title: Abl.3_RNAseq_DMSO_day2_rep2

first: Saumya

last: Gupta

state: MA

address: 450 Brookline Ave

department: Cancer Biology

institute: Dana-Farber Cancer Institute

description: Cell line Abl.3, in growth medium with 1% DMSO, cells collected on Day 2, Library preparation replicate 2

data_processing:
Reference preparation: we created two custom parental genomes (“pseudogenomes” (Crowley et al., 2015; Zou et al., 2014); or “pseudocontigs” when we have phasing by contigs) for each organism, we used a custom script (all custom software tools mentioned are available on Github https://github.com/gimelbrantlab/QCumber). We also created a vcf-file with one allele considered as a reference (maternal 129S1 or first phased allele) and the other as an alternative allele. Only heterozygous sites were used.
Alignment: RNA-seq reads were aligned with STAR aligner v.2.5.4a (Dobin et al., 2013) on each of two pseudogenomes, with default threshold on quality of alignment. Only uniquely alligned reads were used.
Allele assigment. Given vcf file with heterozygous positions, coverage over each SNP was calculated using custom script based on samtools mpileup and parsed to obtain the table with allelic counts. Data processing steps from read alignment to allelic counts were based on the GATK pipeline (Castel et al., 2015).
Allele counting per gene. All exons belonging to the same gene were merged into a single gene model based on GTF file (RefSeq GTF files, GRCm38.68 and GRCh37.63, were downloaded from Ensemble ftp://ftp.ensembl.org/pub/release-68/gtf/ (Zerbino et al., 2018)), excluding overlapping regions that belong to multiple genes. This step was based on the GATK pipeline (Castel et al., 2015).
AI point estimate per gene obtained as a proportion of maternal gene counts to total allelic gene counts
RNA abundance was measured using DESeq2
Assembly: mm10
Supplementary files format and content: allelic counts, allelic imbalance with confidence intervals, tpm (transcripts per million) values for RNA abundance

source_name: immortalised B cells

growth_protocol: Immortalised B-cell llines  were cultured in Roswell Park Memorial Institute medium (Gibco), containing 15% FBS (Sigma), 1X L-Glutamine (Gibco), 1X Penicillin/Streptomycin (Gibco) and 0.1% β-mercaptoethanol (Sigma).

extract_protocol:
RNA was extracted from cells using a magnetic bead-based protocol using Sera-Mag SpeedBeadsTM (GE Healthcare). Isolated RNA was DNase-treated with RQ1 DNase (Promega).
Library preparation was done using SMARTseqv4 kit (Clonetech), starting with 10ng input RNA for each library according to manufacturers’ instructions.

treatment_protocol: Cells were grown in grown in growth media. On Day 2, live cells were collected after sucrose gradient centrifugation (Histopaque®-1077, Sigma, Cat 10771), and RNA was prepared for RNAseq.

value: 129S1/SvImJ x CAST/EiJ F1

tag: drug treatment

value: control

tag: library replicates

tag: Sex

value: male


title: Abl.4_RNAseq_DMSO_day2_rep2

status: Public on Oct 06 2022

type: SRA

city: Boston

country: USA

description: Cell line Abl.4, in growth medium with 1% DMSO, cells collected on Day 2, Library preparation replicate 2

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

tag: strain

value: female


title: Abl.3_RNAseq_DMSO_day2_rep1

description: Cell line Abl.3, in growth medium with 1% DMSO, cells collected on Day 2, Library preparation replicate 1


title: Abl.4_RNAseq_DMSO_day2_rep1

description: Cell line Abl.4, in growth medium with 1% DMSO, cells collected on Day 2, Library preparation replicate 1


title: Abl.2_RNAseq_DMSO_day2_rep1

description: Cell line Abl.2, in growth medium with 1% DMSO, cells collected on Day 2, Library preparation replicate 1


title: Abl.2_RNAseq_DMSO_day2_rep2

description: Cell line Abl.2, in growth medium with 1% DMSO, cells collected on Day 2, Library preparation replicate 2


title: WT-3 [RNA-seq]

status: Public on Oct 04 2022

city: Hong Kong

first: Xin

last: DENG

address: 1/F, Block 1, To Yuen Building, City University of Hong Kong, 31 To Yuen Street, Kowloon Tong

department: Department of Biomedical Sciences

country: Hong Kong

institute: City University of Hong Kong

description:
paired-end RNA-seq data of Pseudomonas sringae 1448A
(MM) WT control for PSPPH_1568-1 PSPPH_2510-1 PSPPH_2601-1 PSPPH_2604-1 PSPPH_3386-1 PSPPH_3454-1 PSPPH_3906-1 PSPPH_3877-1 PSPPH_3041-1 PSPPH_3628-1 PSPPH_2377-1 PSPPH_3736-1

data_processing:
ChIP-seq reads were mapped to the Pseudomonas sringae 1448A (NC_005773.3) using Bowtie2 (version 2.3.4.3). Only the uniquely mapped reads were kept for the subsequent analyses. Bam files were transformed into bigWig files by deeptools (version 3.1.3)
RNA-seq reads were mapped to the Pseudomonas sringae 1448A (NC_005773.3) using STAR (version STAR_2.6.1). Only the uniquely mapped reads were kept for the subsequent analyses. The gene differential expression analysis was performed using DESeq2 R package.
Assembly: Pseudomonas sringae 1448A NC_005773.3
Supplementary files format and content: narrowPeak files were generated from ChIP-seq, containing significant peaks. xlsx files were generated from RNA-seq, containing the differential expressional genes.

organism: Pseudomonas savastanoi pv. phaseolicola 1448A

source_name: liquid culture in minimal medium

growth_protocol: ChIP-(KB with Rif and Spec) ; RNA-seq(KB or MM with Rif)

extract_protocol:
TruSeq RNA sample prep kit protocol (Illumina)
TruSeq RNA sample prep kit protocol (Illumina)
The ChIP assay was performed as previously described.For the FLAG-tagged plasmids, the open reading frame of each regulator coding gene was amplified by PCR from Psph1448A genome and cloned into pHM1 plasmid. Wild-type Psph1448A containing empty pHM1 or pHM1-RR-FLAG was cultured in 20 ml KB medium until mid-log phase (OD600 = 0.6). Cultured bacteria were then reated with 1% formaldehyde for 10 min at 28 ℃. Cross-linking was stopped by the addition of 125 mM glycine. Bacterial pellets were washed twice with a Tris buffer (20 mM Tris-HCl [pH 7.5] and 150 mM NaCl), re-suspended in 500 μl IP buffer (50 mM HEPES–KOH [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and mini-protease inhibitor cocktail (Roche), and then subjected to sonication to produce 100-300 bp DNA fragments. Insoluble cellular debris was removed by centrifugation at 4 ℃ and the supernatant was used as the input sample in IP experiments. Both control and IP samples were washed with protein A beads (General Electric) and then incubated with 50 μl agarose- conjugated anti-FLAG antibodies (Sigma) in IP buffer. Washing, crosslink reversal, and purification of the ChIP DNA were conducted. DNA fragments (150-250 bp) were selected for library construction and sequencing libraries were prepared using the NEXTflex™ ChIP-Seq Kit (Bioo Scientific). The libraries were sequenced using the HiSeq 2000 system (Illumina). Two biological replications have been performed for all ChIP-seq experiments. ChIP-seq reads were mapped to the Pseudomonas syringae pv. phaseolicola 1448A genome (NC_005773.3) using Bowtie2 (version 2.3.4.3). Uniquely mapped reads were retained for the subsequent analyses. Binding peaks (q < 0.01) were identified using MACS2 software (version 2.1.0). Reproducible binding sites in both replicates were identified by BEDtools software (version 2.27.1)and used for the following analysis. Subsequently, the enriched loci for each regulator were annotated using the R package ChIPpeakAnno (version 3.18.2) . Target genes were annotated by peaks located in gene promoters defined as upstream of transcription start site (TSS), overlapping with TSS or encompassing the whole gene.
To examine the effect of histidine kinases on the transcriptome, 2 ml of mid-log-phase (OD600 = 0.6) bacterial cultures or 2 ml cultures of mid-log-phase induced in MM with 6 hours (Psph1448A and transcriptional regulator mutant strains) were collected by centrifugation (12,000 rpm, 4 ℃). A RNeasy mini kit (Qiagen) was used for subsequent RNA purification with DNaseI (NEB) treatment. After removing rRNA by using the MICROBExpress kit (Ambion), mRNA was used to generate the cDNA library according to the NEBNext® UltraTM II RNA Library Prep Kit protocol (Illumina), which was then sequenced using the HiSeq 2000 system (Illumina). Two biological replications have been performed for all RNA-seq experiments. For each RNA-seq sample, raw reads were mapped to the Pseudomonas sringae pv. phaseolicola 1448A genome (NC_005773.3) using STAR (version 2.6.1), and uniquely mapped reads were kept for further analyses. Differentially expressed genes were identified using DESeq2 (BH-adjusted P < 0.05 and log2 Fold Change| > 0.5).
NEXTflex™ ChIP-Seq Kit (Bioo Scientific)  and TruSeq RNA sample prep kit protocol (Illumina)

treatment_protocol:
grow until mid-log phase in KB, then transfer to MM for 6 h
grow until mid-log phase

value: wild type

tag: condition

value: liquid culture in minimal medium


title: WT-4 [RNA-seq]


title: WT-8 [RNA-seq]

description:
paired-end RNA-seq data of Pseudomonas sringae 1448A
(MM) WT control for PSPPH_4451-1 PSPPH_0641-1 PSPPH_3473-1 PSPPH_2262-1 PSPPH_3295-1 PSPPH_4001-1 PSPPH_5172-1 PSPPH_4827-1 PSPPH_4381-1 PSPPH_5115-1 PSPPH_3961-1 PSPPH_1874-1 PSPPH_2483-1 PSPPH_4852-1 PSPPH_1907-1 PSPPH_3360-1 PSPPH_4256-1 PSPPH_4074-1


title: WT-7 [RNA-seq]


title: WT-5 [RNA-seq]

description:
paired-end RNA-seq data of Pseudomonas sringae 1448A
(MM) WT control forPSPPH_2185-1 PSPPH_0253-1 PSPPH_0070-1 PSPPH_0147-1 PSPPH_0483-1 PSPPH_0805-1 PSPPH_1371-1 PSPPH_1905-1 PSPPH_2021-1 PSPPH_2083-1 PSPPH_1479-1 PSPPH_1194-1 PSPPH_1729-1


title: PSPPH-5115-1 [RNA-seq]

description: paired-end RNA-seq data of Pseudomonas sringae 1448A

value: PSPPH-5115 mutant


title: PSPPH-5172-1 [RNA-seq]

value: PSPPH-5172 mutant


title: WT-2 [RNA-seq]

description:
paired-end RNA-seq data of Pseudomonas sringae 1448A
(MM) WT control for PSPPH_1480 PSPPH_3276 PSPPH_4095 PSPPH_3446 PSPPH_4481 PSPPH_3550 PSPPH_4416 PSPPH_2606 PSPPH_4003


title: PSPPH-5172-2 [RNA-seq]


title: PSPPH-4827-2 [RNA-seq]

value: PSPPH-4827 mutant


title: WT-1 [RNA-seq]


title: PSPPH-4852-2 [RNA-seq]

value: PSPPH-4852 mutant


title: PSPPH-4451-2 [RNA-seq]

value: PSPPH-4451 mutant


title: PSPPH-4416-2 [RNA-seq]

value: PSPPH-4416 mutant


title: Human meningoma, QM110

status: Public on Oct 10 2022

city: San Francisco

first: William

last: Chen

state: CA

address: 1450 3rd Street, HD 402

institute: UCSF

library_source: transcriptomic single cell

data_processing:
Transcript quantification was performed with Kallisto (v0.46.2)
Assembly: GRCh38.96.gtf
Supplementary files format and content: TSV files containing abundance outputs from Kallisto, including estimated counts and TPM

organism: Homo sapiens

source_name: Meningioma

extract_protocol:
Frozen meningiomas were mechanically lysed using a TissueLyser II (QIAGEN) according to the manufacturer’s instructions. RNA were extracted from lysed tissue using the AllPrep DNA/RNA/miRNA Universal Kit (#80224, QIAGEN). RNA quality were initially assessed using a NanoDrop One (Thermo Fisher Scientific). RNA samples with 260/280 values less than 1.8 or 260/230 values less than 1.6 were cleaned using the RNA Cleanup protocol from the RNeasy Mini Kit (#74106, QIAGEN).
library preparation was performed using the TruSeq Standard mRNA Kit (#20020595, Illumina)

tag: tissue

value: Meningioma


title: PSPPH-4481-2 [RNA-seq]

value: PSPPH-4481 mutant


title: PSPPH-4451-1 [RNA-seq]


title: PSPPH-4416-1 [RNA-seq]


title: PSPPH-4827-1 [RNA-seq]


title: EAE Non immunized Input 1

city: REHOVOT

first: Steffen

last: Jung

address: Herzl

department: Immunology and Regenerative Biology

country: Israel

institute: The Weizmann Institute of Science

description: Source data Fig 2C.xlsx

data_processing:
UTAP analysis (https://doi.org/10.1186/s12859-019-2728-2)
Demultiplexing
Low-quality trimmng
UMI counting
Normalization
Assembly: mm10
Supplementary files format and content: Matrix table with normalized gene counts for every gene above 5 reads number and every sample

source_name: microglia

growth_protocol: Mice were maintained on a 12 hour light/dark cycle, with food and water provided ad libitum, and maintained under specific pathogen-free (SPF) conditions.

