at 254 nm in a Spectrolinker UV Crosslinker. Cells were then scraped from culture dishes, washed once with PBS, pelleted by centrifugation at 1500g for 4 min, and flash-frozen in liquid nitrogen for storage at -80 °C.
Cells were lysed in 1 mL lysis buffer (50mM Tris pH 7.5, 100mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1X Promega PIC). For CLIP samples, RNA was digested with Ambion RNase I (1:3,000 dilution) to achieve a size range of 100-500 nucleotides in length. We used a fixed RNase condition for all experiments to enable comparison between proteins. Lysates were precleared by mixing with Protein G beads (Dynabeads) for 30 minutes at 4°C. Target proteins were immunoprecipitated overnight at 4°C from 10 million cells with 5 μg of antibody coupled to 40 μL of Protein G beads in 100 μL lysis buffer. Antibodies were pre-coupled to beads for 1 hour at room temperature with mixing, followed by 3 washes with lysis buffer to remove unbound protein. After immunoprecipitation, beads were washed four times with high salt wash buffer (50 mM TrisHCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) and four times with wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20). RNA and protein were eluted by incubating in NLS elution buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 2% N-lauroylsacrosine, 2.5 mM TCEP) supplemented with 100 mM DTT at 50°C for 20 minutes. Samples were then run through an SDS-PAGE gel and transferred to a nitrocellulose membrane using the iBlot transfer system. The region 70 kDa above the molecular weight of the protein of interest was isolated and treated with Proteinase K (NEB), followed by purification with RNA Clean & Concentrate-5 (Zymo). Anti-V5 antibody (Bethyl, A190-120A) was used for all CLIP experiments.  For CLAP samples, cells were resuspended in 1 mL of cold lysis buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with 1X Protease Inhibitor Cocktail (Promega), 200 U of RiboLock (NEB), 20 U of TURBO DNase (Ambion), and 1X manganese/calcium mix (0.5mM CaCl2, 2.5 mM MnCl2). Samples were incubated on ice for 10 minutes and then at 37°C for 10 minutes at 1150 rpm shaking on a ThermoMixer (Eppendorf). Lysates were cleared by centrifugation at 15000g for 2 minutes and the supernatant was collected for capture to HaloLink Resin (Promega). For each CLAP capture, 200 μL of 25% HaloLink Resin (50 μL of HaloLink Resin total) was used per 10 million cells. Resin was washed three times with 2 mL of 1X TBS (50 mM Tris pH 7.5, 150 mM NaCl) and incubated in 1X Blocking Buffer (50 mM HEPES, pH 7.5, 10 μg/mL Random 9-mer, 100 μg/mL BSA) for 20 minutes at room temperature with continuous rotation. After the incubation, resin was washed three times with 1X TBS. Cleared lysate was mixed with 50 μL of HaloLink Resin and incubated at 4°C overnight with continuous rotation. The captured protein bound to resin was washed three times with lysis buffer at room temperature and then three times at 90°C for 3 minutes while shaking at 1200 rpm with each of the following buffers: 1X ProK/NLS buffer (50 mM HEPES, pH 7.5, 2% NLS, 10 mM EDTA, 0.1% NP-40, 10 mM DTT), high salt buffer (50 mM HEPES, pH 7.5, 10 mM EDTA, 0.1% NP-40, 1M NaCl), 8M urea buffer (50 mM HEPES, pH 7.5, 10 mM EDTA, 0.1% NP-40, 8 M Urea), and Tween buffer (50 mM HEPES, pH 7.5, 0.1% Tween 20, 10 mM EDTA). After the last wash, samples were centrifuged at 7500g for 30 seconds and supernatant was discarded. For elution, HaloLink Resin was resuspended in 100 μL of ProK/NLS buffer + 10 μL of Proteinase K (NEB) and incubated at 50°C for 20 minutes while shaking at 1200 rpm. Elutions were then transferred to microspin cups (Pierce, Thermo Fisher), centrifuged at 2000g for 30 seconds, and purified with RNA Clean and Concentrate-5 (Zymo).

