title: U2OS_HA_eCLIP_CBL0137_RBRIIIMUT_input

description: Library name: U2OS_HA_eCLIP_CBL0137_RBRIIIMUT_input

data_processing:
For seCLIP experiments, the analysis was performed according to the Skipper workflow (Boyle et al 2023). Fastq files of CLIP and input samples were normalized to sequencing depth using seqtk (*). Per condition, the same input subsampled using different random seeds was provided. For samples spiked with cDNA, reads mapping to the DNA spike-in were identified using STAR (Dobin et al. 2013). Accordingly, fastq files were normalized according to the number of reads mapping to the spike-in in each sample (*#). All other samples represent multiplexed eCLIPs that were demultiplexed using UMItools (Smith et al. 2017) and used without further normalization. Subsequently, samples were trimmed utilizing skewer (Jiang et al. 2014) and extracts unimolecular identifiers (UMIs) using fastp (Chen et al 2018) and aligned to the human genome version GRCh38 for all MYC seCLIP and eCLIP experiments or to the murine genome version GRCm38 for TLR3 seCLIPs using STAR (Dobin et al. 2013). PCR duplicates were removed by using UMItools (Smith et al. 2017). To identify genomic regions with enriched signal in the CLIP over the SMI input, the automated Skipper analysis pipeline was used. The GENCODE version 38 gene annotations were obtained from gencodegenes.org. Transcripts were filtered based on total RNA-seq data in K562 cells with a minimum expression level of one transcript per million. A custom parse_gff.R script (Boyle et al 2023) was used to define RNA feature and transcript types (CDS_START ‘Start codon’, CDS_STOP ‘Stop codon’, CDS ‘CDS’ (of protein coding genes), UTR5 ‘5’ UTR’, UTR3 ‘3’ UTR’, EXON_SMALL ‘ncRNA’ (including snoRNA, snRNA, scaRNA, scRNA, miRNA, vault_RNA, rRNA, rRNA_pseudogene, Mt_rRNA, Mt_tRNA, ribozyme, sRNA, misc_RNA), INTRON ‘Intron’, EXON_LNCRNA ‘lncRNA’ (including lncRNA, processed_transcript), EXON_MRNA ‘Other’ (including Immunoglobulin (Ig) variable chain and T-cell receptor (TcR) genes, retaind_intron, non_stop_decay, protein_cidubg_LoF, nonsense_mediated_decay, TEC), PSEUDO ‘Pseudogene’ (including inactivated immunoglobulin genes, pseudogene, polymorphic_pseudogene, translated_pseudogene, translated_unprocessed_pseudogene, pseudogene, transcribed_unprocessed_pseudogene, transcribed_processed_pseudogene, transcribed_unitary_pseudogene, unitary_pseudogene, processed_pseudogene, unprocessed_pseudogene), PRIMIRNA ‘pri-miRNA’, SS3_ADJ ‘3’ splice site adj. (100 bp intronic region downstream of the 3’ splice site), SS3_ADJ ‘5’ splice site adj. (100 bp intronic region upstream of the 5’ splice site), SSB_ADJ ‘3’&5’ splice site adj. (within a 100 bp intronic region upstream of 3’ splice site and downstream of a 5’ splice site), SS3_PROX ‘3' splice site prox. (500 bp intronic region downstream of the 3’ splice site), SS5_PROX ‘5’ splice site prox.' (500 bp intronic region upstream of the 5’ splice site), SSB_PROX '3’&5’ splice site prox.' (within a 500 bp intronic region upstream of 3’ splice site and downstream of a 5’ splice site)). Each feature was tiled into evenly spaced windows of maximum 100 nucleotides in length and then tested for enrichment in immunoprecipitated over SMI samples using a beta-binomial distribution that accounts for overdispersion in read counts and GC bias. For the analysis of TLR3 seCLIPs by Skipper, statistical thresholds calculated from the fit of the beta-binomial distribution were derived from the automated Skipper pipeline (only windows passing a 20% false discovery rate in both replicates of each experiment were considered enriched), while for MYC seCLIP experiments the statistical thresholds were customized. Within individual replicates, windows that passed a p value threshold p<0.05 were considered enriched windows. Of these, windows that were identified in both replicates and passed a 20% FDR in at least one of the replicates were designated as reproducibly enriched windows. Windows that were called in more than 17% of all K562 and HepG2 ENCODE eCLIP samples were blacklisted and removed. For calling TLR3 reproducible enriched windows in the TLR3-IPs, reproducible enriched windows identified in IgG and NXL controls were blacklisted additionally. Reproducible windows were fine mapped by centering them around the local maximum of binding enrichment per window and adjusted to a fixed size of 75 nt. For RNA motif identification the HOMER script findMotifsGenome.pl (http://homer.ucsd.edu/homer/) was run with the following options: -preparsedDir <directory> -size given -rna -nofacts -S 20 -len 5,6,7,8,9 -nlen 1 -bg <background windows>. A random RNA background was specified by selecting random 75 nt windows belonging to the same RNA feature group. To visualize similarities in binding characteristics to different RNA transcript types in a t-SNE plot, performed MYC seCLIP data was pooled with ENCODE reference RBP eCLIP data, and plotted together in a two-dimensional space using the Rtsne package (https://lvdmaaten.github.io/tsne/). Several random seeds were tested to confirm a reproducible association of the queried protein with a certain feature type. Enrichment odds ratios (ORs), represented by the enrichment of observed CLIP enrichment over a baseline CLIP enrichment per RNA feature type, were calculated based on the call_enriched_windows.R script (Boyle et al 2023). For metagenes and heatmaps, log2FCs of CLIP/input were determined based on deduplicated bam files using bamCompare and plotted using deeptools (Ramirez et al. 2014) using the –sortUsingSamples and --averageTypeSummaryPlot mean options. Intersections between different sets of reproducible enriched windows were generated and visualized using intervene (Khan et al. 2014). Publicly available ENCODE eCLIP and control datasets were downloaded and log2 ratios of CLIP over SMIs were calculated using bamCompare and plotted over called windows of interest using deeptools (Ramirez et al. 2014).
Assembly: GRCh38 (MYC/HA seCLIPs/eCLIPs), GRCm38 (TLR3 seCLIPs in KPC cells), GRCm39 (MYC eCLIPs in KPC cells)
Supplementary files format and content: bedgraph or bw files