extract_protocol:
Mice were anesthetized with Pental (1:2 in PBS) and were perfused with PBS. Samples were homogenized on ice in ice-cold homogenization buffer (50 mM Tris, pH 7.4, 100 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 1:100 protease inhibitor (Sigma), 200 units/ml RNasin (Promega) and 0.1 mg/ml cycloheximide (Sigma) in RNase free DDW) 10% w/v with a Dounce homogenizer (Sigma) until the suspension was homogeneous. To remove cell debris, 1 ml of the homogenate was transferred to an Eppendorf tube and was centrifuged at 10,000g and 4°C for 10 min. Supernatants were transferred to a fresh Eppendorf tube on ice, then 10μl was removed for 'input' analysis and 10µg of anti-HA antibody (H9658, Sigma) or 10µg of mouse monoclonal IgG1 antibody (Sigma, Cat# PP100) was added to the supernatant, *followed by 4 h of incubation with slow rotation in a cold room at 4 °C. Meanwhile, Dynabeads Protein G (Thermo Fisher Scientific), 100 μl per sample, were equilibrated to homogenization buffer by washing three times. At the end of 4 h of incubation with antibody, beads were added to each sample, followed by incubation overnight in cold room at 4 °C. After not more than 12 h, samples were washed three times with high-salt buffer (50 mM Tris, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 1:200 protease inhibitor, 100 units/ml RNasin and 0.1 mg/ml cycloheximide in RNase free DDW), 5 min per wash in a cold room on a rotator. At the end of the washes, beads were magnetized and excess buffer was removed, 100µl Lysis Buffer  was added to the beads (Ambion Life Technologies). For Sort+aHA IP samples (RiboTag IP after sorting), 30,000 microglial cells were sorted directly into 100µl Lysis Buffer (Ambion Life Technologies) containing 10µg of anti-HA antibody (H9658, Sigma) or 10µg of mouse monoclonal IgG1 antibody (Sigma, Cat# PP100), the rest of IP was continued as above from *.
t cells were sorted from whole brain
PolyA-tailed mRNA was captured using the Dynabeads mRNA DIRECT kit (Ambion Life Technologies) according to manufacturer’s guidelines. RNA-seq libraries were constructed used a derivation of MARS-seq as described in Jaitin et al. (2014) adapted for bulk cell populations.
Mars-seq (DOI: 10.1126/science.1247651)

treatment_protocol: All mice were treated with Tamoxifen as follows: 5mg of tamoxifen (Sigma, diluted in corn oil) were administered to mice by gavage 4 times every other day at the age of 4-6 weeks.

value: F1 of C57BL/6 * SJL

tag: genotype

tag: organ

value: brain

tag: cell type

value: microglia

tag: immunoprecipitation antibody

tag: treatment

value: relapsing remmiting EAE


title: WT-6 [RNA-seq]


title: PSPPH-5115-2 [RNA-seq]


title: PSPPH-4381-2 [RNA-seq]

value: PSPPH-4381 mutant


title: PSPPH-4852-1 [RNA-seq]


title: PSPPH-4381-1 [RNA-seq]


title: PSPPH-4074-1 [RNA-seq]

value: PSPPH-4074 mutant


title: PSPPH-4256-1 [RNA-seq]

value: PSPPH-4256 mutant


title: PSPPH-4095-1 [RNA-seq]

value: PSPPH-4095 mutant


title: PSPPH-4003-1 [RNA-seq]

value: PSPPH-4003 mutant

value: liquid culture in minimal medium


title: PSPPH-4481-1 [RNA-seq]


title: PSPPH-3736-1 [RNA-seq]

value: PSPPH-3736 mutant


title: PSPPH-4001-1 [RNA-seq]

value: PSPPH-4001 mutant


title: PSPPH-4003-2 [RNA-seq]


title: PSPPH-3628-2 [RNA-seq]

value: PSPPH-3628 mutant


title: PSPPH-4001-2 [RNA-seq]


title: PSPPH-4256-2 [RNA-seq]


title: PSPPH-4074-2 [RNA-seq]


title: PSPPH-3550-1 [RNA-seq]

value: PSPPH-3550 mutant


title: PSPPH-3961-2 [RNA-seq]

value: PSPPH-3961 mutant


title: PSPPH-4095-2 [RNA-seq]


title: PSPPH-3473-2 [RNA-seq]

value: PSPPH-3473 mutant


title: PSPPH-3877-2 [RNA-seq]

value: PSPPH-3877 mutant


title: PSPPH-3877-1 [RNA-seq]


title: PSPPH-3906-2 [RNA-seq]

value: PSPPH-3906 mutant


title: PSPPH-3961-1 [RNA-seq]


title: PSPPH-3550-2 [RNA-seq]


title: PSPPH-3454-2 [RNA-seq]

value: PSPPH-3454 mutant


title: PSPPH-3906-1 [RNA-seq]


title: PSPPH-3473-1 [RNA-seq]


title: PSPPH-3446-2 [RNA-seq]

value: PSPPH-3446 mutant


title: PSPPH-3736-2 [RNA-seq]


title: PSPPH-3628-1 [RNA-seq]


title: PSPPH-3454-1 [RNA-seq]


title: PSPPH-3446-1 [RNA-seq]


title: PSPPH-3360-1 [RNA-seq]

value: PSPPH-3360 mutant


title: PSPPH-3386-2 [RNA-seq]

value: PSPPH-3386 mutant


title: PSPPH-3360-2 [RNA-seq]


title: PSPPH-3295-2 [RNA-seq]

value: PSPPH-3295 mutant


title: PSPPH-3276-1 [RNA-seq]

value: PSPPH-3276 mutant


title: PSPPH-3276-2 [RNA-seq]


title: PSPPH-3295-1 [RNA-seq]


title: PSPPH-3041-2 [RNA-seq]

value: PSPPH-3041 mutant


title: PSPPH-2606-2 [RNA-seq]

value: PSPPH-2606 mutant


title: PSPPH-2604-1 [RNA-seq]

value: PSPPH-2604 mutant

value: liquid culture in minimal medium


title: PSPPH-2606-1 [RNA-seq]


title: PSPPH-3041-1 [RNA-seq]


title: PSPPH-2604-2 [RNA-seq]


title: SK-N-DZ_Enhancer_deletion_WT_P3E12

city: La Jolla

first: Jesse

middle: R

last: Dixon

address: 10010 N. Torrey Pines Rd.

institute: Salk Institute for Biological Studies

data_processing:
RNA-seq data was aligned using STAR to the hg38 reference genome. PCR duplicate reads identified using STAR.
Processed RNA-seq files (bigWig) were generated by first generating bedGraph files from STAR. Separate bedGraph files were generated for reads aligning to the positive and negative strands. These were normalized for read depth (reads per million). Negative stranded bedGraph values were multiplied by -1 for display purposed. Bedgraphs were converted to bigwig files using wigToBigWig from UCSC.
Processed RNA-seq files (*rpkm.txt) were generated by first generating gene counts over Gencode genes using HTSeq. Expression was then normalized for gene length and total read counts (RPKM).
Assembly: hg38
Supplementary files format and content: bigWig files
Supplementary files format and content: text files (gene model RPKM)

molecule: polyA RNA

source_name: Engineered SK-N-DZ cell line

growth_protocol: Cell lines were grown in recommended media and passaged with TrypLE.

extract_protocol:
For RNA, cells were harvested in Trizol according to the manufacturers instrutions. After homogenization, samples were stored at -80C until further processing. RNA was isolate using the Purelink RNA mini kit with on column Dnase I treatment. RNA integrity was evaluated using RNA tapestation.
Stranded mRNA-seq libraries were constructed using the Illumina stranded mRNA seq kit according to the manufacturers instructions. Libaries were sequenced on the NextSeq 500 platform.

treatment_protocol: No treatments were performed

tag: cell line

value: SK-N-DZ

tag: experiment type

value: Stranded mRNA-seq

value: Engineered t(1;8) and deletion chr1:49,045,908- 49,063,390, different allele

tag: media

value: DMEM + 10% FBS + P/S + NEAA


title: SK-N-DZ_Enhancer_deletion_WT_P3H2


title: SK-N-DZ_C1C8_MYC_GFP_Hi-C

library_source: genomic

data_processing:
Hi-C reads were aligned to the hg38 reference genome using BWA and processed with custom perl scripts (available from https://github.com/dixonlab/HiC_alignment_basic_scripts).
Processed Hi-C files were generated by first converting aligned bam files to contact files using custom perl scripts (available from https://github.com/dixonlab/HiC_alignment_basic_scripts). *.hic or *.cool files were generated using juicer (*.hic files) or cooler (*.cool files).
Assembly: hg38
Supplementary files format and content: *hic files
Supplementary files format and content: *cool files

molecule: genomic DNA

extract_protocol:
For Hi-C, cells were fixed with 1% formaldehyde for 10 minutes at room temperture, quenched with 0.2M Glycine for 5 minutes. Adherent cells were fixed on the tissue culture plate and removed using a cell scraper. Suspension cells were fixed in suspension. After fixation and quenching, cells were pelleted, washed 1X with DPBS, pelleted again, and frozen on dry ice. Samples were stored in -80C until further processing.
In situ Hi-C was performed accoring to Rao et al. Cell 2014 using the MboI restriction enzyme. Libraries were sequenced on the NextSeq 500 platform.
MboI in situ Hi-C

value: MboI in situ Hi-C

value: Engineered t(1;8) and MYC-GFP reporter


title: PSPPH-2601-2 [RNA-seq]

value: PSPPH-2601 mutant


title: SK-N-DZ_Enhancer_deletion_WT_P3D6


title: SK-N-DZ_Enhancer_deletion_WT_P3C11


title: PSPPH-2601-1 [RNA-seq]


title: PSPPH-2510-2 [RNA-seq]

value: PSPPH-2510 mutant


title: SK-N-DZ_Enhancer_deletion_WT_P3B8


title: PSPPH-3386-1 [RNA-seq]


title: SK-N-DZ_Enhancer_deletion_WT_P3B5


title: SK-N-DZ_Enhancer_deletion_WT_P2F2


title: PSPPH-2377-2 [RNA-seq]

value: PSPPH-2377 mutant


title: PSPPH-2483-1 [RNA-seq]

value: PSPPH-2483 mutant


title: PSPPH-2483-2 [RNA-seq]


title: PSPPH-2377-1 [RNA-seq]


title: SK-N-DZ_Enhancer_deletion_Myc_P3F3

value: Engineered t(1;8) and deletion chr1:49,045,908- 49,063,390, same allele


title: PSPPH-2262-2 [RNA-seq]

value: PSPPH-2262 mutant


title: PSPPH-2262-1 [RNA-seq]


title: SK-N-DZ_Enhancer_deletion_Myc_P3D4


title: PSPPH-2083-2 [RNA-seq]

value: PSPPH-2083 mutant


title: SK-N-DZ_Enhancer_deletion_Myc_P2F3


title: PSPPH-2185-1 [RNA-seq]

value: PSPPH-2185 mutant


title: PSPPH-1907-1 [RNA-seq]

value: PSPPH-1907 mutant


title: PSPPH-2185-2 [RNA-seq]


title: PSPPH-2021-2 [RNA-seq]

value: PSPPH-2021 mutant


title: SK-N-DZ_Enhancer_deletion_Myc_P1F7


title: PSPPH-2021-1 [RNA-seq]

value: liquid culture in minimal medium


title: PSPPH-1905-1 [RNA-seq]

value: PSPPH-1905 mutant


title: PSPPH-1874-2 [RNA-seq]

value: PSPPH-1874 mutant


title: PSPPH-1907-2 [RNA-seq]


title: PSPPH-2083-1 [RNA-seq]


title: PSPPH-2510-1 [RNA-seq]


title: PSPPH-1905-2 [RNA-seq]


title: PSPPH-1729-1 [RNA-seq]

value: PSPPH-1729 mutant


title: PSPPH-1729-2 [RNA-seq]


title: PSPPH-1874-1 [RNA-seq]


title: SK-N-DZ_Enhancer_deletion_WT_P2D1

value: SK-N-DZ

value: Stranded mRNA-seq

value: Engineered t(1;8) and deletion chr1:49,045,908- 49,063,390, different allele

value: DMEM + 10% FBS + P/S + NEAA


title: SK-N-DZ_Enhancer_deletion_WT_P2E2


title: PSPPH-1568-2 [RNA-seq]

value: PSPPH-1568 mutant


title: PSPPH-1371-1 [RNA-seq]

value: PSPPH-1371 mutant


title: PSPPH-1479-2 [RNA-seq]