value: Human embryonic kidney cells


title: Untreated rep2

value: untreated


title: D2 Foxp1cKO + ControlAAV #2


title: LG0812 Alec 17d rep 1


title: D2 Foxp1cKO + ControlAAV #1


title: YAM1_WT_Gs_CHX_2

source_name: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0

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

value: Glucose starvation + CHX


title: YAM1_WT_Gs_CHX_1


title: mLND3-1

value: Mediastinal lymph node


title: YAM1_WT_CHX_2

value: Control - CHX


title: LG0812 ctrl rep 2

value: untreated 0.1% DMSO


title: TET2_CLAP_Rep1


title: D2 Foxp1CTL + ControlAAV #3

value: D2 Foxp1CTL


title: LG0812 Alec 17d rep 3


title: YAM1_WT_CHX_1


title: LG0812 ctrl rep 3


title: LG0812 ctrl rep 1


title: PSPC1_CLAP_Rep2


title: D2 Foxp1CTL + ControlAAV #1


title: SUZ12_NoMixing_CLAP


title: TH021 Osim 7d rep 3

value: EGFR-mutant

value: Osimertinib 10 mg/kg 7d


title: PSPC1_CLAP_Rep1


title: Renal LEC, cisplatin, scRNAseq

city: Bryan

first: Joseph

middle: M.

last: Rutkowski

address: 8447 Riverside Parkway

department: Medical Physiology

institute: Texas A&M University School of Medicine

data_processing:
Sample demultiplexing, barcode processing, and gene count were conducted with CellRanger V6.0.1. Briefly, single-cell RNA sequencing reads were processed using 10x Genomics cloud analysis. Briefly, fastq files were uploaded to the 10x Genomics Analysis cloud and aligned to the reference mouse transcriptome (mm10) using Cell Ranger Count v6.0.1. a digital gene expression matrix, including raw unique molecular identifier (UMI) counts were assigned to each cell in the respective sample. (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: kidney

extract_protocol:
Kidneys were minced into small pieces and placed into 5mL digestion buffer (15mg/mL Collagenase D (Roche Applied Science, 11088858001), 10mg/mL Collagenase Type 4 (Gibco, 17-105-019), 2 mg/mL Dispase II (Roche Applied Biosciences, 4942078001), BSA Fraction V (Fisher Scientific, BP1605-100), and HBSS (ThermoFisher Scientific, 14025092). The samples were then incubated in a 37°C water bath with manual agitation every 10 minutes. Digestion was halted by adding 10mL of ice cold HBSS. Digested samples were filtered through both 70µm and 40 µm filters. Endothelial cells were enriched magnetically using a mouse CD31 biotinylated antibody (BD Pharmingen, 553371) and a cleavable Biotin Binder Kit (Invitrogen, 11533D). After the linker was cleaved, the isolated cells were subsequently enriched using an anti-mouse podoplanin (Pdpn) biotinylated antibody (BAF3244, R&D Systems) as above. CD31+Pdpn+ cells were placed into ice cold RPMI 1640 + 10% FBS prior to FACS confirmation of CD45-CD31+Pdpn+ cells. Cell fractions were resuspended in in RPMI1640 +10% FBS, counted, and diluted to a concentration of ~1,000 viable cells/μL.
Library preparation was performed in accordance with the manufacturer's instructions. Renal LEC samples were mixed with master mix, paritioning oil, and gel then loaded onto the Chromium X Controller using the Chromium Next GEM Single Cell 3’ Reagent Kit V3.1. Sample cDNA quality was checked using the Agilent High Sensitivity D5000 ScreenTape assay on the Agilent 4200 TapeStation System (Agilent Technologies, G2991BA). cDNA samples were then quantified and normalized prior to the library sequencing. Libraries were sequenced on a NovaSeq S4 Flow cell according to 10x Genomics recommendations.
scRNA-seq. Libraries were sequenced on a NovaSeq S4 Flow cell according to 10x Genomics recommendations.