source_name: Bone

extract_protocol:
seCLIP (single-end enhanced cross-linking and immunoprecipitation) sequencing was performed as described in Van Nostrand et al. and Blue et al. All seCLIP experiments were conducted in duplicates with one pooled size-matched inputs per condition. Per IP sample, 30-40 million cells were crosslinked with UV-C light (254 nm, 2 min, 400 mJ/ cm2). Not UV-crosslinked cells were used as negative control. Cells were lysed in seCLIP lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% (vol/vol) Igepal CA-630, 0.1% (vol/vol) SDS and 0.5% (wt/vol) sodium deoxycholate) supplemented with a protease inhibitor cocktail (Sigma-Aldrich) and 440 U/ml murine RNase inhibitor (NEB), and 10% of crosslinked murine NIH 3T3 cells were added to each sample as spike-in (*). Samples were sonicated using the M220 Focused-ultrasonicator (Covaris) for 3 min. Next, samples were treated with 40 U RNase I (Thermo Fisher Scientific) and 10 U Turbo DNase for 5 min at 37  °C shaking at 1200 rpm. After centrifugation at 15,000 g for 10 min at 4 °C, supernatants were added to pre-coupled protein A/G Dynabeads (62.5 µl each per IP) to the antibody of interest (10 µg per sample) and incubated rotating at 4 °C overnight. 2% v/v of the lysate (20 µl) including beads were taken as SMInput, both for the RNA gel and for the diagnostic western blot. The remaining lysate was magnetically separated and washed 2 times with high-salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1% (vol/vol) Igepal CA-630, 1 mM EDTA, 0.1% (vol/vol) SDS and 0.5% (wt/vol) sodium deoxycholate), followed by a transition wash which was conducted by adding 500 µl of high-salt wash buffer, mixing and then adding 500 µl of wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% (vol/vol) Tween-20 and 5 mM NaCl). Three more washes with wash buffer were conducted, before the immunoprecipitated RNA was dephosphorylated on-beads using the FastAP and PNK enzymes. Therefore, 1X FastAP buffer (Thermo Fisher Scientific), 80 U murine RNase inhibitor (NEB), 4 U Turbo DNase (NEB) and 3 U FastAP enzyme (Thermo Fisher Scientific) were added in a total volume of 50 µl and incubated for 20 min at 37 °C shaking at 1,200 rpm. Without removing the phosphorylation mix, 150 µl PNK master mix (1x PNK7 buffer (700 mM Tris-HCl pH 7, 100 mM MgCl2), 40 U PNK4 enzyme) were added and incubated for another 20 min at 37 °C (shaking at 1,200 rpm). Samples were then washed with 200 µl seCLIP high-salt wash buffer once, followed by a transition wash and three further washings with seCLIP wash buffer. 10% of the IP samples were saved for biotin-labeling to visualize the RNA cross-linked to the protein of interest. To the remaining RNA, InvRiL19 RNA adapters (IDT) were ligated by incubating the samples with 25 µl 3’RNA linker mix (1.2x RNA ligase buffer (no DTT), 1.2 µM ATP, 3.6% DMSO, 0.024% v/v Tween-20, 18% w/v PEG 8000, 0.8 U murine RNase inhibitor (NEB), 72 U T4 RNA ligase high-concentration enzyme) rotating at RT for 75 min. Beads were then washed as follows: 1x wash buffer, transition wash to high-salt wash buffer, 1x high-salt wash buffer, transition wash to wash buffer, 3x wash buffer. After the final wash, 20% per IP sample was kept for the diagnostic western blot. To all IP and SMInput samples, NuPAGE LDS buffer (Thermo Fisher Scientific) containing DTT (98 mM, freshly prepared) was added before samples were incubated at 70 °C for 10 min. After magnetically separating the beads from the lysates, RNA-protein complexes were separated by gel electrophoresis using 8% Bis-Tris gels, and transferred onto nitrocellulose or methanol-activated PVDF membranes for the RNA gels and western blots, respectively. The diagnostic western blot was used as a guide to cut out the region of the membrane starting from the observed size of the protein of interest and extending until the maximal expected size of the RNA-protein complexes (but at least ~75 kDa larger). Membrane pieces were then sliced into ~2 mm x 2 mm squares and transferred into a fresh tube. To digest the protein, membranes were incubated with 24 U proteinase K (Roth) in PKS buffer for 20 min at 37 °C followed by an incubation at 50 °C for another 20 min. RNA was extracted using the RNA Clean and Concentrator-5 kit (Zymo) following the manufacturer’s instructions, and RNA was eluted in 10 µl H2O. Next, SMInput samples were dephosphorylated by incubating each SMInput RNA sample with 20 µl of dephosphorylation master mix (1x FastAP buffer (Thermo Fisher Scientific), 40 U murine RNase inhibitor (NEB), 2 U FastAP enzyme (Thermo Fisher Scientific) incubating at 37 °C for 20 min shaking at 1,200 rpm, followed by another 20 min incubation at 37 °C (1,200 rpm) in PNK master mix (1.05x PNK7 buffer, 5.26 mM DTT, 2 U Turbo DNase (NEB), 40 U T4 PNK enzyme). RNA was purified using the Clean & Concentrator 5 kit (Zymo) as described before. To append the SMInput RNA with 3’ RNA adapters, 1.5 µl 100% DMSO and 0.5 µl InvRil19 adapter were added per sample and incubated at 65 °C for 2 min. 13.5 µl of SMInput RNA ligation master mix (0.976x RNA ligase buffer (NEB), 0.976 mM ATP, 2.93% DMSO, 0.02% v/v Tween-20, 14.63 w/v PEG 8000, 12 U murine RNase