value: PSPPH-1479 mutant


title: PSPPH-1480-2 [RNA-seq]

value: PSPPH-1480 mutant


title: PSPPH-1568-1 [RNA-seq]


title: PSPPH-0805-2 [RNA-seq]

value: PSPPH-0805 mutant


title: PSPPH-1371-2 [RNA-seq]


title: PSPPH-1194-2 [RNA-seq]

value: PSPPH-1194 mutant


title: PSPPH-1479-1 [RNA-seq]


title: PSPPH-0641-1 [RNA-seq]

value: PSPPH-0641 mutant


title: PSPPH-0805-1 [RNA-seq]


title: PSPPH-1480-1 [RNA-seq]


title: PSPPH-0641-2 [RNA-seq]


title: PSPPH-0483-2 [RNA-seq]

value: PSPPH-0483 mutant


title: PSPPH-0147-1 [RNA-seq]

value: PSPPH-0147 mutant


title: PSPPH-0147-2 [RNA-seq]

value: liquid culture in minimal medium


title: PSPPH-0253-1 [RNA-seq]

value: PSPPH-0253 mutant


title: SK-N-DZ_602g1_P1C9_RNA

data_processing:
Hi-C reads were aligned to the hg38 reference genome using BWA and processed with custom perl scripts (available from https://github.com/dixonlab/HiC_alignment_basic_scripts).
Processed Hi-C files were generated by first converting aligned bam files to contact files using custom perl scripts (available from https://github.com/dixonlab/HiC_alignment_basic_scripts). *.hic or *.cool files were generated using juicer (*.hic files) or cooler (*.cool files).
RNA-seq data was aligned using STAR to the hg38 reference genome. PCR duplicate reads identified using STAR.
Processed RNA-seq files (bigWig) were generated by first generating bedGraph files from STAR. Separate bedGraph files were generated for reads aligning to the positive and negative strands. These were normalized for read depth (reads per million). Negative stranded bedGraph values were multiplied by -1 for display purposed. Bedgraphs were converted to bigwig files using wigToBigWig from UCSC.
Processed RNA-seq files (*rpkm.txt) were generated by first generating gene counts over Gencode genes using HTSeq. Expression was then normalized for gene length and total read counts (RPKM).
Engineered cell lines were given new formatted names in the main text. Formatted_names.xlsx here provides the links between engineered cell line names in raw data and formatted names in the manuscript.
Assembly: hg38
Supplementary files format and content: *hic files
Supplementary files format and content: *cool files
Supplementary files format and content: bigWig files
Supplementary files format and content: text files (gene model RPKM)
Supplementary files format and content: Formatted_names.xlsx

extract_protocol:
For Hi-C, cells were fixed with 1% formaldehyde for 10 minutes at room temperture, quenched with 0.2M Glycine for 5 minutes. Adherent cells were fixed on the tissue culture plate and removed using a cell scraper. Suspension cells were fixed in suspension. After fixation and quenching, cells were pelleted, washed 1X with DPBS, pelleted again, and frozen on dry ice. Samples were stored in -80C until further processing.
For RNA, cells were harvested in Trizol according to the manufacturers instrutions. After homogenization, samples were stored at -80C until further processing. RNA was isolate using the Purelink RNA mini kit with on column Dnase I treatment. RNA integrity was evaluated using RNA tapestation.
In situ Hi-C was performed accoring to Rao et al. Cell 2014 using the MboI restriction enzyme. Libraries were sequenced on the NextSeq 500 platform.
Stranded mRNA-seq libraries were constructed using the Illumina stranded mRNA seq kit according to the manufacturers instructions. Libaries were sequenced on the NextSeq 500 platform.
MboI in situ Hi-C
Stranded mRNA-seq

value: Engineered t(3;8)


title: SK-N-DZ_s4g2_D2_RNA

value: Engineered t(10;8)


title: SK-N-DZ_797g1_A11_RNA

value: SK-N-DZ

value: Engineered t(6;8)


title: SK-N-DZ_797g1_P1H9_RNA

value: Stranded mRNA-seq


title: PSPPH-0070-1 [RNA-seq]

value: PSPPH-0070 mutant


title: SK-N-DZ_602g1_P3E1_RNA

value: DMEM + 10% FBS + P/S + NEAA


title: PSPPH-1194-1 [RNA-seq]


title: PSPPH-0070-2 [RNA-seq]


title: SK-N-DZ_s4g1_A9_RNA


title: SK-N-DZ_s4g1_B7_RNA


title: PSPPH-0483-1 [RNA-seq]


title: PSPPH-0253-2 [RNA-seq]


title: SK-N-DZ_S4g1_F6_RNA


title: SK-N-DZ_s3g2_P1B9_RNA

value: Engineered t(15;8)


title: SK-N-DZ_797g1_A11_Hi-C

value: MboI in situ Hi-C


title: SK-N-DZ_s4g2_D2_Hi-C


title: SK-N-DZ_602g1_P1C9_Hi-C


title: SK-N-DZ_797g1_P1H9_Hi-C


title: SK-N-DZ_s4g1_A9_Hi-C


title: SK-N-DZ_S3g2_P2F2_Hi-C


title: SK-N-DZ_s3g2_P1B9_Hi-C


title: SK-N-DZ_C5BC8_P4C11_RNA

value: Engineered t(5;8)


title: SK-N-DZ_S3g2_P2F2_RNA


title: SK-N-DZ_S4g1_F6_Hi-C


title: SK-N-DZ_1002g1_P1F6_RNA

value: Engineered t(11;8)


title: SK-N-DZ_1002g2_P2B3_RNA


title: SK-N-DZ_s1g1_B10_RNA

value: Engineered t(16;8)


title: SK-N-DZ_602g1_P3E1_Hi-C


title: SK-N-DZ_BD1g1_P2A8_RNA


title: SK-N-DZ_s1g1_B2_Hi-C


title: SK-N-DZ_BD1g2_P1F7_RNA


title: SK-N-DZ_s3g2_P1D1_Hi-C


title: SK-N-DZ_s3g2_P1D1_RNA


title: SK-N-DZ_BD1g2_P1F7_Hi-C


title: SK-N-DZ_BD1g1_P2A8_Hi-C


title: SK-N-DZ_s1g1_B2_RNA


title: SK-N-DZ_1002g1_P1A7_RNA


title: SK-N-DZ_C12BC8_P1D3_RNA

value: Engineered t(12;8)


title: SK-N-DZ_1002g1_P1F6_Hi-C


title: SK-N-DZ_s4g1_B7_Hi-C


title: SK-N-DZ_s1g1_B10_Hi-C


title: SK-N-DZ_1002g1_P1A7_Hi-C


title: SK-N-DZ_1002g2_P2B3_Hi-C


title: SK-N-DZ_C5BC8_P4C11_Hi-C


title: Ptcy s2 ATAC-Seq

status: Public on Oct 01 2022

city: Seattle

first: Scott

middle: N

last: Furlan

address: 1100 Fairview Ave N

institute: CRD

data_processing:
cellranger arc 1.0.1 makefastq
cellranger arc 1.0.1 count
macs2 callpeaks (VERSION)
signac (VERSION)
Assembly: GRCh38
Supplementary files format and content: Raw counts

source_name: CD4+ and CD8+ T

growth_protocol: B6 recipients were transplanted with 5 x 10^6 BM with 0.5 x 10^6 CD4 + 0.5 x 10^6 CD8 T cells from C3H.SW donors (alloBMT).

extract_protocol:
CD4 and CD8 T cells were FACS sorted from BM (4 mice were pooled per group) at 14 days post-transplant
cDNA and fragments from transposase accessible gDNA were generated using 10X Multiome platform per manufacturers instructions

treatment_protocol: Half of the alloBMT recipients were treated with 50 mg/kg cyclophosphamide on D+3 and D+4 after transplantation (PTCy).

value: BM T cells

value: alloBMT with PT-Cy

tag: timepoint

value: D+14 post-SCT

value: C57Bl/6 with C3HSW donor


title: Ptcy s2 RNA-Seq


title: Retina_2

city: Aarhus

first: Lin

last: Lin

address: Høegh-Guldbergs Gade 10

department: Department of Biomedicine

country: Denmark

institute: Aarhus University

data_processing:
Cell Ranger 3.0.2 (10x Genomics) was used to process the raw sequencing data of polyA RNA libraries. The sequencing data from  nuclear RNA libraries was filtered, and the gene expression matrix was obtained using DNBelab C Series scRNA analysis software (MGI).
Assembly: Sscrofa11.1
Supplementary files format and content: matrix files

organism: Sus scrofa domesticus

extract_protocol:
The fresh tissues were collected, immediately placed on ice, and processed within 30 minutes.
The ployA RNA libraries were prepared following the manufacturer’s instructions of GemCode Single Cell 3’ Gel Bead and Library kit (v3 Chemistry ) from 10x Genomics, Inc. (Pleasanton, CA). The nuclear RNA library was operated on a DNBelab C4 device (MGI).

value: Retina


title: Liver_4

molecule: nuclear RNA

value: Liver


title: Retina_1


title: SK-N-DZ_C12BC8_P1D3_Hi-C

value: SK-N-DZ


title: AlloSCT s1 RNA-Seq

value: alloBMT


title: w_v24_3

status: Public on Sep 27 2022

city: Basel

first: DBM Bioinformatics

last: Core Facility

state: BS

address: Hebelstrasse 20

department: Departement of Biomedicine

country: Switzerland

institute: University of Basel

data_processing:
Read quality was assessed with the FastQC tool (version 0.11.5). Reads were mapped to the mouse genome (UCSC version mm10) with STAR (version 2.5.2a) with default parameters, except filtering out reads mapping to more than 10 genomic locations (outFilterMultimapNmax=10), reporting only one hit in the final alignment for multimappers (outSAMmultNmax=1) and filtering reads without evidence in the spliced junction table (outFilterType="BySJout").
Gene expression data analysis was performed using the R software (version 3.5). Read alignment quality was evaluated using the qQCReport function of the R Bioconductor package QuasR (version 1.18). Gene expression was quantified using the qCount function of QuasR as the number of reads (5'ends) overlapping with the exons of each gene assuming an exon union model (using the UCSC knownGenes annotation downloaded on 2015-12-18).
The R Bioconductor package edgeR (version 3.24.3) was used for differential gene expression analysis. Between samples normalization was done using the TMM method. Only genes with CPM (counts per million reads mapped) values more than 1 in at least 4 samples (the number of biological replicates) were retained. An generalized linear model including a genotype effect, an activation effect, and a replicate effect (nested within genotype) was fitted to the raw counts (function glmFit), and differential expression was tested using likelihood ratio tests (function glmLRT). P-values were adjusted by controlling the false-discovery rate (Benjamini-Hochberg method) and genes with a FDR lower than 1% were considered differentially expressed.
Gene set enrichment analysis was performed with the function camera from the edgeR package (using the default parameter value of 0.01 for the correlations of genes within gene sets) using gene sets from the curated gene set collection of the Molecular Signature Database (MSigDB v6.0). We considered only sets containing more than 10 genes and gene sets with a false discovery rate lower than 1% were considered significant.
Assembly: mm10
Supplementary files format and content: gzipped text file with tab-separated counts for each gene; each row contains the annotation and raw counts per gene; columns correspond to: 1. Entrez Gene ID, 2. gene length (exon union model), 3.-60. read counts per gene and sample.
Supplementary files format and content: gzipped text file with tab-separated counts falling on introns for each gene; each row contains the annotation and intronic counts per gene; columns correspond to: 1. Entrez Gene ID, 2. gene length (intron union model), 3.-60. read counts per gene and sample.

source_name: CD4+ T cells

growth_protocol: Naïve CD4+ T cells were isolated from cell suspensions of pooled lymph nodes and spleen from 10 mice (male and female). Isolation was performed with StemCell mouse naïve CD4+ T cell isolation kit according to manufacturer’s instructions.

extract_protocol:
2.5*10^5 cells were washed with PBS and resuspend in 200µl TRI Reagent. RNA was extracted from Trizol-samples with a Zymo Direct-zol kit which includes DNAse treatment.
RNA quality was assessed with a Fragment Analyzer (Advanced Analytical) and RNA-seq library preparation was performed using Illumina Truseq stranded kit. Sequencing was performed on an Illumina NexSeq 500 machine to produce single-end 76-mers reads. All steps were performed at the Genomics Facility Basel (ETH Zurich).

treatment_protocol: Cells were cultured for 24 or 48 hours on plates coated with a combination of anti-CD3 (0.25 or 0.5µg per ml PBS for very weak and weak activation samples respectively) and anti-CD28 (0.2µg per ml PBS) mAb to mimic activation and co-activation signals. Plates were washed with PBS before plating the cells in complete T cell medium supplied with 50U IL2/ml.