value: kidney

value: Lymphatic endothelial cells

value: 10-12 weeks of age

value: cisplatin


title: YAM1_WT_Gs_1

value: Glucose starvation - Mock


title: Merged_HEK293T_Input


title: TH021 Osim 7d rep 2

value: Lung cancer patient-derived xenograft


title: YAM1_WT_2

value: Control - Mock


title: Renal LEC, saline, scRNAseq

value: saline


title: TH021 Osim 7d rep 1


title: TH021 ctrl rep 3


title: YAM1_WT_1


title: HaloYY1_CLAP

value: Human embryonic kidney cells


title: EZH2_NoMixing_CLAP


title: TH021 ctrl rep 2


title: E12.5 PGC Fanca Rep3

city: Cambridge

first: Alastair

last: Crisp

address: Francis Crick Avenue

country: United Kingdom

institute: MRC Laboratory of Molecular Biology

data_processing:
Raw reads quality trimmed using trim_galore with q 30
Mapping to GRCm38 using hisat2
Differential expression analysis with cuffdiff
Gene set enrichment analysis with g:Profiler
Assembly: GRCm38 mouse genome
Supplementary files format and content: tab delimited cuffdiff output files of gene expression

source_name: PGC

extract_protocol:
total RNA was extracted using the RNeasy Mini Kit (QIAGEN)
RNA sequencing libraries were prepared using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® kit and NEBNext® Poly(A) mRNA Magnetic Isolation module kit following the manufacturer’s instructions. For hepatocytes, sequencing libraries were prepared using the NEBNext® UltraTM II RNA Library Prep Kit for Illumina® kit following the manufacturer’s instructions. Subsequently, all libraries were ligated to NEBNext® Multiplex Oligos for Illumina® (Dual Index Primer Set I)

value: PGC

value: Fanca-


title: upf1_CHX_2

description: Ribosome protected RNA

data_processing:
UMI were extracted from read with umi_tools extract
Reads were trimmed using cutadapt --trim-n --match-read-wildcards -u 4 -n 4 -a AAGATCGGAAGAGC -a AAAAAAAA -e 0.2 --nextseq-trim 20 -m 20 -q 20
Reads were mapped on S. cerevisiae reference genome (R64-2-1), 2-micron plasmid and ERCC sequences, using HISAT2 V2.2.0, with default parameters and maximum intron size of 5000.
rRNA reads were discarded
PCR duplicates were removed using umi_tools dedup
Uniquely mapped reads with a length of 25-35 nt were kept
Ribotricer v1.3.1 was used to annotate candidates ORF and compute phase for each condition
Assembly: R64-2-1
Supplementary files format and content: tab delimited file, with read count per ORF and phase score per condition

growth_protocol: Yeast cells were grown to mid-log phase (OD600 0,5) at 30°C in YPD.

extract_protocol:
For each sample, 250 ml of cells were harvested by centrifugation at room temperature and directly frozen in liquid nitrogen after supernatant removal. Cells were lysed in 1X lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 30 mM MgCl2) supplemented by 2X cOmplete Protease Inhibitor Cocktail (Roche) and ribosome protected fragments (RPFs) were prepared as previously described (Baudin-Baillieu et al., 2016. Methods Mol Biol), with the following modifications. Polysomes were purified on sucrose cushion and then digested with Rnase I (Ambion, 5 units/UA260). Ribo-depletion was performed using in-house designed oligonucleotides.
Libraries were then prepared from 10 ng of RPFs using the D-Plex Small RNA-Seq kit for Illumina (Diagenode) and the D-Plex Unique Dual Indexes for Illumina – set A (Diagenode).

treatment_protocol: Cells were treated or not with CHX for 15 minutes.