inhibitor, 36 U T4 RNA ligase high-concentration enzyme) were added and samples were incubated at RT for 60 min on a rotating wheel. To purify the RNA, 15 µl MyOne Silane Dynabeads per sample were washed ones with RLT buffer (Qiagen), resuspended in RLTW buffer (RLT + 0.025% v/v Tween-20) and added to the RNA. An equal volume of 100% EtOH was added and samples were incubated at RT for 10 min, before samples were washed three times with 80% v/v EtOH. After the final wash, beads were tried and subsequently eluted in 9.5 µl of TT elution buffer (10 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 0.01% (vol/vol) Tween-20). All IP and SMInput samples were next reverse transcribed using the SuperScript III enzyme. For that, 1 µl of 5 µM InvAR17 RT primer  and 1 µl of 10 mM dNTPs were added per RNA sample and incubated for 2 min at 65 °C. 10 µl of reverse transcription master mix (1x first strand buffer (Invitrogen), 5 mM DTT, 8 U murine RNase inhibitor (NEB), 120 U Superscript III enzyme (Invitrogen)) were added to each sample and incubated at 55 °C for 20 min. 2.5 µl ExoSAP-IT (Thermo Fisher Scientific) were used to remove unincorporated dNTPs and RT primers (37 °C, 15 min) before 1 µl of 0.5 M EDTA was added. To remove RNA stands, 3 µl of 1 M sodium hydroxide were added and incubated at 70 °C for 10 min. 3 µl of 1 M hydrochloric acid were added to neutralize the sodium hydroxide. RNAs were again purified using MyOne Silane beads as described earlier. After drying, 2.5 µl of 5’ adapter master mix (4.66 µM InvRand3Tr3 adapter and 7.77% DMSO in TT elution buffer) were directly added to the beads and incubated at 70 °C for 2 min before 7.8 µl of ligation master mix (0.097% RNA ligase buffer (NEB), 1.94 mM DTT, 0.97 mM ATP, 0.019% v/v Tween-20, 17.48% PEG 8000, 30 U T4 RNA ligase high-concentration enzyme (NEB), 5’ deadenylase enzyme (NEB)) and incubated at RT overnight. RNA was subsequently purified using MyONE silane beads as described earlier. To estimate the number of cycles required for the final PCR, qPCRs were conducted. For multiplexing of eCLIPs, each RNA adapters containing unique barcodes (RNA_A01, RNA_B06, RNA_C01, RNA_D08, RNA_X1A, RNA_X1B) based on 44 were ligated to the RNA after immunoprecipitation. At least one third of each sample was mixed and further processed as a pool. SMI were separately run through the gel and barcoded RNA adapters were ligated after RNA isolation from the nitrocellulose membranes. As for the IP samples, inputs were merged to a pool and processed together. For total input samples, 20 µl of lysate were taken before the immunoprecipitation. Using the eCLIP adapter strategy 44, the in-line barcode on read 1 was later used to demultiplex the samples bioinformatically. Samples labeled with * contain 10% murine spike-in (NIH 3T3) that has not been used for further analysis. For fractionation eCLIPs (+) cells were cross-linked as described above. Cells were then fractionated as described in Nojima et al. Briefly, cells were resuspended in ice-cold HLB+N buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2 and 0.5% (vol/vol) NP-40) and incubated for 5 min on ice. The lysates were then resuspended in 1 ml ice-cold HLB-NS buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2, 0.5% (vol/vol) NP-40 and 10% (wt/vol) sucrose) and centrifuged at 420 g for 5 min. The cytosolic fraction in the supernatant was removed and the nuclear pellet was resuspended in ice-cold NUN1 buffer (20 mM Tris-HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, and 50% (vol/vol) glycerol), followed by the addition of ice-cold NUN2+E buffer (20 mM HEPES-KOH (pH 7.6), 300 mM NaCl, 0.2 mM EDTA, 7.5 mM MgCl2, 1% (vol/vol) NP-40, 1 M urea, and 3% Empigen (Sigma, Cat. No. 30326)). After incubation for 5 min on ice, samples were centrifuged at 300 g for 1 min at 4°C and the nucleoplasmic supernatant was separated from the chromatin pellet. The chromatin pellet was washed twice with ice-cold PBS. Immunoprecipitations were performed in seCLIP Lysis Buffer. All subsequent steps were performed as described for eCLIP multiplex sequencing.
cDNA libraries were amplified using a three-step, six cycle amplification followed by a two-step amplification bringing each sample to its calculated number of cycles. The final libraries were either purified using an agarose gel-based strategy cutting out gel pieces corresponding to 175-300 bp in size and purifying the DNA using the Qiagen MiniElute gel extraction kit following the manufacturer’s instructions. Alternatively, samples were cleaned using a two-sided selection with SPRI beads (0.7x right-sided, followed by a 1x left-sided selection). The quality and quantity of the libraries were assessed using the Fragment Analyzer (Agilent). To account for different cycle number used during final PCR amplification (e.g. for IgG controls), DNA spike-in containing adaptors for the Illumina index primers used in the final PCR was generated and equal amounts were added to the immunoprecipitation samples prior to the final PCR for indicated experiments (*#), resulting in different relative ratios of CLIP:DNA spike-in in the final libraries as observed upon measurement using the fragment analyzer.