value: wt

tag: activation

value: CD4+ T cells

value: very weak


title: l_v48_2

value: T1792 delta/delta


title: w_v48_3


title: t_v48_3

value: T1792 tg/tg


title: t_v48_4


title: t_v48_2


title: w_v48_4


title: l_v48_4


title: w_v24_2


title: w_v48_2


title: w_v24_4


title: t_v48_1


title: l_v48_1


title: w_v24_1


title: w_v48_1


title: AlloSCT s1 ATAC-Seq


title: t_v24_1


title: t_v24_3


title: t_v24_4


title: l_v24_4


title: t_v24_2


title: l_v24_3


title: l_v48_3


title: w_w48_2

value: weak


title: t_n_4

value: naive


title: w_n_2


title: w_n_1


title: w_w48_3


title: w_w48_4


title: l_v24_2


title: w_w48_1


title: t_w48_4


title: l_w48_3


title: t_w48_1


title: t_w24_2


title: t_w24_3


title: w_w24_1


title: w_w24_2


title: t_w24_4


title: l_w24_3


title: l_w24_2


title: w_w24_4


title: l_w48_2


title: t_w24_1


title: w_w24_3


title: l_w24_4


title: l_w24_1


title: t_w48_3


title: w_n_4


title: l_v24_1


title: t_w48_2


title: l_w48_4


title: l_w48_1


title: RNAseq_Hek293_qko_mbd_dmso_3

first: Dirk

last: Schuebeler

address: Fabrikstrasse 24

institute: Friedrich Miescher Institute for Biomedical Research

data_processing:
Illumina RTA 2.4.1 (NextSeq 500) and bcl2fastq2 v2.17 or lllumina RTA 3.4.5 (NovaSeq 6000) and bcl2fastq2 v2.20 was used for basecalling and demultiplexing.
Assembly: mm10 or hg19
Supplementary files format and content: Alignments (first read for paired-end) overlapping the opposite strand of any exon of a known gene (gene annotation from the Bioconductor package TxDb.Mmusculus.UCSC.mm10.knownGene, version 3.10.0 or TxDb.Hsapiens.UCSC.hg19.knownGene, version (v3.2.2) ) were counted using the qCount function from the QuasR Bioconductor package (Gaidatzis et al, Bioinformatics, 2015) with default parameters.
Supplementary files format and content: totalRNA_knownGene_mm10_esc_siRNA_treated.csv
Supplementary files format and content: totalRNA_knownGene_hg19_hek293.csv

extract_protocol: Sequencing libraries were prepared from purified RNA in triplicates using TruSeq stranded total RNA Library Prep (Illumina)

value: human embryonic kidney cells

value: MBD quadruple knock-out

value: kidney; Embryo


title: RNAseq_Hek293_qko_mbd_dmso_1


title: l_n_2


title: l_n_4


title: t_n_2


title: t_n_1


title: l_n_1


title: RNAseq_Hek293_qko_mbd_aza_3


title: RNAseq_Hek293_qko_mbd_dmso_2


title: RNAseq_Hek293_qko_mbd_aza_2


title: RNAseq_Hek293_wt_aza_3

value: wild-type


title: RNAseq_Hek293_wt_dmso_3


title: RNAseq_ESC_tko_mbd_siRNA_mbd4_3

description: totalRNA_knownGene_mm10_esc_siRNA_treated.csv

source_name: esc

value: ebryonic stem cells

value: MBD triple knock-out

value: HA36 cells (mixed 129-C57Bl/6 strain)


title: RNAseq_Hek293_wt_dmso_2


title: RNAseq_ESC_tko_mbd_siRNA_mbd4_1


title: RNAseq_Hek293_wt_aza_2


title: RNAseq_ESC_tko_mbd_siRNA_cntrl_3


title: RNAseq_Hek293_wt_dmso_1


title: RNAseq_ESC_tko_mbd_siRNA_cntrl_2


title: RNAseq_ESC_tko_mbd_siRNA_cntrl_1


title: RNAseq_Hek293_wt_aza_1


title: t_n_3


title: RNAseq_Hek293_qko_mbd_aza_1


title: RNAseq_ESC_tko_mbd_siRNA_mbd4_2


title: KLF4_ChIP_delta862-1253_rescue_Etop_R2

status: Public on Sep 23 2022

city: Kunming

first: Ping

last: Zheng

address: Qingsong Road 21

country: China

institute: Kunming Institute of Zoology, Chinease Academy of Sciences

data_processing:
Basecalls performed using CASAVA version 2.19
For RNA-Seq, reads were mapped to mm10 genome using tophat2 (version 2.0.8) with default parameters. Differentialy expressed genes were determined using cuffdiff (version 2.1.1);ChIP-seq reads were aligned to the mm10 genome assembly using Bowtie version 2.1.0.
Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) were calculated using cufflinks version 2.1.1
Peaks were called using MACS version 2.2.7.1 with the following setting: qvalue=0.01.
Assembly: mm10
Supplementary files format and content: files include FPKM values for each Sample
Supplementary files format and content: peak text files

source_name: mouse embryonic stem cells

growth_protocol: Mouse ESCs were routinely maintained in DMEM/F12 supplemented with 20% Knockout serum replacement (Thermo Fisher,, A3181502), 104 unit/mL recombinant mouse leukemia inhibitory factor (Millipore, ESG1107), 2mM L-glutamine (Sigma; G8540-25g), penicillin (100 U/mL)/streptomycin (100 μg/mL) (Gibco; 15140-122), 100 μM beta-mercaptoethanol (Sigma; M7522), 1×MEM non-essential amino acids solution (Gibco; 11140-035).

extract_protocol:
For RNA-Seq, RNA were isolated using Trizol. For ChIP-Seq,cell lysates were clarified from sonicated nuclei and KLF4 or P53-DNA complexes were isolated with antibody. KLF4 antibody:vendor:R&D systems; cat#:AF3158; lot#:WRR0719041; P53 antibody:P53(S15）:Vendor:CST; cat#:9284; lot#: 21.
RNA libraries were prepared for sequencing using standard Illumina protocols;For ChIP-Seq, Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303).

treatment_protocol: Cells were treated or untreated with 10 uM etoposide.

value: Lnc956 knockout and overexpression of fragment of Lnc956 which 862-1253nt was deleted.

value: 10 uM etoposide for 3 hours


title: KLF4_ChIP_delta862-1253_rescue_Etop_R1


title: KLF4_ChIP_FL_rescue_R2

value: Lnc956 knockout and overexpression of Full length of Lnc956

value: untreated


title: KLF4_ChIP_FL_rescue_R1

value: Lnc956 knockout and overexpression Full length of Lnc956


title: KLF4_ChIP_FL_rescue_Etop_R2


title: KLF4_ChIP_lnc956_KO_Etop_R2

value: Lnc956 knockout


title: KLF4_ChIP_FL_rescue_Etop_R1


title: KLF4_ChIP_delta862-1253_rescue_R1


title: KLF4_ChIP_lnc956_KO_R2


title: KLF4_ChIP_lnc956_KO_Etop_R1


title: KLF4_ChIP_WT_Etop_R2

value: wild type


title: KLF4_ChIP_WT_con_rep2


title: Control

status: Public on Sep 26 2022

city: Seoul

first: Duk Ki

last: Kim

address: Daehakro 103

country: South Korea

institute: Seoul National University of Hospital

description: Single cell RNA sequencing data

data_processing:
the Cell Ranger toolkit provided by 10X Genomics was applied to align reads and produce the gene-cell unique molecular identifier (UMI) matrix using the reference genome GRCm38.
Assembly: GRCm38

source_name: Whole live cells of Pan02 tumor

growth_protocol: After Pan02 cancer cell injection to pancreas of mice, all mice were housed in a ventillated temperature-controlled environment (median temperature 23C, humidity 65%), under a 12-hour light/dark cycle, with a free access to food and water. Mouse cages were changed weekly.

extract_protocol:
After sacrificng mice, tumors were resected from mice, and digested with Dnase, Hyaluronidase, Collagenase. Digested tissues were minced, filtered through a 70-um cell strainer, and made into single-cell suspensions. Single-cell suspensions prepared from Pan02 samples were stained with a Live/Dead fixable dye and sorted by live cells using a BD Aria III sorter.
Libraries were prepared using the Chromium controller according to the 10X Single Cell 3' v3 protocol (10x Genomics). The cell suspensions were diluted in nuclease-free water to obtain a cell count of 10,000. The cell suspension was mixed with master mix and loaded with Single-cell 3? Gel Beads and Partitioning Oil into a Single-cell 3' Chip. Transcripts from single cells were uniquely barcoded and reverse-transcribed within droplets. cDNA molecules were pooled, and the cDNA pool was subjected to end-repair, single "A" base addition, and adapter ligation. The products were then purified and enriched via PCR to create the final cDNA library. The purified libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA) and qualified using the Agilent Technologies 4200 TapeStation (Agilent Technologies).

treatment_protocol: Gemcitabine or Ate-Grab with Gemcitabine were injected to mice 5 times every 3 days after tumor size up to 50-100mm^3

value: FACS sorted live cells of naive Pan02 tumor.

value: No treatment


title: KLF4_ChIP_lnc956_KO_R1


title: KLF4_ChIP_WT_Etop_R1


title: Lnc956_KO_and_P53_KD_Etop_0h_rep2

value: Lnc956 knockout and P53 knockdown


title: Lnc956_KO_and_P53_KD_Etop_9h_rep1

treatment_protocol: Cells were treated or untreated with 1 uM etoposide.

value: 1 uM etoposide for 9 hours


title: KLF4_ChIP_WT_con_rep1


title: Lnc956_KO_and_P53_KD_Etop_3h_rep1

value: 1 uM etoposide for 3 hours


title: Lnc956_KO_and_P53_KD_Etop_6h_rep2

value: 1 uM etoposide for 6 hours


title: Lnc956_KO_and_P53_KD_Etop_6h_rep1


title: Lnc956_KO_and_P53_KD_Etop_9h_rep2

value: 10 uM etoposide for 9 hours


title: KLF4_ChIP_delta862-1253_rescue_R2


title: P53_KD_Etop_9h_rep2

value: P53 knockdown


title: Lnc956_KO_and_P53_KD_Etop_0h_rep1


title: CT-2

city: Shanghai

first: ying

last: yan

address: 320 Yueyang Road, Shanghai

institute: Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences

data_processing:
Libraries were prepared according to the Illumina TruSeq protocol
Sequenced reads pooled pooled  for deep sequencing by using Illumina Hiseq Xten (2 x 150) platforms
Adaptor sequences and read sequences on both ends with  Phred quality scores below 30 were trimmed. Trimmed reads were then mapped with the Hisat2  algorithm (Hisat2 v2.1.0) to target sequences
Gene expression levels were quantified by the software  package HTSeq (v0.6.1p1).
Assembly: GRCm38
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample

source_name: CT, liver

growth_protocol: Male C57BL/6J mice aged about 8-12 weeks were used.

extract_protocol:
Total RNA was isolated by using Trizol reagent from liver
Libraries were prepared according to the Illumina TruSeq protocol and pooled for deep sequencing by using Illumina Hiseq Xten (2 x150) platforms

treatment_protocol: No treatment.

value: liver

tag: gender

value: male


title: CT-3


title: P53_KD_Etop_6h_rep1

value: 10 uM etoposide for 6 hours


title: P53_KD_Etop_6h_rep2


title: P53_KD_Etop_3h_rep1


title: P53_KD_Etop_0h_rep2


title: Lnc956_KO_Etop_9h_rep1


title: P53_KD_Etop_0h_rep1


title: Lnc956_KO_Etop_6h_rep1


title: Lnc956_KO_Etop_9h_rep2


title: CT-1


title: Lnc956_KO_and_P53_KD_Etop_3h_rep2


title: Lnc956_KO_Etop_6h_rep2


title: Lnc956_KO_Etop_3h_rep2

value: 10 uM etoposide for 3 hours


title: Lnc956_KO_Etop_3h_rep1


title: WT_Etop_9h_rep2


title: Lnc956_KO__Etop_0h_rep2


title: P53_KD_Etop_9h_rep1


title: WT_Etop_6h_rep2


title: WT_Etop_6h_rep1


title: Control replicate 2

status: Public on Sep 22 2022

city: Caparica

first: Daniel

last: Sobral

address: Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia - Universidade Nova de Lisboa, Campus de Caparica

department: Life Sciences

country: Portugal

institute: UCIBIO - FCT NOVA

data_processing:
RNA sequencing reads were aligned against the Drosophila melanogaster BDGP release 6 genome using the STAR aligner (2.7.2a)
Gene-level counts were obtained using featureCounts (v1.6.3); parameters: -s 2 -p -B -P -C
ChIPseq reads aligned to BDGP release 6 genome using Bowtie2
bigWig files were obtained from RNAseq and ChIPseq alignments transformed with the deeptools (3.5.0) bamcoverage tool with CPM normalization
Assembly: BDGP6
Supplementary files format and content: bigWig

organism: Drosophila melanogaster

source_name: fixed chromatin from oreR ovaries

growth_protocol: Ovaries from 2 days old females (after pupae eclosion) were isolated

extract_protocol:
whole ovaries fixed 10 min. in 2% paraformaldehyde, sonicated in Qsonica Q800R3 chilled-bath sonicator (4ºC, 20sec on, 20sec off, 20 min, 50% amplitude)
Takara bio ThruPLEX DNA-seq kit