value: MATa his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 upf1*::kanMX4

value: CHX


title: E12.5 PGC Fanca Rep2


title: EWSR1_CLAP_Rep2


title: upf1_CHX_1

description: Ribosome protected RNA


title: D2 Foxp1CTL + ControlAAV #2

value: Dorsal Striatum


title: E12.5 PGC Fanca Rep1


title: EWSR1_CLAP_Rep1


title: E12.5 PGC ERCC1 Rep3


title: TH021 ctrl rep 1

value: untreated 0.1% DMSO


title: E12.5 PGC ERCC1 Rep2


title: upf1-2

description: Ribosome protected RNA


title: CHTOP_CLAP_Rep2


title: upf1-1

description: Ribosome protected RNA


title: E12.5 PGC ERCC1 Rep1


title: EED_NoMixing_CLAP


title: WT_CHX_2

description: Ribosome protected RNA

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


title: TH107 Osim 3d rep 2

source_name: Lung cancer organoid

value: Lung cancer organoid

value: Osimertinib 1 uM 3d


title: YAM1_WT_Gs_2


title: TH107 Osim 9d rep 2

value: Osimertinib 1 uM 11d


title: CHTOP_CLAP_Rep1


title: E12.5 PGC WT Rep3

value: WT


title: WT_CHX_1

description: Ribosome protected RNA


title: TH107 Osim 3d rep 1


title: E12.5 PGC WT Rep2


title: TH107 ctrl 3d rep 2

value: untreated 0.1% DMSO 3d


title: Brain, B-NHL + NT (control)

city: Freiburg

first: Gianni

last: Monaco

address: Breisacher Straße

institute: Freiburg Medical Center

description: 10x Genomics

data_processing:
The demultiplexing was carried out using in-built Illumina software on Next-seq 1000.
Barcode processing, gene counting and aggregation were made using the Cell Ranger software v6.1.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10

source_name: Brain

extract_protocol:
Mice were treated with B-NHL cells and 7 days later   and NT or CD19 CAR T cells treatment. On day 10, mice were perfused with cold HBSS and brains were extracted and flash frozen. A small portion of Cortex tissue was excised from the CNS tissue and enriched for myeloid cells with modified Frankenstein’s protocol. Briefly, CNS tissue was homogenized in Nuclei EZ lysis buffer (Millipore Sigma) and upon incubation was filtere through 70µm filter. The suspension was centrifuged at 600g for 5 min at 4°C. The pellet was washed with nuclei wash and resuspension buffer (1x PBS, 1.0% BSA, 0.2 U/μl RNase Inhibitor) and centrifuged at 600g for5min at 4°C.Following this, Nuclei were stained for DAPI, NeuN and Olig 2 markers and sorted by Flow cytometry.
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: Brain

value: Microglia, neuron, Oligodencrocytes

value: B-NHL + NT T cell therapy (control)


title: Brain, B-NHL+ CAR T cell therapy

description: 10x Genomics

value: B-NHL + CD19 CAR T cell therapy


title: E12.5 PGC WT Rep1


title: MDA-MB-231 cells, Ig_231_A_72h, rep4

city: Bethesda

first: Thomas

middle: Joshua

last: Meyer

state: Maryland

address: 41 Center Drive, Building 41, Room B620

department: National Cancer Institute

institute: National Institutes of Health

description:
Sample_Roberts_56
This sample did not pass QC, a clear outlier, and was excluded from final analysis downstream of filtering low counts.

data_processing:
84 mRNA-seq samples were pooled and sequenced on NextSeq using Illumina TruSeq Stranded mRNA Library Prep and paired-end sequencing. This analysis used 16 of those samples.
Reads of the samples were trimmed for adapters and low-quality bases using Trimmomatic software before alignment with the reference genome (Human - hg38) and the annotated transcripts using STAR.
The gene expression quantification analysis was performed for all samples using STAR/RSEM tools.
Low count genes were filtered and two samples (Sample_Roberts_38 & Sample_Roberts_56) was identified as a technical outlier. These two samples were removed from downstream analysis, leaving a total of 14 samples.
Counts were transformed into CPM and quantile normalized using Limma voom followed by differential expression of genes analysis in 4 contrast comparisons.
GSEA analysis was performed using fGSEA and a preranked methodology, using the t-statistic from the DEG as the ranking variable.
Assembly: Release 30 (GRCh38.p12)
Supplementary files format and content: Comma-separated values (.csv) files containing a matrix of normalized batch corrected expression, a matrix of DEG results, and a matrix of GSEA results produced in this study.