value: Bone

value: Osteosarcoma cell line

value: MYC-AID, pInd20 RBRIIIMUTMYC-HA

value: doxycycline (1 µg/ml, 24 h), auxin (500 µM, 24 h), CBL0137 (5 µM, 2 h)


title: SLE_Control HD A0221161

status: Public on Feb 06 2026

type: RNA

city: Durham

first: James

middle: M

last: Ward

state: NC

address: 111 TW Alexander Dr

department: Integrative Bioinformatics

country: USA

institute: NIEHS

hyb_protocol: Hybridization was performed following the standard Affymetrix protocol.

scan_protocol: Fluorescence intensities were scanned with a GeneArray Scanner (Affymetrix), and image analysis was performed with GCOS (Affymetrix).

data_processing: Data were proessed in R using fRMA signal extraction and normalization. Statistical comparisons were performed using limma un-paired moderated t-tests.

label: biotin

molecule: total RNA

organism: Homo sapiens

source_name: circulating cells from peripheral blood

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 100ng total RNA.

extract_protocol: Serum and RNA, collected in PAXgene tubes (Becton-Dickinson, Palo Alto, CA), were processed immediately following blood draw, and stored at -80 degrees C.

tag: cohort

value: HD


type: SRA

city: New York

first: Zihan

last: Xu

state: NY

address: 1230 York Ave

institute: The Rockefeller University

description:
Library name: PerturbFate pooled screen Tri-omics New RNA-120
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad

library_source: other

data_processing:
For PerturbFate Dual-omics libraries, BCL files were demultiplexed into FASTQ files using P7 PCR barcodes. Each FASTQ file contained all RNA and ATAC reads from the same indexing PCR well. During barcode extraction, reads from the oligo-dT/randomN RNA and ATAC modalities were split into separate files based on modality-specific adapter sequence matching. Read1 matching the "TG" sequence at the 41–42 nt position was classified as transcriptome data. Oligo-dT-originated RNA reads required at least 10 consecutive T bases at positions 51–60 nt, while randomN-originated RNA reads did not exhibit this pattern. ATAC read pairs were identified by the Tn5 ME sequence at the 41–50 nt position of Read1.
For the PerturbFate Dual-omics RNA reads, cell barcodes and UMI sequences from Read1 were appended to the headers of each Read2 FASTQ file, and only Read2 was used for subsequent steps. Poly(A) sequences, adapter sequences, and low-quality bases were trimmed from Read2 of oligo-dT reads, while 14 additional bases (potential priming hexamer and UMI sequences) were removed from randomN reads using Trim_Galore (0.6.7). After this step, oligo-dT and randomN reads were combined for further processing. STAR (2.7.9a) was used to map reads to the hg38 reference genome (GRCh38.p13, GENCODE). Reads with a mapping score ≥30 were selected using SAMtools (1.13). Single-cell deduplication was performed based on UMI sequences and alignment locations, and retained reads were split into SAM files per cell. Finally, single-cell gene-to-cell raw count matrices were constructed by assigning reads to genes.
For the PerturbFate Dual-omics ATAC reads, cell barcodes from RNA Read1 were appended to the headers of both ATAC Read1 and Read2 FASTQ files. Adapter sequences were removed in paired-end mode using Trim_Galore (0.6.7). STAR (2.7.9a) was used to map read pairs to the hg38 reference genome (GRCh38.p13, GENCODE). Picard (2.27.4) was used to remove PCR duplicates, and TSV files containing genomic locations and full cell barcodes were generated for SnapATAC2 (2.6.1)
sgRNA reads were matched against constant sequences in Read1 and Read2, allowing a maximum of one mismatch. For each filtered read pair, the cell barcode, sgRNA sequence, and UMI were extracted from designed positions. Extracted sgRNA sequences with at most one mismatch from the sgRNA library were corrected. UMI sequences were used for deduplication by collapsing identical UMIs for each individual sgRNA under a unique cell barcode. Cells with sgRNA UMI counts exceeding 5 were retained, and the sgRNA × cell count matrix was constructed.
For PerturbFate Tri-omics, most computational steps were the same as described above, with minor modifications. In this protocol, nascent and pre-existing transcriptomes from the same cell were amplified separately using distinct P7 PCR barcodes and processed as separate cells (two groups of reads with distinct cell barcodes). These were matched after single-cell gene-to-cell raw count matrices were generated. Additionally, because ATAC DNA underwent secondary tagmentation, the pipeline was modified to process single-end Read1 of ATAC fragments for chromatin accessibility quantification. The sgRNA library from the same cell was amplified from both nascent and pre-existing transcriptome PCR wells but shared a unique cell barcode for matching, achieved by adding the same barcoded sgRNA inner P7-U6 primer to paired nascent and pre-existing transcriptome PCR wells.
Assembly: hg38
Supplementary files format and content: RDS file contains a sparse matrix that can be read into R
Supplementary files format and content: csv file include meta data of cells
Supplementary files format and content: h5ad file include an anndata object containing a peak x count matrix with cell meta data

molecule: other

source_name: A375 cell line

growth_protocol: A375-dCas9-BFP-KRAB cells were transduced with a sgRNA library at a low MOI and BFP+ GFP+ double positive cells were sorted out. Cells were cultured for additional 6 days with either DMSO or 1uM Vemurafenib treatment.

extract_protocol:
After trypsinization, cells in each 6cm dish were collected into a 15ml falcon tube and kept on ice. Cells were spun down at 300xg for 5 minutes (4 °C) and washed once in 3ml ice-cold PBS. Every 1M cells were fixed with 1ml ice-cold 1% FA in PBS for 10 minutes on ice. For every 1mL cell suspension, 20μL of 10% Triton X-100 (0.2% final) was added to the tube to permeabilize cells for 3 min on ice. Spin down tubes at 500g for 3 min at 4 ̊C and quickly aspirate the supernatant. Cells are resuspended in 500uL of PBSI, and mix with 500uL 100 mM Tris-HCl pH8 with 5 μL of 10% Triton-X100. Spin down tubes at 500g for 5 min at 4 ̊C.
Fixed cells underwent Tn5 tagmentation in bulk, reverse transcription in bulk, two rounds of indexing ligation, and finally indexing PCR. SgRNA libraries were further separated and enriched by another round of PCR. A step-by-step experimental protocol of PerturbFate library preparation is included in the supplementary material of the publication.

treatment_protocol: On day6, before the end of the screen, 2.5ml of the original media from each plate was mixed with 2.5uL of 200mM 5EU dissolved in DMSO and was put back for nascent RNA metabolic labeling. After 1 hour of treatment, cells were harvested for PerturbFate profiling.

tag: cell line

value: A375 cell line

tag: cell type

value: Melanoma

tag: genotype

value: BRAF V600E

tag: treatment

value: DMSO or Vemurafenib


title: WT CTL Diet S4

city: La Jolla

first: Leigh-Ana

last: Rossitto

state: CA

address: 9500 Gilman Drive

department: Neurosciences

institute: UC San Diego

description:
Library name: Sample4
10x Genomics

library_source: transcriptomic single cell

data_processing:
A custom reference genome was constructed using CellRanger mkref (ver. 9.0.1) to add the hTauP301S-1N4R transgene into the GRCm39 (2024-A) genome assembly as hTau.80 Fastq files were aligned to our custom PS19 genome using the CellRanger Count (ver. 9.0.1) pipeline using default settings and including introns.
Assembly: GRCm39 and the hTauP301S-1N4R transgene
Supplementary files format and content: H5 files with the raw and filtered feature matrices