treatment_protocol: For negative control or Sfmbt depleted ovaries, we used the nanos-Gal4 driver for germ line-specific expression of, respectively, UAS-mCherry RNAi (negative control) or UAS-Sfmbt RNAi (Sfmbt RNAi-1)

value: fixed sonicated chromatin precipitated with rabbit anti-GFP (Invitrogen A-11122)

tag: replicate

value: untagged


title: WT_Etop_9h_rep1


title: GFP Sfmbt replicate 1

source_name: fixed chromatin from GFP-Sfmbt ovaries

value: GFP-Sfmbt (knock-in)


title: Endometriosis Patient 9 - Ectopic Adjacent-2

status: Public on Sep 21 2022

city: Farmington

middle: F

last: Flynn

state: CT

address: 10 Discovery Drive

institute: The Jackson Laboratory

data_processing:
FASTQ files for all samples were generated and demultiplexed using Illumina bcl2fastq 2.20.0.422
A filtered digital gene expression matrix was generated for each single cell gene expression library against the 10X Genomics GRCh38 reference build (version 3.0.0, GRCh38.93) using 10X Genomics CellRanger count version 3.1.0 for all samples.
A digital counts matrix was generated for each single cell hashtag library using CITE-Seq-Count (https://github.com/Hoohm/CITE-seq-Count), version 1.4.5. This produces a matrix of counts with hashtag labels for rows (available via BioLegend TotalSeq-A antihuman HashTags https://www.biolegend.com/totalseq) and cell barcodes for columns.
For all single cell samples, R1 contains a 16bp cell barcode + 12bp UMI barcode.  For all samples, R2 contains a 91bp biological read.
For all bulk RNA-seq samples, data was aligned to the same 10X Genomics GRCh38.93 human reference using nf-core/rnaseq v1.4.2 with the STAR aligner using Nextflow 20.07.1 (build 5412).
In order to further safeguard patients' genomic identities, SNVs relative to the reference genome are masked in all bam files (BAMboozle v0.5.0).
Assembly: GRCh38.93
Supplementary files format and content: Transcriptomic and HashTag (HTO) counts matrices in Matrix Market CSR format.  Rows correspond to genes in the order provided in genes.tsv/features.tsv and columns correspond to cell-barcodes in the order provided in barcodes.tsv.

growth_protocol: Organoid libraries (EOR01/EOR03) were grown from remaining single cells suspension from tissue dissociation, in 100% Matrigel dome,with expansion media described in Boretto et al. (2019).

extract_protocol:
Single cells were collected through cold active protease dissociation protocol, viable cell sorting strategy. 12,000 single cells were loaded into one lane of a 10X Genomics Chromium controller, targetted 6,000 barcoded single cells. Single cell capture, barcoding and library preparation were performed using the 10X Chromium platform, version 3 chemistry. cDNA and libraries were checked for quality on Agilent 4200 Tapestation, quantified by KAPA qPCR, and sequenced on an Illumina NovaSeq 6000 (either an S2 or S4 flow cell) targetting 100,000 raw read pairs per cell.  For Cell Hashing libraries, cells from each input sample were labeled with BioLegend TotalSeq-A anti-human hashtags prior to loading onto the 10X Genomics Chromium system and were sequenced by pooling the gene expression and hashtag libraries at a 20:1 ratio before sequencing on an Illumina NovaSeq 6000, targetting 100k read pairs per cell. For Bulk libraries, fresh tissue were either preserved in RNA Later or flash frozen before extraction. Frozen tissues were submitted to the Genome Technologies core service at the Jackson Laboratory for RNA extraction, and sequenced on NovaSeq6000 (Illumina) with SP 100 cycles single-end reads kit resulting in, on average, 42.7 million reads per sample.
10X Genomics Chromium v3 chemistry

treatment_protocol: Endometriosis patients receiving oral contraceptive treatment were subject to this study.  Endometrial, peroteonial, ovarian tissue were collected immediately upon surgerical ressection.

value: Endometriosis

tag: sample location

value: Ectopic Adjacent

value: Peritoneum

tag: method

value: scRNA-seq

tag: library type

value: Gene Expression


title: GFP Sfmbt replicate 2


title: Control replicate 1


title: P53_KD_Etop_3h_rep2


title: Pho replicate 2

source_name: fixed chromatin from yw ovaries

value: fixed sonicated chromatin precipitated with rabbit anti-Pho (Brown et al. 2003)


title: Endometriosis Patient 9 - Ectopic Adjacent-1

tag: condition


title: Pho replicate 1


title: Endometriosis Patient 4 - Ectopic

description: Endometriosis Patient 4 - Ectopic

value: Ectopic

tag: tissue


title: Endometriosis Patient 9 - Ectopic

description: Endometriosis Patient 9 - Ectopic


title: Endometriosis Patient 5 - Ectopic Adjacent


title: Endometriosis Patient 4 - Ectopic Adjacent


title: Lnc956_KO_Etop_0h_rep1

tag: genotype

value: Lnc956 knockout

value: untreated


title: Endometriosis Patient 3 - Ectopic Adjacent


title: Endometriosis Patient 2 - Ectopic

description: Endometriosis Patient 2 - Ectopic


title: Endometriosis Patient 2 - Ectopic Adjacent


title: Endometriosis Patient 5 - Ectopic

description: Endometriosis Patient 5 - Ectopic


title: Endometriosis Patient 1 - Ectopic

description: Endometriosis Patient 1 - Ectopic


title: ID_006715_WT_shock_O2_C

status: Public on Sep 20 2022

city: Würzburg

first: Falk

last: Ponath

address: Josef-Schneider-Straße 2

country: Germany

institute: Helmholtz Institute for RNA-based Infection Research (HIRI)

description: samples were grown to mid-exponetial phase and treated according column "treatment"

data_processing:
Reads from the RNA-seq experiments were trimmed and filtered using the FASTX toolkit (v.0.10.1; http://hannonlab.cshl.edu/fastx_toolkit). Mapping was performed using READemption (v.1.01)(Förstner et al., 2014) against the genome sequence for F. nucleatum subsp. nucleatum ATCC 23726 (NZ_CP028109.1) downloaded from the National Center for Biotechnology Information (NCBI). Differential gene expression analysis was carried using DEseq2 (v.1.18.01) (Love et al., 2014). All analysis for each was carried for biological triplicates and considered significant with a false-discovery rate ≤ 0.05.All genes had to additionally show a fold change either ≤ 1 or ≥ -1 to be marked as significantly differentially expressed.
Assembly: NZ_CP028109.1
Supplementary files format and content: wiggle

source_name: Fusobacterium nucleatum subsp. nucleatum ATCC 23726 - clean deletion of sRNA FoxI

growth_protocol: Growth was performed in Columbia broth at 37°C inside an anaerobic chamber. Pre-cultures were inoculated with a signel colony. After 24h, the working cultures were inoculated at a dilution 1:50 and samples taken during the mid-exponential growth phase.

extract_protocol:
Total RNA was extracted using the hot phenol protocol. Bacterial cultures were grown to the desired OD600, mixed with 0.2 volumes of STOP solution (95 % ethanol, 5 % phenol) and snap-frozen at – 80 °C if not directly processed. The bacterial solution was centrifuged for 20 min, 4500 rpm at 4 °C and the supernatant was completely discarded. Cells were suspended in 600 μl of 10 mg/ml lysozyme in TE buffer (pH 8.0) and incubated at 37 °C for 10 min. Next, 60 μl of 10 % w/v SDS was added and everything mixed by inversion. Samples were incubated in a water bath at 64 °C, 1-2 min before adding 66 μl 3 M NaOAc, pH 5.2. Next, 750 μl acid phenol (Roti-Aqua phenol) was added, followed by incubation for 5 min. at 64 °C. Samples were briefly placed on ice to cool before centrifugation for 15 min, 13,000 rpm at 4 °C. The aqueous layer was transferred into a 2 ml phase lock gel tube (Eppendorf), 750 μl chloroform (Roth, #Y015.2) was added, and everything centrifuged for 12 min, 13,000 rpm at room temperature. For ethanol precipitation, the aqueous layer was transferred to a fresh tube, 2 volumes of 30:1 mix (EtOH:3 M NaOAc, pH 6.5) was added and incubated overnight at -20 °C. Precipitated RNA was harvested by centrifugation, washed with cold 75 % v/v ethanol and air-dried. Purified RNA was resuspended in 50 µl RNase-free water and stored at -80 °C.
The cDNA library preparation was carried by Vertis (Munich, Germany). The first step consisted out of rRNA removal. The rRNA depleted RNA was then fragmented via ultrasound (1 pulse of 30 seconds; 4 °C). The fragmented RNA was then used to ligate an adapter to the 3’ end of the molecules. For first-strand cDNA synthesis, the M-MLV reverse transcriptase was used in conjunction with the introduced 3’-adapter serving as a primer. After purification, 5’ Illumina TruSeq sequencing adapters were ligated to the cDNA. The resulting cDNA was used as input for during PCR amplification (10 – 20 ng µl-1). The amplified cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and further evaluated via capillary electrophoresis. The cDNA was pooled further purified to include only cDNA from 200 – 600 bp using a preparative agarose gel. The finished pooled libraries were sequenced by the Core unit SysMed (University of Würzburg) using an Illumina NextSeq 500 system and 75 bp read length.

treatment_protocol: see description of samples

value: 20 min. exposure to oxygen

tag: growth phase

value: mid-exponetial phase

value: wildtype


title: Endometriosis Patient 1 - Ectopic Adjacent


title: ID_006714_WT_shock_O2_B

organism: Fusobacterium nucleatum subsp. nucleatum ATCC 23726


title: ID_006713_WT_shock_O2_A


title: Endometriosis Patient 3 - Ectopic

description: Endometriosis Patient 3 - Ectopic


title: ID_006709_WT_shock_polyB_C

source_name: Fusobacterium nucleatum subsp. nucleatum ATCC 23726

value: 20 min + 400ng/ml polymyxin B


title: 2-3

city: little rock

first: Dongzheng

last: Gai

state: AR

address: 4301 W Markham St

institute: UAMS

data_processing:
The raw sequencing data (fastq files) were preprocessed using CellRanger software (10x Genomics) version 6 with reference genome of Mus musculus version mm10 to demultiplex for cell and transcript and generate count table.
The count table were load in to R through Seurat version 4 package for further analysis.
Principal component analysis (PCA) was then performed on significantly variable genes of the remained high-quality cells. The results of individual sample were used for data integration across samples using reciprocal PCA method to minimize technical differences between samples.
Then the nitration results were used as input for clustering using Louvain algorithm with multilevel refinement and Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP). The gene specific markers of each cluster were identified using the FindMarkersAll function with MAST method test statistics. The expression pattern of selected gene markers across cell cluster were plot as a heat map to assess the quality of cell clustering result.
To visualize some of gene markers simultaneously in kernel joint density estimatiscon, Nebulosa package was used. Based on the kernel joint density of Adgr1 and Fcgr3, we sub select cells that have high value of the kernel joint density for subclsutering analysis to study the cellular heterogeneity of Macrophage cells. Gene-set enrichment analysis of marker genes was performed on Gene Ontology annotation using piano package.
Assembly: mmp10
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample

source_name: Mice bone marrow mononuclear cells

extract_protocol:
The RNA were prepared using the Chromium Single Cell 3′ Reagent Kits (v3.1) follow the standard protocol.
The libraries were prepared using the Chromium Single Cell 3′ Reagent Kits (v3.1) follow the standard protocol.

tag: strain

value: Mice bone marrow mononuclear cells

tag: age


title: Endometriosis Patient 10 - Ectopic Ovary

value: Ectopic Ovary

value: Ovary


title: ID_006707_WT_shock_polyB_A

tag: treatment


title: ID_006703_WT_shock_ctrl_C


title: ID_006701_WT_shock_ctrl_A


title: ID_006708_WT_shock_polyB_B


title: ID_006702_WT_shock_ctrl_B


title: SKOV3_shNC_2

status: Public on Sep 19 2022

city: Jinan

first: Zixiang

last: Wang

state: Shandong

address: No.44 Wenhuaxi Road

department: Cheeloo College of Medicine

institute: Shandong University

description: SKOV3_NC1_NC2_NC3VSSH1_SH2_SH3.all_genes_fpkm_expression.tar.gz

data_processing:
Sequenced reads were generated by base calling using the Illumina standard pipeline
Raw reads were filtered to obtain clean reads after removal of reads containing adapter and at low quality
High-quality reads were mapped to the human reference genome hg38 using HISAT2 software
Alignment was independently performed for reads from each sample, and reads mapping to more than three genomic sites were discarded
Featurecounts and DESeq2 were used to perform differential gene expression analysis for mRNAs by calculating counts
Assembly: hg38
Supplementary files format and content: Gene FPKM of all the samples was provided as a compressed tar.gz file.

extract_protocol:
Total RNA was extracted using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Cells were harvested 72h after doxycycline induction.