source_name: MDA-MB-231 cells, Control  antibody

growth_protocol: MD-MB-231 cells were cultured using SmartDish™  with  AggreWell™ EB Formation Medium from Stem cell technologies

extract_protocol:
Total RNA will be prepared using total RNA extraction from BIOLINE #BIO-52073
Illumina TruSeq Stranded mRNA Library Prep and paired-end sequencing

treatment_protocol: The cells were treated with Celgene Antibody CC90002, or the Control Anibody, or SIRPα-Fc for 72hrs

value: Cancer cell

value: IgG control


title: E12.5 HSC Fanca Rep3

source_name: HSC

value: HSC

value: Fanca-


title: MDA-MB-231 cells, Ig_231_A_72h, rep3


title: H3122 Alec 14d VT104 rep 3

source_name: Lung cancer

value: Lung cancer

value: drug-tolerant persister

value: Alectinib 500 nM 14d, VT104 1 uM 72h


title: TH107 ctrl 3d rep 1


title: E12.5 HSC Fanca Rep2


title: TH107 Osim 9d rep 1


title: upf1_CHX 2

data_processing:
Reads were trimmed using Trim Galore
Reads were mapped on the S. cerevisiae reference genome (R64-2-1, including the 2-micron plasmid) and ERCC spike-in RNA sequences, using HISAT2 V2.2.0, with default parameters and maximum intron size of 5000.
Gene count was obtained with featureCounts v2.0.0.
Gene count was normalized using estimatesizefactorsformatrix() function from DESeq2 package (R software), using snoRNA counts.
Normalized densities were computed for each sample as : normalized gene count / gene length
Assembly: R64-2-1
Supplementary files format and content: tab delimited file, containing normalized tag/nt for each sample

growth_protocol: Yeast cells were grown to mid-log phase (OD600 0.5) at 30°C in YPD medium.

treatment_protocol: Cells were treated for 15 minutes with cycloheximide (100 μg/ml, final concentration) or with an equal volume of DMSO.

value: MATa his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0 upf1*::kanMX4

value: CHX


title: upf1_CHX 1


title: MDA-MB-231 cells, Ig_231_A_72h, rep2


title: H3122 Alec 14d VT104 rep 2


title: E12.5 HSC Fanca Rep1


title: upf1_DMSO 2

value: DMSO


title: upf1_DMSO 1


title: E12.5 HSC ERCC1 Rep3


title: E12.5 HSC ERCC1 Rep1


title: MDA-MB-231 cells, Si_231_A_72h, rep4

source_name: MDA-MB-231 cells, SIRPαFc

value: Sirp alpha Fc recombinant protein


title: H3122 Alec 14d VT104 rep 1


title: H3122 Alec 14d rep 3

value: Alectinib 500 nM 14d


title: MDA-MB-231 cells, Ig_231_A_72h, rep1


title: WT-1

description: Ribosome protected RNA

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


title: WT_CHX 2


title: MDA-MB-231 cells, Si_231_A_72h, rep3


title: WT-2

description: Ribosome protected RNA


title: E12.5 HSC WT Rep3

value: WT


title: WT_CHX 1


title: H3122 Alec 14d rep 1


title: MDA-MB-231 cells, Si_231_A_72h, rep2


title: E12.5 HSC WT Rep2


title: PC9-persister-Osim 9d VT104 rep 3

value: Osimertinib 2 uM 9d, VT104 1 uM 72h


title: MDA-MB-231 cells, Si_231_A_72h, rep1


title: E12.5 HSC WT Rep1


title: PC9-persister-Osim 9d VT104 rep 2


title: Adult Hepatocyte ERCC1p53 Rep3

source_name: Hepatocyte

value: Hepatocyte


title: WT_DMSO 2


title: MDA-MB-231 cells, Cg_231_A_72h, rep4

source_name: MDA-MB-231 cells, CC90002 antibody (CG)

value: CD47 blocking antibody


title: upf1_ANS_2

treatment_protocol: Cells were treated for 30 minutes with anisomycin (100 μg/ml, final concentration) or with an equal volume of DMSO.