molecule: nuclear RNA

organism: Mus musculus

source_name: Hippocampus

extract_protocol:
Frozen tissues were dounce-homogenized in lysis solution - 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM TrisCl (pH 8), 10% Triton, and 0.1 M DTT with Protector RNase Inhibitor (Roche, 03335402001). Then, nuclei isolation was performed with an iodixanol gradient. To remove the nuclear layer, 1000 μL was removed from between the 30% and 40% iodixanol layers with a P1000 and collected. 10 μL of nuclei suspension was used to count nuclei using Trypan blue. Then, the nuclei were resuspended in wash buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM TrisCl, 0.1% Tween-80), and centrifuged at 500xg for 10 min at 4ºC. Supernatant was removed, and nuclei were resuspended once more in wash buffer with 1% BSA to achieve a concentration of 1200 nuclei/μL.
Approximately 20,000 nuclei/sample were used for GEM generation, barcoding, and cDNA libraries using the Chromium GEM-X Single Cell 3' Kit v4 and corresponding protocol. Pooled libraries were sequenced using the NovaSeq X Plus 25B in order to achieve >20,000 reads/nuclei.

treatment_protocol: WT v hTau+ PS19 mice on either ketone ester (KE) or control diets

tag: tissue

value: Hippocampus

value: WT

tag: Sex

value: male

value: .Diet


city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

country: Germany

institute: University of Wuerzburg

description: Library name: U2OS_HA_eCLIP_CBL0137_EtOH_control_input

library_source: transcriptomic

growth_protocol: Cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin

treatment_protocol: Treatment as indicated

value: MYC-AID, merge


title: SLE_Control HD A0221153


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-119
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: U2OS_HA_eCLIP_CBL0137_IgG_control_input

description: Library name: U2OS_HA_eCLIP_CBL0137_IgG_control_input


title: hTau KE Diet S3

description:
Library name: Sample3
10x Genomics

value: hTau


title: SLE_Control HD A0221137


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-121
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-116
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-117
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-118
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: U2OS_HA_eCLIP_CBL0137_RBRIIIMUT_R2

description: Library name: U2OS_HA_eCLIP_CBL0137_RBRIIIMUT_R2


title: U2OS_HA_eCLIP_CBL0137_WT_input

description: Library name: U2OS_HA_eCLIP_CBL0137_WT_input

value: MYC-AID, pInd20 WTMYC-HA


title: P3 digit, diet control

city: Dallas

first: Sneha

last: Korlakunta

state: TX

address: 6000 Harry Hines Blvd

department: Surgery

institute: UT Southwestern Medical Center

description:
Library name: Diet-Control
10x Genomics

data_processing:
Cellranger pipeline (v6.1.2) was used for demultiplexing and counting as recommended by 10X Genomics (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/mkfastq)
Assembly: GRCm39-2024-A
Supplementary files format and content: Tab-separated value files and matrix files

source_name: P3 digit

extract_protocol:
Digits from ten mice were pooled for each sample. A scalpel blade was used to sever hindlimb digits 2-4 at the proximal end of the 2nd phalangeal element. Each digit was divided down the midline along its length to maximize the exposure of internal tissue surface area to media. A single cell suspension was generated following these techniques: Briefly, digit tissue was subjected to enzymatic digestion using Collagenase I at 3 mg/mL (Thermo Fisher Scientific, 17100-017), Collagenase II at 2 mg/mL (Thermo Fisher Scientific, 17101-015), and Dispase at 3mg/mL (MilliporeSigma, D4693) per 1mL of DMEM (Thermo Fisher Scientific, 11960044) for 45 minutes at 37°C under constant agitation at 150 rpm. Digestions were then quenched with DMEM containing 10% FBS (Gibco, 10437028), filtered through 40um mesh and pelleted via centrifugation for 5min at 400xG at 4⁰C.Supernatant was removed and red blood cells were lysed by resuspending the pellets with 2ml of ACK Lysis Buffer (Gibco A10492-01) and incubating for 2 minutes at room temp. Cell viability was assessed with Trypan blue exclusion on a Countess III (Thermo Fisher Scientific) automated counter and only samples with >75% viability were processed for further sequencing.
Single Cell 3' v3

value: P3 digit

tag: age

value: 6-8 weeks

value: female

tag: strain

value: No treatment

tag: batch


title: SLE_Control HD A0221124


title: SLE_Control HD A0221136


title: hTau KE Diet S1

description:
Library name: Sample1
10x Genomics


title: hTau CTL Diet S2

description:
Library name: Sample2
10x Genomics


title: SLE_Control HD A0221123


title: U2OS_HA_eCLIP_CBL0137_RBRIIIMUT_R1

description: Library name: U2OS_HA_eCLIP_CBL0137_RBRIIIMUT_R1

value: Bone

value: Osteosarcoma cell line

value: MYC-AID, pInd20 RBRIIIMUTMYC-HA

value: doxycycline (1 µg/ml, 24 h), auxin (500 µM, 24 h), CBL0137 (5 µM, 2 h)