tag: cell line

tag: cell type

value: serous ovarian cancer cell line

tag: transfection

value: shNC


title: A2780_shBUD31_2

description: A2780_NC1_NC2_NC3VSSH1_SH2_SH3.all_genes_fpkm_expression.tar.gz


title: A2780_shNC_2


title: SKOV3_shBUD31_1


title: SKOV3_shBUD31_3


title: SKOV3_shNC_3


title: A2780_shBUD31_3


title: A2780_shNC_1


title: HEYA8_BUD31_1

description: HEYA8_NC1_NC2_NC3VSOE1_OE2_OE3.all_genes_fpkm_expression.tar.gz


title: SKOV3_shNC_1


title: A2780_shNC_3


title: HEYA8_NC_2

value: NC


title: SKOV3_shBUD31_2


title: HEYA8_BUD31_2


title: A2780_shBUD31_1


title: HEYA8_NC_1


title: ATACseq adult alveolar macrophages in vitro MxCre Cebpb KO IFNa rep3 [AM_MxCre_CebpbKO_IFNa_3]

status: Public on Sep 16 2022

city: Turku

first: alexander

last: mildner

address: Tykistökatu 6A

department: Biomedicine

country: Finland

institute: University of Turku / Medicity

data_processing:
RNA-seq:
We used fastuniq to collapse PCR duplicates
STAR v2.6.1a was used to align unique reads to the mouse genome (GRCm38).
Gene expression was quantified using featureCounts v1.6.3 and the Gencode vM12 reference
ATAC-seq:
ATAC-seq reads were aligned to the mouse genome v. GRCm38 using the BWA-MEM algorithm implemented in the bwa program (Li, 2013), v. 0.7.17.
peaks were called with MACS2 v. 2.2.7.1
Peak calling was done with the R package DiffBind v. 2.14
Differential binding was done using the R package DESeq2 v. 1.26
Peaks were annotated using ChIPseeker v. 1.22.1
Assembly: GRCm38
Supplementary files format and content: RNA-seq: gene counts (featureCounts) as csv file
Supplementary files format and content: ATAC-seq: Annotation file peak_annotation.xlsx contains the PrimaryIDs and the output of ChIPseeker. Count matrix count_matrix.xlsx contains the counts collected from DiffBind.
ChIP-seq:
ChIPmentation data was analyzed with the Pigx pipeline for ChIP-seq data, which comprised a read trimming with Trim Galore, read mapping to the mouse genome mm10 with Bowtie2 and peak calling with MACS2 (R. Stark, G. B. R. P. version, 2011). Peaks were called relative to input sample background.
Resulting peaks were filtered by FDR with a cutoff of 1e-05 and merged using Diffbind while retaining only those peaks found in both replicates
Peaks were annotated to genes using GREAT
A coverage map of the peaks was prepared with deeptools.
Supplementary files format and content: ChIP-seq: sample peaks called relative to input sample background by MACS

source_name: in vitro cultured alveolar macrophages adult

extract_protocol:
bronchoalveolar lavage with 5x 1ml PBS with 2mM EDTA and 1% FBS
For the preparation of ATAC-seq libraries 15,000-20,000 cells were used as described earlier (Lara-Astiaso et al., 2014; Buenrostro et al., 2013). Briefly, nuclei were isolated by lysis with cold lysis buffer (10mM Tris-HCl pH7.4, 10mM MgCl2, 0.1% Igepal CA-630) and centrifuged for 25min at 500xg and 4°C with low acceleration and brake using a swing-out rotor. The supernatant was carefully removed and nuclei were resuspended in 25μl reaction buffer containing 2μl of Tn5 transposase and 12.5μl of TD buffer (Nextera DNA library preparation kit; Illumina). The mix was incubated at 37°C for 1hr. Afterwards, the DNA was purified by adding 5μl of cleanup buffer (900mM NaCl, 30mM EDTA), 2μl of proteinase K (NEB) and 2μl of 5% SDS. After an incubation phase of 30min at 40°C, the tagmented DNA was enriched using AMPure XP beads (Beckman Coulter). The DNA was PCR-amplified with indexed primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems). After the PCR, tagmented DNA fragments were selected for fragments smaller than 600bp and purified using AMPure XP beads. Libraries were subjected to a second PCR amplification with Kapa HiFi Hotstart ready mix (Kapa Biosystems), indexing primers and an appropriate number of reaction cycles depending on the library concentration. Library sizes were determined using a TapeStation (Agilent Technologies) and measurement of DNA concentration was performed with a Qubit fluorometer (Life Technologies). Libraries where sequenced with an average of 20 million reads per sample on a NextSeq500 system (Illumina).
RNA-Seq:Cells for RNA-Seq or ATAC-Seq were isolated by FACS. Cells for RNA-Seq (500-20,000 sorted cells) were lysed with 50-200µl of lysis/binding buffer (Life Technologies), frozen on dry ice and stored at -80°C until further use. The mRNA was purified using a Dynabeads mRNA DIRECT Purification Kit (Life Technologies) according to the manufacturer’s guidelines. Reverse transcription was performed with MARS-seq barcoded RT primers and the Affinity Script cDNA Synthesis Kit (Agilent) in a 10µl reaction volume.
RNA-Seq:mRNA was subjected to reverse transcription using an Affinity Script cDNA Synthesis Kit (Agilent) and MARS-seq barcoded RT primers. Samples were analyzed by qPCR and up to 8 samples with similar Ct values were combined. Pooled samples were then treated with Exonuclease I (New England BioLabs (NEB)) and incubated at 37°C for 30min and 80°C for 10min followed by a 1.2X AMPure XP beads (Beckman Coulter) cleanup. Second strand synthesis of the cDNA was performed using a second strand synthesis kit (NEB) at 16°C for 2h and samples were cleaned with 1.4X volumes of AMPure XP beads. Afterwards samples were subjected to in vitro transcription (IVT) with a HiScribe T7 RNA Polymerase kit (NEB) at 37°C for 13-16h. The DNA template was removed by Turbo DNase I (Life Technologies) treatment at 37°C for 15min and the samples were cleaned with 1.2X volumes of AMPure XP beads. Subsequently, the RNA was fragmented at 70°C using RNA fragmentation buffer (Invitrogen), after 3min the reaction was stopped with Stop buffer (Invitrogen) and samples were purified by 2X SPRI cleanup. Afterwards T4 RNA ligase (NEB) was used to ligate the fragmented RNA to the MARS-seq ligation adapter (22°C 2h) and samples were purified with a 1.5X AMPure XP beads cleanup. In a second reverse transcription reaction (reaction volume 20µl) using the MARS-seq RT2 primer and an Affinity Script cDNA Synthesis Kit (Agilent Technologies) the RNA was transcribed into cDNA followed by another 1.5X AMPure XP beads purification. As final step the library was amplified in a nested PCR with P5_Rd1 and P7_Rd2 primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems) and purified with a 0.7X AMPure XP beads cleanup. The fragment size of the final libraries was measured using a TapeStation (Agilent Technologies) and library concentrations were quantified with a Qubit fluorometer (Life Technologies). The samples were sequenced using a NextSeq 500 system (Illumina).
ATAC-seq: For the preparation of ATAC-seq libraries 15,000-20,000 cells were used as described earlier (Lara-Astiaso et al., 2014; Buenrostro et al., 2013). Briefly, nuclei were isolated by lysis with cold lysis buffer (10mM Tris-HCl pH7.4, 10mM MgCl2, 0.1% Igepal CA-630) and centrifuged for 25min at 500xg and 4°C with low acceleration and brake using a swing-out rotor. The supernatant was carefully removed and nuclei were resuspended in 25μl reaction buffer containing 2μl of Tn5 transposase and 12.5μl of TD buffer (Nextera DNA library preparation kit; Illumina). The mix was incubated at 37°C for 1hr. Afterwards, the DNA was purified by adding 5μl of cleanup buffer (900mM NaCl, 30mM EDTA), 2μl of proteinase K (NEB) and 2μl of 5% SDS. After an incubation phase of 30min at 40°C, the tagmented DNA was enriched using AMPure XP beads (Beckman Coulter). The DNA was PCR-amplified with indexed primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems). After the PCR, tagmented DNA fragments were selected for fragments smaller than 600bp and purified using AMPure XP beads. Libraries were subjected to a second PCR amplification with Kapa HiFi Hotstart ready mix (Kapa Biosystems), indexing primers and an appropriate number of reaction cycles depending on the library concentration. Library sizes were determined using a TapeStation (Agilent Technologies) and measurement of DNA concentration was performed with a Qubit fluorometer (Life Technologies). Libraries where sequenced with an average of 20 million reads per sample on a NextSeq500 system (Illumina).
ChIP-seq: Preparation of ChIPmentation samples was performed according to Schmidl et al., 2015. 2M AMs were BAL-isolated from C57BL/6 mice and fixed in a 1% PFA solution (in 10% FBS/PBS). The fixation reaction was quenched on ice by addition of glycine (125mM final concentration) and the samples were centrifuged at 300xg for 5min at 4°C. Subsequently cell pellets were washed twice with cold PBS and 1X Protease Inhibitor (Roche), snap-frozen in liquid nitrogen and stored at -80°C until further processing. The samples were thawed on ice and resuspended in Chromatin Prep Buffer (High Sensitivity Chromatin Preparation kit, Active Motif) with 1X Protease Inhibitor (Roche) before nuclei were released using a dounce homogenizer with a tight pestel (Active Motif) and centrifuged at 4°C and 1250xg for 5min. Nuclei were then resuspended in cold Sonication Buffer (0.25% SDS, 10mM Tris-HCl pH 8.0, 2mM EDTA, 1X Protease Inhibitor) and pipetted to facilitate nuclei disruption. The chromatin was sonicated using a Diagenode Bioruptor (12 cycles: 20sec on, 30sec off) to achieve a fragment size ranging from 200-500bp. 1% of the sonicated chromatin was used for the INPUT sample, the rest was incubated overnight with pre-washed A Dynabeads (Thermofisher; previously blocked with 0.1% BSA) and 4μg of anti-C/EBPβ antibody (C-19; Santa Cruz Biotechnology). The following day, the beads were washed twice with RIPA-LS (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 140mM NaCl, 0.1% SDS, 0.1% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with RIPA-HS (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 500mM NaCl, 0.1% SDS, 0.1% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with RIPA-LiCl (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 250mM LiCl, 0.1% SDS, 0.5% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with 10mM Tris-HCl pH 8.0 and finally resuspended and incubated in the Tagmentation Solution (0.25% Tagmentation Buffer, 2mM Tn5 (Illumina)) for 2min at 37°C. The tagmentation reaction was stopped on ice by adding cold RIPA-LS. Later, beads were washed twice in RIPA-LS, twice in 1X TE and finally resuspended in ChIP Elution Buffer (10mM Tris-HCl pH8.0, 5mM EDTA pH8.0, 300mM NaCl, 0.4% SDS). Samples were de-crosslinked overnight and purified on the following day using AMPure XP beads (NEB). Libraries were amplified using KAPA HiFi Hotstart ready mix (Kapa Biosystems) and indexing primers published by Schmidl et al. 2015 (22) and finally purified using AMPure XP beads (NEB). Library sizes were determined using a TapeStation (Agilent Technologies) and measurement of DNA concentration was performed with a Qubit fluorometer (Life Technologies). Libraries were sequenced with an average of 30 million reads per sample on a NextSeq500 system (Illumina).

value: Cebpbtm1.1Elgaz/J crossed with Tg(Mx1-cre)1Cgn/J

tag: strain background

value: MxCre+ Cebpb flox/flox

value: bronchoalveolar lavage

value: AMs were isolated by BAL, seeded in RPMI complete medium in non-tissue culture treated 12-well plates and incubated at 37°C to allow the AMs to adhere. After 4h the medium was exchanged to remove dead cells, debris and cells that were not adherent. AMs were then cultured in RPMI complete medium supplemented with 20ng/ml CSF2 (Stemcell Technologies) for 7 days to expand the cells. Every 2 to 3 days the medium was replaced. At day 7 cells were treated with 1000units/ml of mouse IFNα (Miltenyi) to induce Cre-mediated recombination. IFNα treatment was repeated twice in two-day intervals. At day 14 all cells were harvested for analysis.