value: ANS


title: PC9-persister-Osim 9d VT104 rep 1


title: H3122 Alec 14d rep 2


title: Adult Hepatocyte ERCC1p53 Rep2


title: MDA-MB-231 cells, Cg_231_A_72h, rep1

value: Cancer cell


title: PC9-persister-Osim 9d rep 3

value: Osimertinib 2 uM 9d


title: upf1_ANS_1


title: upf1_DMSO_2


title: PC9-persister-Osim 9d rep 2


title: MDA-MB-231 cells, Cg_231_A_72h, rep2


title: upf1_DMSO_1


title: Adult Hepatocyte ERCC1 Rep3


title: PC9-persister-Osim 9d rep 1


title: MDA-MB-231 cells, Ut_231_A_72h, rep4

source_name: MDA-MB-231 cells, Untreated

value: UT


title: WT_ANS_2


title: Adult Hepatocyte ERCC1 Rep2


title: MDA-MB-231 cells, Ut_231_A_72h, rep3


title: PC9-YAP-S94A-rep3

value: Lung cancer

value: YAP-S94A OverExpression

value: untreated


title: Adult Hepatocyte ERCC1 Rep1


title: MDA-MB-231 cells, Ut_231_A_72h, rep2

description:
Sample_Roberts_38
This sample did not pass QC, a clear outlier, and was excluded from final analysis downstream of filtering low counts.


title: Adult Hepatocyte p53 Rep3


title: WT_ANS_1


title: WT_DMSO 1


title: PC9-YAP-S94A-rep2


title: MDA-MB-231 cells, Cg_231_A_72h, rep3


title: PC9-YAP-S94A-rep1


title: Adult Hepatocyte ERCC1p53 Rep1


title: Adult Hepatocyte p53 Rep2


title: WT_DMSO_1


title: Adult Hepatocyte p53 Rep1


title: PC9-YAP-5SA-rep3

value: YAP-5SA OverExpression


title: PC9-YAP-5SA-rep2


title: Adult Hepatocyte WT Rep3


title: PC9-YAP-5SA-rep1


title: WDR5_CLAP_Rep2

city: Pasadena

first: Jimmy

last: Guo

address: 1200 E California Blvd

institute: Caltech

value: Human embryonic kidney cells


title: Adult Hepatocyte WT Rep2


title: Adult Hepatocyte WT Rep1


title: WDR5_CLAP_Rep1


title: CTCF_ChIP

data_processing:
Paired-end DNA sequencing reads were aligned to the appropriate reference genome (mm9 for mouse and hg19 for human) using Bowtie2 v2.3.1 with the default parameters and with the following deviations. We used a local alignment search (--local) and disabled searching for discordant alignments (--no-discordant).
Assembly: mm9, hg19
Supplementary files format and content: bigwig files were generated at 50nt bins using deepTools