title: P3 digit, SAR131675 diet

description:
Library name: Diet
10x Genomics

value: Dietary SAR131675


title: U2OS_HA_eCLIP_CBL0137_WT_R2

description: Library name: U2OS_HA_eCLIP_CBL0137_WT_R2


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-115
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad

value: A375 cell line

value: Melanoma

value: BRAF V600E

value: DMSO or Vemurafenib


title: SLE_Control HD A0221118

value: HD


title: U2OS_HA_eCLIP_CBL0137_WT_R1

description: Library name: U2OS_HA_eCLIP_CBL0137_WT_R1


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-114
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-113
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, GMT, 12-20wk, rep2

city: Houston

first: Lanlan

last: Shen

state: Texas

address: 1100 Bates Ave

department: Pediatrics

institute: Baylor College of Medicine

description: Library name: GMT13w2

library_source: genomic

data_processing:
Metagenomic samples were quality-controlled and trimmed for adaptors using fastp (v0.23.1).
clean data was aligned to the host database using Bowtie2 (v2.5.4) with the parameter: --end-to-end,--sensitive,-I 200, and- X 400, and host-derived sequences were removed.
Using Kraken2 (v2.1.3), microbial sequences (including bacteria, archaea, fungi, and viruses) were compared against a database built with kraken2-build and the species annotation results were corrected using Braken (v2.9).
Assembly: Host reads removed using Mus musculus GRCm39 (mm39).Microbial species annotation performed with Kraken2/Bracken using RefSeq microbial genomes.
Supplementary files format and content: text file include abundance of taxonomic annotation for each sample

molecule: genomic DNA

source_name: fecal

extract_protocol:
Genomic DNA (200 ng) was randomly sheared into ~350 bp fragments using a Covaris ultrasonic disruptor.
The integrity and insert size distribution of the resulting libraries were assessed with AATI analysis. Libraries meeting the expected insert size were further quantified using quantitative PCR (qPCR), and only those with an effective concentration greater than 3 nM were retained.

value: fecal

value: Gut microbiota transfer (GMT)


title: U2OS_HA_eCLIP_CBL0137_EtOH_control

description: Library name: U2OS_HA_eCLIP_CBL0137_EtOH_control

value: MYC-AID, merge


title: SLE_Control HD A0221114


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-112
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, GMT, 12-20wk, rep3

description: Library name: GMT13w3


title: SLE_Control HD A0221113


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-111
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, GMT, 12-20wk, rep1

description: Library name: GMT13w1


title: U2OS_IgG_ChIPseq_input_r2

description: Library name: U2OS_IgG_ChIPseq_r2

data_processing:
ChIP-Rx samples were mapped separately to the human GRCh38 genome and to the murine GRCm39 genome using Bowtie2 with -N 1 parameters. Subsequently, aligned reads were spike-normalized by dividing the number of mapped reads aligned to GRCh38 by the number of reads mapped to GRCm39 for each sample and multiplying this ratio with the smallest number of reads aligned to GRCm39 for any sample. For murine samples, human reads mapped to the t2t genome were used for spike normalization. For ChIPseqs performed using the HA antibody (*), samples were read normalized.
Assembly: GRCh38
Supplementary files format and content: bedgraph files for each condition and replicate.

extract_protocol:
To crosslink cells following treatment, 1% v/v formaldehyde was added to the cell culture medium and incubated 5 min rotating at RT. 125 mM glycine were added to stop the reaction and incubated for 5 min rotating at RT. Cells were then harvested in ice-cold PBS containing protease and phosphatase inhibitors (Sigma Aldrich). Cells were centrifuged (1,500 rpm, 20 min, 4°C) and the pellet was resuspended in ChIP lysis buffer I (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40). For spike-normalization, 10% murine NIH3T3 cells were added based on the cell number. After 20 min incubation on ice, nuclei were pelleted by centrifugation (1,500 rpm, 20 min, 4°C) and subsequently lysed by incubation in ChIP lysis buffer II (10 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) for 10 min on ice. The chromatin was fragmented using the Covaris Focused Ultrasonicator M220 (50 min; cycles/burst, 200; peak power, 75; duty factor, 10), and the required fragment size of 150-300 bp was validated by agarose gel electrophoresis. The fragmented chromatin was collected by centrifugation (14,000 rpm, 20 min, 4°C). For each IP, 100 µl of each Protein A and Protein G-coupled Dynabeads (Thermo Fisher Sientific) were pre-incubated overnight with 15 µg antibody in the presence of 5 mg/mL BSA at 4°C. Subsequently, beads were added to the chromatin and incubated overnight at 4°C on a rotating wheel. Immunoprecipitated protein-chromatin complexes were washed 3 times each with ice-cold ChIP wash buffer I (20 mM Tris pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), ChIP wash buffer II (20 mM Tris pH 8.1, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), ChIP wash buffer III (10 mM Tris pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate; including a 5 min incubation at 4°C), and once with ice-cold TE buffer. Protein-chromatin complexes were eluted twice by incubating in 150 µl elution buffer 100 mM NaHCO3, 1% SDS) for 15 min at RT. To remove potential RNA contaminations, IP and input samples were treated with RNase A for 1 h at 37°C. Samples were de-crosslinked by incubation at 65°C shaking overnight, and then treated with Proteinase K for 2 h at 45°C to degrade the protein. The amount of DNA was quantified using Quant-iT PicoGreen dsDNA assay (Thermo Fisher Scientific)
Libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) following manufacturer’s instructions.