title: ATACseq adult alveolar macrophages in vitro MxCre IFNa rep2 [AM_MxCre_IFNa_2]

value: MxCre+


title: ATACseq adult alveolar macrophages in vitro MxCre Cebpb KO IFNa rep2 [AM_MxCre_CebpbKO_IFNa_2]


title: ChIPseq adult alveolar macrophages wildtype rep2 - anti-C/EBPb [AM_WT_CEBPb_2]

source_name: alveolar macrophages adult

extract_protocol:
bronchoalveolar lavage with 5x 1ml PBS with 2mM EDTA and 1% FBS
Preparation of ChIPmentation samples was performed according to Schmidl et al., 2015. 2M AMs were BAL-isolated from C57BL/6 mice and fixed in a 1% PFA solution (in 10% FBS/PBS). The fixation reaction was quenched on ice by addition of glycine (125mM final concentration) and the samples were centrifuged at 300xg for 5min at 4°C. Subsequently cell pellets were washed twice with cold PBS and 1X Protease Inhibitor (Roche), snap-frozen in liquid nitrogen and stored at -80°C until further processing. The samples were thawed on ice and resuspended in Chromatin Prep Buffer (High Sensitivity Chromatin Preparation kit, Active Motif) with 1X Protease Inhibitor (Roche) before nuclei were released using a dounce homogenizer with a tight pestel (Active Motif) and centrifuged at 4°C and 1250xg for 5min. Nuclei were then resuspended in cold Sonication Buffer (0.25% SDS, 10mM Tris-HCl pH 8.0, 2mM EDTA, 1X Protease Inhibitor) and pipetted to facilitate nuclei disruption. The chromatin was sonicated using a Diagenode Bioruptor (12 cycles: 20sec on, 30sec off) to achieve a fragment size ranging from 200-500bp. 1% of the sonicated chromatin was used for the INPUT sample, the rest was incubated overnight with pre-washed A Dynabeads (Thermofisher; previously blocked with 0.1% BSA) and 4μg of anti-C/EBPβ antibody (C-19; Santa Cruz Biotechnology). The following day, the beads were washed twice with RIPA-LS (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 140mM NaCl, 0.1% SDS, 0.1% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with RIPA-HS (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 500mM NaCl, 0.1% SDS, 0.1% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with RIPA-LiCl (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 250mM LiCl, 0.1% SDS, 0.5% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with 10mM Tris-HCl pH 8.0 and finally resuspended and incubated in the Tagmentation Solution (0.25% Tagmentation Buffer, 2mM Tn5 (Illumina)) for 2min at 37°C. The tagmentation reaction was stopped on ice by adding cold RIPA-LS. Later, beads were washed twice in RIPA-LS, twice in 1X TE and finally resuspended in ChIP Elution Buffer (10mM Tris-HCl pH8.0, 5mM EDTA pH8.0, 300mM NaCl, 0.4% SDS). Samples were de-crosslinked overnight and purified on the following day using AMPure XP beads (NEB). Libraries were amplified using KAPA HiFi Hotstart ready mix (Kapa Biosystems) and indexing primers published by Schmidl et al. 2015 (22) and finally purified using AMPure XP beads (NEB). Library sizes were determined using a TapeStation (Agilent Technologies) and measurement of DNA concentration was performed with a Qubit fluorometer (Life Technologies). Libraries were sequenced with an average of 30 million reads per sample on a NextSeq500 system (Illumina).
RNA-Seq:Cells for RNA-Seq or ATAC-Seq were isolated by FACS. Cells for RNA-Seq (500-20,000 sorted cells) were lysed with 50-200µl of lysis/binding buffer (Life Technologies), frozen on dry ice and stored at -80°C until further use. The mRNA was purified using a Dynabeads mRNA DIRECT Purification Kit (Life Technologies) according to the manufacturer’s guidelines. Reverse transcription was performed with MARS-seq barcoded RT primers and the Affinity Script cDNA Synthesis Kit (Agilent) in a 10µl reaction volume.
RNA-Seq:mRNA was subjected to reverse transcription using an Affinity Script cDNA Synthesis Kit (Agilent) and MARS-seq barcoded RT primers. Samples were analyzed by qPCR and up to 8 samples with similar Ct values were combined. Pooled samples were then treated with Exonuclease I (New England BioLabs (NEB)) and incubated at 37°C for 30min and 80°C for 10min followed by a 1.2X AMPure XP beads (Beckman Coulter) cleanup. Second strand synthesis of the cDNA was performed using a second strand synthesis kit (NEB) at 16°C for 2h and samples were cleaned with 1.4X volumes of AMPure XP beads. Afterwards samples were subjected to in vitro transcription (IVT) with a HiScribe T7 RNA Polymerase kit (NEB) at 37°C for 13-16h. The DNA template was removed by Turbo DNase I (Life Technologies) treatment at 37°C for 15min and the samples were cleaned with 1.2X volumes of AMPure XP beads. Subsequently, the RNA was fragmented at 70°C using RNA fragmentation buffer (Invitrogen), after 3min the reaction was stopped with Stop buffer (Invitrogen) and samples were purified by 2X SPRI cleanup. Afterwards T4 RNA ligase (NEB) was used to ligate the fragmented RNA to the MARS-seq ligation adapter (22°C 2h) and samples were purified with a 1.5X AMPure XP beads cleanup. In a second reverse transcription reaction (reaction volume 20µl) using the MARS-seq RT2 primer and an Affinity Script cDNA Synthesis Kit (Agilent Technologies) the RNA was transcribed into cDNA followed by another 1.5X AMPure XP beads purification. As final step the library was amplified in a nested PCR with P5_Rd1 and P7_Rd2 primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems) and purified with a 0.7X AMPure XP beads cleanup. The fragment size of the final libraries was measured using a TapeStation (Agilent Technologies) and library concentrations were quantified with a Qubit fluorometer (Life Technologies). The samples were sequenced using a NextSeq 500 system (Illumina).
ATAC-seq: For the preparation of ATAC-seq libraries 15,000-20,000 cells were used as described earlier (Lara-Astiaso et al., 2014; Buenrostro et al., 2013). Briefly, nuclei were isolated by lysis with cold lysis buffer (10mM Tris-HCl pH7.4, 10mM MgCl2, 0.1% Igepal CA-630) and centrifuged for 25min at 500xg and 4°C with low acceleration and brake using a swing-out rotor. The supernatant was carefully removed and nuclei were resuspended in 25μl reaction buffer containing 2μl of Tn5 transposase and 12.5μl of TD buffer (Nextera DNA library preparation kit; Illumina). The mix was incubated at 37°C for 1hr. Afterwards, the DNA was purified by adding 5μl of cleanup buffer (900mM NaCl, 30mM EDTA), 2μl of proteinase K (NEB) and 2μl of 5% SDS. After an incubation phase of 30min at 40°C, the tagmented DNA was enriched using AMPure XP beads (Beckman Coulter). The DNA was PCR-amplified with indexed primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems). After the PCR, tagmented DNA fragments were selected for fragments smaller than 600bp and purified using AMPure XP beads. Libraries were subjected to a second PCR amplification with Kapa HiFi Hotstart ready mix (Kapa Biosystems), indexing primers and an appropriate number of reaction cycles depending on the library concentration. Library sizes were determined using a TapeStation (Agilent Technologies) and measurement of DNA concentration was performed with a Qubit fluorometer (Life Technologies). Libraries where sequenced with an average of 20 million reads per sample on a NextSeq500 system (Illumina).
ChIP-seq: Preparation of ChIPmentation samples was performed according to Schmidl et al., 2015. 2M AMs were BAL-isolated from C57BL/6 mice and fixed in a 1% PFA solution (in 10% FBS/PBS). The fixation reaction was quenched on ice by addition of glycine (125mM final concentration) and the samples were centrifuged at 300xg for 5min at 4°C. Subsequently cell pellets were washed twice with cold PBS and 1X Protease Inhibitor (Roche), snap-frozen in liquid nitrogen and stored at -80°C until further processing. The samples were thawed on ice and resuspended in Chromatin Prep Buffer (High Sensitivity Chromatin Preparation kit, Active Motif) with 1X Protease Inhibitor (Roche) before nuclei were released using a dounce homogenizer with a tight pestel (Active Motif) and centrifuged at 4°C and 1250xg for 5min. Nuclei were then resuspended in cold Sonication Buffer (0.25% SDS, 10mM Tris-HCl pH 8.0, 2mM EDTA, 1X Protease Inhibitor) and pipetted to facilitate nuclei disruption. The chromatin was sonicated using a Diagenode Bioruptor (12 cycles: 20sec on, 30sec off) to achieve a fragment size ranging from 200-500bp. 1% of the sonicated chromatin was used for the INPUT sample, the rest was incubated overnight with pre-washed A Dynabeads (Thermofisher; previously blocked with 0.1% BSA) and 4μg of anti-C/EBPβ antibody (C-19; Santa Cruz Biotechnology). The following day, the beads were washed twice with RIPA-LS (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 140mM NaCl, 0.1% SDS, 0.1% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with RIPA-HS (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 500mM NaCl, 0.1% SDS, 0.1% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with RIPA-LiCl (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 250mM LiCl, 0.1% SDS, 0.5% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with 10mM Tris-HCl pH 8.0 and finally resuspended and incubated in the Tagmentation Solution (0.25% Tagmentation Buffer, 2mM Tn5 (Illumina)) for 2min at 37°C. The tagmentation reaction was stopped on ice by adding cold RIPA-LS. Later, beads were washed twice in RIPA-LS, twice in 1X TE and finally resuspended in ChIP Elution Buffer (10mM Tris-HCl pH8.0, 5mM EDTA pH8.0, 300mM NaCl, 0.4% SDS). Samples were de-crosslinked overnight and purified on the following day using AMPure XP beads (NEB). Libraries were amplified using KAPA HiFi Hotstart ready mix (Kapa Biosystems) and indexing primers published by Schmidl et al. 2015 (22) and finally purified using AMPure XP beads (NEB). Library sizes were determined using a TapeStation (Agilent Technologies) and measurement of DNA concentration was performed with a Qubit fluorometer (Life Technologies). Libraries were sequenced with an average of 30 million reads per sample on a NextSeq500 system (Illumina).

tag: chip antibody

value: C/EBPb (C-19, Santa Cruz Biotechnology)


title: ChIPseq adult alveolar macrophages wildtype rep1 - input DNA [AM_WT_input_1]


title: chimera_10w alveolar macrophages CD45.1-WT rep3 [lung_1.1_10w_3 ]