extract_protocol:
Mouse embryonic stem cell lines (pSM33) were crosslinked in either 1% formaldehyde or 1% formaldehyde and 2 mM DSG (Thermo #2059). Briefly, cells were washed with room temperature 1X PBS and then incubated in crosslinker solution in PBS at room temperature with gentle rocking for 45 minutes. Cells were then washed with room temperature 1X PBS. After washing, a 1% formaldehyde solution was then added on top of cells and further incubated at room temperature for 10 minutes. To quench the formaldehyde crosslinking, 2 mL of a 2.5M Glycine solution was added to each plate and incubated for an additional 5 minutes. After quenching, cells were washed three times in 1X cold PBS. After the last wash, 7.5 mL of scraping buffer (1X PBS + 0.5% BSA) was added to cells. Cells were then scraped using a rubber policeman, aliquoted into 10 million cell aliquots, and flash frozen until ChIP was performed.
To solubilize chromatin and fragment DNA, cells were lysed and then sonicated. To begin nuclear fractionation, cells were resuspended in 1 mL of Gagnon HLB Buffer + 1X PIC and incubated on ice for 10 minutes. Samples were briefly vortexed and centrifuged at 1250g for 3 minutes at 4°C. Samples were then resuspended in 600 μL of mammalian lysis buffer (1% TritonX-100, 0.1% sodium deoxycholate, 0.1% SDS, 150mM NaCl, and 50mM HEPES pH 7.5) + PIC and transferred to 15 mL Diagenode conical tubes. Following transfer, cells were sonicated using a Bioruptor waterbath sonicator (in 15 mL tubes with adapters) at max intensity for 30 seconds, followed by either 30 seconds of rest for 27 cycles (1% formaldehyde samples) or 30 seconds of rest for 36 cycles (1% formaldehyde + DSG samples). Samples were then transferred to 1.5 mL tubes and cleared of insoluble material by pelleting at 13000 rpm for 10 minutes at 4°C. Supernatants were mixed with 1800 μL HBSS (Thermo Scientific) + 1X PIC and 2400 μL 2X RIPA + 1X PIC. Lysate was incubated overnight with Invitrogen M-280 Sheep Anti-Rabbit IgG Dynabeads coupled with 5 μg of H3K27me3 antibody (Active Motif, 39155) or 5 μg of Goat anti-V5 antibody (Bethyl, A190-119A). Samples were washed with 1 mL of low salt buffer, high salt buffer, LiCl wash buffer, and finally TE buffers. Samples were reverse-crosslinked and Proteinase K-digested overnight at 65°C, and DNA was subsequently purified with Zymo Clean and Concentrate.  Post reverse-crosslinking ChIP libraries were constructed as previously described. Briefly, purified DNA was end repaired and dA-tailed using 1X NEBNext Ultra II End Repair/dA-Tailing Module (NEB, E7546L). DNA adaptors were then ligated to each sample and cleaned up using 0.7X SPRI (AMPure XP) followed by a repeat clean-up with 1X SPRI. PCR amplification was achieved using primers that add the indexed full Illumina adaptor sequences.

tag: chip antibody

value: Cat #A190-119Afrom Bethyl


title: PC9-YAP-WT-rep3

value: YAP-WT OverExpression


title: CTCF_CLAP_Rep2


title: PC9-YAP-WT-rep1


title: Adult HSC ERCC1p53 Rep3

value: HSC


title: CTCF_CLAP_Rep1


title: PC9-YAP-WT-rep2


title: Adult HSC ERCC1p53 Rep1


title: WT_DMSO_2

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

value: DMSO


title: PC9-EV-rep3

value: Empty Vector


title: MDA-MB-231 cells, Ut_231_A_72h, rep1


title: Adult HSC ERCC1 Rep2


title: PC9-EV-rep2


title: PC9-EV-rep1


title: MOUNTSINAI_11

city: New York

first: Aarthi

last: Ramakrishnan

state: New York

address: 1425 Madison Avenue

institute: Icahn School of Medicine at Mount Sinai

data_processing:
Raw sequencing reads from mice were mapped to hg38 using HISAT2.
Counts of reads mapping to genes were obtained using htseq-counts software against Ensembl v90 annotation.
Differential expression was done using the DESeq2 package.
Assembly: hg38
Supplementary files format and content: tab-delimited text files

extract_protocol:
To isolate RNA from cultured GBM aggregates, the solid Matrigel was first dissolved by addition of Cultrex Organoid Harvesting Solution (OHS; R&D systems). For each condition, 30 GBM aggregates were taken up with a 1 ml micropipette with cut-off tip from the 96-well U-wells and collected in a 6-well dish, to which 5 ml Cultrex OHS was added for 30 min and placed on a shaker in a 4°C cold room. Aggregates were collected in a 15 ml tube and pelleted by spinning at 1,000 rpm for 5 minutes, then 350 µl RLT buffer of the RNAeasy kit (Qiagen) was added to lyse aggregates by grinding them up with a Doune homogenizer. The lysed RNA in RLT buffer was stored at -80°C until further processing. To prepare cDNA libraries for Illumina sequencing from lysed RNA of GBM aggregates, total RNA was isolated with the RNeasy Micro kit.
The cDNA libraries were synthesized with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina by using100 ng RNA as input for each replicate sample.

value: Brain

value: UNB

value: GBM

value: Hypoxia


title: MOUNTSINAI_12


title: WDR5_ChIP


title: Adult HSC ERCC1 Rep1


title: H1975-B10 d9

value: lung cancer cell line

value: Osimertinib 2 uM


title: MOUNTSINAI_10


title: Adult HSC p53 Rep3


title: MOUNTSINAI_9

value: Normoxia


title: H1975-B10 t0

value: Lung cancer


title: Pax_12h_1

city: Callaghan

first: Dylan

last: Kiltschewskij

state: NSW

address: Room MS616, Level 6, Medical Sciences Building, University Drive

department: School of Biomedical Sciences and Pharmacy

country: Australia

institute: The University of Newcastle

data_processing:
Read 1 (sample barcodes and unique molecular identifiers) and Read 2 (poly(A) cDNA inserts) were demultiplexed and aligned to the reference genome using STAR with soft clipping enabled, as follows: STAR --runMode alignReads --outSAMmapqUnique 60 --runThreadN 8 --outSAMunmapped Within --soloStrand Forward --quantMode GeneCounts --outBAMsortingThreadN 8 --genomeDir /STAR_Index --soloType CB_UMI_Simple --soloCBstart 1 --soloCBlen 12 --soloUMIstart 13 --soloUMIlen 9 --soloCellFilter None --soloCBwhitelist barcodefile.txt --soloFeatures Gene --outSAMattributes NH HI nM AS CR UR CB UB GX GN sS sQ sM --outFilterMultimapNmax 1 --readFilesCommand zcat --outSAMtype BAM SortedByCoordinate --outFileNamePrefix bamfiles --readFilesIn R2.fastq.gz R1.fastq.gz
A read-count matrix was assembled utilising BRBseqTools-1.6.jar as follows: java -jar BRBseqTools-1.6.jar ExtractReadCountMatrix -b bamfilesAligned.sortedByCoord.out.bam -c barcodefile_brbseq.txt -gtf star_index/Homo_sapiens.GRCh38.90.gtf -o count_matrix
Processing of raw reads and pair-wise differential gene expression were conducted via EdgeR (version 3.36.0).
Assembly: Ensembl GRCh38, release 90
Supplementary files format and content: Tab-delimited text file containing raw gene-level read-counts across all samples

source_name: SU-DIPG-VI

growth_protocol: Neurosphere cultures were maintained in Tumor Stem Media (TSM) consisting of DMEM/F12 and neurobasal A, supplemented with B27 supplement minus vitamin A, human-bFGF (20 ng/ml), human-EGF (20 ng/ml), human-PGDF-AA (10ng/ml), human-PDGF-BB (10 ng/ml) and heparin (10 ng/ml) at time of use. When confluent, neurospheres were dissociated using Stempro Acctuase for serial culture or use in in vitro assays. No antibiotics were added. Cells maintained at 5% C02 and 37 degrees Celsius.

extract_protocol:
Total RNA was extracted via RNeasy Plus Micro Kit (Qiagen) as per the manufacturer's instructions.
RNA libraries for BRB-seq were prepared via the AlitheaGenomics Mercurius BRB-seq kit for 96 samples (V.0.1.61), as per the manufacturer's instructions.
BRB-seq (Bulk RNA Barcoding Sequencing)

treatment_protocol: Cells were treated with 0.5µM Paxilisib or 1% DMSO for 6 or 12 hours.

value: SU-DIPG-VI

value: Diffuse intrinsic pontine glioma

value: Paxilisib, 0.5µM

value: 12 hours


title: Pax_12h_2


title: Adult HSC p53 Rep2


title: PC9-C2 d9


title: MOUNTSINAI_8


title: MOUNTSINAI_7


title: PC9-C2 t0

value: untreated


title: MOUNTSINAI_6

value: UCA


title: H358-R RMC 48h rep 3

value: resistant


title: Adult HSC ERCC1 Rep3

value: HSC


title: Adult HSC p53 Rep1


title: Adult HSC WT Rep2

value: WT


title: Pax_6h_2

value: 6 hours


title: Pax_6h_1


title: MOUNTSINAI_5

value: Brain