source_name: donor bone marrow-derived alveolar macrophages adult

extract_protocol:
lung digestion in RPMI complete with 1mM collagenase IV and 2mg/ml DNase I at 37°C for 30 min
mRNA was subjected to reverse transcription using an Affinity Script cDNA Synthesis Kit (Agilent) and MARS-seq barcoded RT primers. Samples were analyzed by qPCR and up to 8 samples with similar Ct values were combined. Pooled samples were then treated with Exonuclease I (New England BioLabs (NEB)) and incubated at 37°C for 30min and 80°C for 10min followed by a 1.2X AMPure XP beads (Beckman Coulter) cleanup. Second strand synthesis of the cDNA was performed using a second strand synthesis kit (NEB) at 16°C for 2h and samples were cleaned with 1.4X volumes of AMPure XP beads. Afterwards samples were subjected to in vitro transcription (IVT) with a HiScribe T7 RNA Polymerase kit (NEB) at 37°C for 13-16h. The DNA template was removed by Turbo DNase I (Life Technologies) treatment at 37°C for 15min and the samples were cleaned with 1.2X volumes of AMPure XP beads. Subsequently, the RNA was fragmented at 70°C using RNA fragmentation buffer (Invitrogen), after 3min the reaction was stopped with Stop buffer (Invitrogen) and samples were purified by 2X SPRI cleanup. Afterwards T4 RNA ligase (NEB) was used to ligate the fragmented RNA to the MARS-seq ligation adapter (22°C 2h) and samples were purified with a 1.5X AMPure XP beads cleanup. In a second reverse transcription reaction (reaction volume 20µl) using the MARS-seq RT2 primer and an Affinity Script cDNA Synthesis Kit (Agilent Technologies) the RNA was transcribed into cDNA followed by another 1.5X AMPure XP beads purification. As final step the library was amplified in a nested PCR with P5_Rd1 and P7_Rd2 primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems) and purified with a 0.7X AMPure XP beads cleanup. The fragment size of the final libraries was measured using a TapeStation (Agilent Technologies) and library concentrations were quantified with a Qubit fluorometer (Life Technologies). The samples were sequenced using a NextSeq 500 system (Illumina).
RNA-Seq:Cells for RNA-Seq or ATAC-Seq were isolated by FACS. Cells for RNA-Seq (500-20,000 sorted cells) were lysed with 50-200µl of lysis/binding buffer (Life Technologies), frozen on dry ice and stored at -80°C until further use. The mRNA was purified using a Dynabeads mRNA DIRECT Purification Kit (Life Technologies) according to the manufacturer’s guidelines. Reverse transcription was performed with MARS-seq barcoded RT primers and the Affinity Script cDNA Synthesis Kit (Agilent) in a 10µl reaction volume.
RNA-Seq:mRNA was subjected to reverse transcription using an Affinity Script cDNA Synthesis Kit (Agilent) and MARS-seq barcoded RT primers. Samples were analyzed by qPCR and up to 8 samples with similar Ct values were combined. Pooled samples were then treated with Exonuclease I (New England BioLabs (NEB)) and incubated at 37°C for 30min and 80°C for 10min followed by a 1.2X AMPure XP beads (Beckman Coulter) cleanup. Second strand synthesis of the cDNA was performed using a second strand synthesis kit (NEB) at 16°C for 2h and samples were cleaned with 1.4X volumes of AMPure XP beads. Afterwards samples were subjected to in vitro transcription (IVT) with a HiScribe T7 RNA Polymerase kit (NEB) at 37°C for 13-16h. The DNA template was removed by Turbo DNase I (Life Technologies) treatment at 37°C for 15min and the samples were cleaned with 1.2X volumes of AMPure XP beads. Subsequently, the RNA was fragmented at 70°C using RNA fragmentation buffer (Invitrogen), after 3min the reaction was stopped with Stop buffer (Invitrogen) and samples were purified by 2X SPRI cleanup. Afterwards T4 RNA ligase (NEB) was used to ligate the fragmented RNA to the MARS-seq ligation adapter (22°C 2h) and samples were purified with a 1.5X AMPure XP beads cleanup. In a second reverse transcription reaction (reaction volume 20µl) using the MARS-seq RT2 primer and an Affinity Script cDNA Synthesis Kit (Agilent Technologies) the RNA was transcribed into cDNA followed by another 1.5X AMPure XP beads purification. As final step the library was amplified in a nested PCR with P5_Rd1 and P7_Rd2 primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems) and purified with a 0.7X AMPure XP beads cleanup. The fragment size of the final libraries was measured using a TapeStation (Agilent Technologies) and library concentrations were quantified with a Qubit fluorometer (Life Technologies). The samples were sequenced using a NextSeq 500 system (Illumina).
ATAC-seq: For the preparation of ATAC-seq libraries 15,000-20,000 cells were used as described earlier (Lara-Astiaso et al., 2014; Buenrostro et al., 2013). Briefly, nuclei were isolated by lysis with cold lysis buffer (10mM Tris-HCl pH7.4, 10mM MgCl2, 0.1% Igepal CA-630) and centrifuged for 25min at 500xg and 4°C with low acceleration and brake using a swing-out rotor. The supernatant was carefully removed and nuclei were resuspended in 25μl reaction buffer containing 2μl of Tn5 transposase and 12.5μl of TD buffer (Nextera DNA library preparation kit; Illumina). The mix was incubated at 37°C for 1hr. Afterwards, the DNA was purified by adding 5μl of cleanup buffer (900mM NaCl, 30mM EDTA), 2μl of proteinase K (NEB) and 2μl of 5% SDS. After an incubation phase of 30min at 40°C, the tagmented DNA was enriched using AMPure XP beads (Beckman Coulter). The DNA was PCR-amplified with indexed primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems). After the PCR, tagmented DNA fragments were selected for fragments smaller than 600bp and purified using AMPure XP beads. Libraries were subjected to a second PCR amplification with Kapa HiFi Hotstart ready mix (Kapa Biosystems), indexing primers and an appropriate number of reaction cycles depending on the library concentration. Library sizes were determined using a TapeStation (Agilent Technologies) and measurement of DNA concentration was performed with a Qubit fluorometer (Life Technologies). Libraries where sequenced with an average of 20 million reads per sample on a NextSeq500 system (Illumina).
ChIP-seq: Preparation of ChIPmentation samples was performed according to Schmidl et al., 2015. 2M AMs were BAL-isolated from C57BL/6 mice and fixed in a 1% PFA solution (in 10% FBS/PBS). The fixation reaction was quenched on ice by addition of glycine (125mM final concentration) and the samples were centrifuged at 300xg for 5min at 4°C. Subsequently cell pellets were washed twice with cold PBS and 1X Protease Inhibitor (Roche), snap-frozen in liquid nitrogen and stored at -80°C until further processing. The samples were thawed on ice and resuspended in Chromatin Prep Buffer (High Sensitivity Chromatin Preparation kit, Active Motif) with 1X Protease Inhibitor (Roche) before nuclei were released using a dounce homogenizer with a tight pestel (Active Motif) and centrifuged at 4°C and 1250xg for 5min. Nuclei were then resuspended in cold Sonication Buffer (0.25% SDS, 10mM Tris-HCl pH 8.0, 2mM EDTA, 1X Protease Inhibitor) and pipetted to facilitate nuclei disruption. The chromatin was sonicated using a Diagenode Bioruptor (12 cycles: 20sec on, 30sec off) to achieve a fragment size ranging from 200-500bp. 1% of the sonicated chromatin was used for the INPUT sample, the rest was incubated overnight with pre-washed A Dynabeads (Thermofisher; previously blocked with 0.1% BSA) and 4μg of anti-C/EBPβ antibody (C-19; Santa Cruz Biotechnology). The following day, the beads were washed twice with RIPA-LS (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 140mM NaCl, 0.1% SDS, 0.1% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with RIPA-HS (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 500mM NaCl, 0.1% SDS, 0.1% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with RIPA-LiCl (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 250mM LiCl, 0.1% SDS, 0.5% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with 10mM Tris-HCl pH 8.0 and finally resuspended and incubated in the Tagmentation Solution (0.25% Tagmentation Buffer, 2mM Tn5 (Illumina)) for 2min at 37°C. The tagmentation reaction was stopped on ice by adding cold RIPA-LS. Later, beads were washed twice in RIPA-LS, twice in 1X TE and finally resuspended in ChIP Elution Buffer (10mM Tris-HCl pH8.0, 5mM EDTA pH8.0, 300mM NaCl, 0.4% SDS). Samples were de-crosslinked overnight and purified on the following day using AMPure XP beads (NEB). Libraries were amplified using KAPA HiFi Hotstart ready mix (Kapa Biosystems) and indexing primers published by Schmidl et al. 2015 (22) and finally purified using AMPure XP beads (NEB). Library sizes were determined using a TapeStation (Agilent Technologies) and measurement of DNA concentration was performed with a Qubit fluorometer (Life Technologies). Libraries were sequenced with an average of 30 million reads per sample on a NextSeq500 system (Illumina).

value: whole lung tissue

value: For the generation of BM chimeras, 8-12 weeks old recipient mice (CD45.1/2) were lethally irradiated. On the following day, the recipients were reconstituted with 5x10^6 BM cells, which were a mixture of 33% WT (CD45.1/1) and 66% CD11c-Cre Cebpbfl/fl (CD45.2/2; CD11cKO) BM cells, by tail vein injection. The animals received an antibiotic treatment with Enrofloxacin in their drinking water for 10 days after irradiation. BM chimeras were analyzed 5 and 10 weeks after transfer.


title: ATACseq adult alveolar macrophages in vitro MxCre Cebpb KO IFNa rep1 [AM_MxCre_CebpbKO_IFNa_1]


title: HEYA8_BUD31_3


title: ATACseq adult alveolar macrophages in vitro MxCre IFNa rep1 [AM_MxCre_IFNa_1]


title: HEYA8_NC_3


title: chimera_10w alveolar macrophages CD45.1-WT rep4 [lung_1.1_10w_4 ]


title: chimera_10w alveolar macrophages CD45.1-WT rep2 [lung_1.1_10w_2 ]


title: chimera_5w alveolar macrophages CD45.1-WT rep2 [lung_1.1_5w_2]


title: ChIPseq adult alveolar macrophages wildtype rep2 - input DNA [AM_WT_input_2]


title: chimera_5w alveolar macrophages CD45.1-WT rep1 [lung_1.1_5w_1]


title: ChIPseq adult alveolar macrophages wildtype rep1 - anti-C/EBPb [AM_WT_CEBPb_1]


title: chimera_5w alveolar macrophages CD45.2-CD11cCre Cebpb KO rep3 [lung_2.2_5w_3]

value: Cebpbtm1.1Elgaz/J crossed with Tg(Itgax-cre,-EGFP)4097Ach/J

value: CD11c +/Cre Cebpb flox/flox


title: chimera_10w alveolar macrophages CD45.2-CD11cCre Cebpb KO rep1 [lung_2.2_10w_1 ]


title: chimera_5w alveolar macrophages CD45.2-CD11cCre Cebpb KO rep4 [lung_2.2_5w_4]


title: chimera_5w alveolar macrophages CD45.1-WT rep3 [lung_1.1_5w_3]

value: wildtype


title: chimera_10w alveolar macrophages CD45.2-CD11cCre Cebpb KO rep2 [lung_2.2_10w_2 ]


title: E18 Ly6Chi lung fetal monocytes Cebpb KO rep4 [KO_Ly6Chi_4 ]

source_name: lung Ly6Chi fetal monocytes E18

value: C/EBPb tm1Pfj

value: mixed background

value: Cebpb -/-


title: chimera_10w alveolar macrophages CD45.2-CD11cCre Cebpb KO rep3 [lung_2.2_10w_3 ]


title: E18 Ly6Chi lung fetal monocytes Cebpb KO rep3 [KO_Ly6Chi_3 ]


title: chimera_5w alveolar macrophages CD45.2-CD11cCre Cebpb KO rep1 [lung_2.2_5w_1]


title: E18 Ly6Chi lung fetal monocytes Cebpb KO rep1 [KO_Ly6Chi_1 ]


title: E18 Ly6Chi lung fetal monocytes Cebpb KO rep2 [KO_Ly6Chi_2 ]


title: chimera_10w alveolar macrophages CD45.1-WT rep1 [lung_1.1_10w_1 ]


title: chimera_5w alveolar macrophages CD45.2-CD11cCre Cebpb KO rep2 [lung_2.2_5w_2]


title: E18 Ly6Chi lung fetal monocytes Cebpb WT rep3 [WT_Ly6Chi_3 ]

value: Cebpb +/+


title: E18 Ly6Chi lung fetal monocytes Cebpb WT rep1 [WT_Ly6Chi_1 ]


title: HFLF_AO_P2

city: Kyoto

first: Shimpei

last: Gotoh

address: 53 Kawaharacho, Shogoin, Sakyo-ku

department: Center for iPS cell Research and Application

country: Japan

institute: Kyoto University

data_processing:
Reads were mapped to GRCh38 and count matrices were generated using Cell Ranger.
Assembly: GRCh38
Supplementary files format and content: Format: H5. Content: filtered_feature_bc_matrix

source_name: iPSC derived alveolar organoids

extract_protocol:
iPSC-derived alveolar organoids were trypsinized, washed twice, filtered through 40 μm strainer and suspended in the alveolarization medium containing 10 μM Y-27632 . Then the single cell suspension of iPSC-derived alveolar organoids was loaded onto a Chromium device (10x Genomics) for generating single cell libraries.
Single cell libraries were generated using Chromium Next GEM Chip G single Cell Kit (PN-1000127) and Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead kit v3.1 (PN-1000128) or Chromium Next GEM Single Cell 3’ kit v3.1 (Dual Index) (PN-1000269). iMES-AO at P2 was processed using Single Index Kit T Set A (PN-1000213), iMES-AO  at P0 and Alveolar organoids with HFLF at P0 and P2 using Dual Index Kit TT Set A (PN-1000215).

value: SFTPC-GFP reporter iPSC (B2-3) and HFLF

tag: culture condition

value: 3D culture


title: iMES_AO_P0

value: SFTPC-GFP reporter iPSC (B2-3) and its parental iPSC (201B7)


title: HFLF_AO_P0


title: CH12_LAM-HTGTS_Irf2bp2_WT_ctrl

status: Public on Sep 15 2022

city: Vienna

first: Tobias

last: Neumann

address: Campus-Vienna-Biocenter 1

country: Austria

institute: IMP

description:
Genomic DNA was extracted and Irf2bp2 genomic region  was biotin isolated and PCR enriched
LAM-HTGTS

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

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

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

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


title: CH12_LAM-HTGTS_Irf2bp2_C13_deltaori_ctrl


title: CH12_LAM-HTGTS_Irf2bp2_C13_restored_ctrl


title: CH12_LAM-HTGTS_Irf2bp2_C13_restored


title: CH12_LAM-HTGTS_Irf2bp2_C13_deltaori


title: E18 Ly6Chi lung fetal monocytes Cebpb WT rep2 [WT_Ly6Chi_2 ]


title: CH12_LAM-HTGTS_Irf2bp2_WT


title: CH12_LAM-HTGTS_Irf2bp2_shMcm


title: CH12_LAM-HTGTS_C13_restored_ctrl

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


title: CH12_LAM-HTGTS_WT_ctrl

value: WT ctrl


title: CH12_LAM-HTGTS_C2_restored_rep3

value: C2 restored


title: CH12_LAM-HTGTS_Irf2bp2_shLacZ_ctrl


title: CH12_LAM-HTGTS_Irf2bp2_shLacZ


title: CH12_LAM-HTGTS_Irf2bp2_shLacZ_hotspots_rep2


title: CH12_LAM-HTGTS_C2_restored_ctrl


title: CH12_LAM-HTGTS_Irf2bp2_shLacZ_hotspots_rep3


title: CH12_LAM-HTGTS_WT_restored_rep2

value: WT restored


title: CH12_LAM-HTGTS_C13_deltaori_ctrl


title: CH12_LAM-HTGTS_C2_restored_rep2


title: CH12_LAM-HTGTS_Irf2bp2_shLacZ_hotspots_rep1


title: CH12_LAM-HTGTS_C2_restored_rep1


title: CH12_LAM-HTGTS_WT_restored_rep3


title: CH12_LAM-HTGTS_C13_restored_rep3

value: C13 restored


title: CH12_LAM-HTGTS_C13_restored_rep2


title: CH12_LAM-HTGTS_C13_restored_rep1


title: CH12_LAM-HTGTS_Irf2bp2_shMcm_ctrl