value: MYC-AID, pInd20 merge

value: doxycycline (1 µg/ml, 24 h); auxin (500 µM, 24 h)


title: fecal, GMT, 12-15wk, rep4

description: Library name: GMT12w4


title: SLE_Control HD A0221100


title: fecal, GMT, 12-15wk, rep2

description: Library name: GMT12w2


title: U2OS_HA_ChIPseq_WT_IP_r2

description: Library name: U2OS_HA_ChIPseq_WT_IP_r2

value: MYC-AID, pInd20 WTMYC-HA


title: U2OS_HA_eCLIP_CBL0137_IgG_control

description: Library name: U2OS_HA_eCLIP_CBL0137_IgG_control


title: SLE_Control HD A0221108


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-108
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: U2OS_HA_ChIPseq_WT_CBL_IP_r2

description: Library name: U2OS_HA_ChIPseq_WT_CBL_IP_r2

value: doxycycline (1 µg/ml, 24 h); auxin (500 µM, 24 h), CBL0137 (5 µM, 2 h)


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-109
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-107
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, GMT, 12-15wk, rep1

description: Library name: GMT12w1


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-110
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: U2OS_HA_ChIPseq_RBRIII_IP_r2

description: Library name: U2OS_HA_ChIPseq_RBRIII_IP_r2

value: Bone


title: fecal, GMT, 12-15wk, rep3

description: Library name: GMT12w3


title: SLE_Control HD A0221068


title: U2OS_HA_ChIPseq_RBRIII_CBL_IP_r2

description: Library name: U2OS_HA_ChIPseq_RBRIII_CBL_IP_r2

value: Osteosarcoma cell line


title: fecal, GMT, 8-15wk, rep4

description: Library name: GMT8w4


title: SLE_Control HD A0221064


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-106
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-105
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, GMT, 8-15wk, rep3

description: Library name: GMT8w3


title: U2OS_HA_ChIPseq_WT_CBL_IP_r1

description: Library name: U2OS_HA_ChIPseq_WT_CBL_IP_r1


title: fecal, GMT, 8-15wk, rep2

description: Library name: GMT8w2


title: SLE_Control HD A0221107


title: SLE_Control HD A0221065


title: SLE_Control HD A0221052


title: U2OS_HA_ChIPseq_WT_IP_r1

description: Library name: U2OS_HA_ChIPseq_WT_IP_r1


title: U2OS_HA_ChIPseq_RBRIII_IP_r1

description: Library name: U2OS_HA_ChIPseq_RBRIII_IP_r1

value: MYC-AID, pInd20 RBRIIIMUTMYC-HA


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-103
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, GMT, 3-15wk, rep4

description: Library name: GMT3w4


title: SLE_Control HD A0221051


title: fecal, GMT, 3-15wk, rep3

description: Library name: GMT3w3


title: U2OS_HA_ChIPseq_input_pool_r1

description: Library name: U2OS_ChIPseq_input_pool_r1

value: doxycycline (1 µg/ml, 24 h); auxin (0/500 µM, 24 h), CBL0137 (0/5 µM, 2 h)


title: U2OS_HA_ChIPseq_RBRIII_CBL_IP_r1

description: Library name: U2OS_HA_ChIPseq_RBRIII_CBL_IP_r1


title: SLE_Control HD A0221055


title: U2OS_HA_ChIPseq_EtOH_Ctrl_IP_r1

description: Library name: U2OS_HA_ChIPseq_EtOH_Ctrl_IP_r1

value: auxin (500 µM, 24 h)


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-104
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad

value: A375 cell line


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-101
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad

value: Melanoma


title: fecal, GMT, 8-15wk, rep1

description: Library name: GMT8w1


title: fecal, GMT, 3-15wk, rep2

description: Library name: GMT3w2


title: 786-O_STAT3_ChIP_Rep2

city: Boston

first: Jeremy

last: Simon

state: MA

address: 450 Brookline Ave

department: Department of Data Science

institute: Dana-Farber Cancer Institute

description: Library name: 786-O_STAT3_ChIP_Rep2

data_processing:
ChIP-seq data for HKC8 (STAT3, H3K4me1, H3K4me3, H3K27ac, input control) and 786-O (STAT3, IgG control) were processed with nf-core/chipseq v2.1.0 using the hg38 reference and GENCODE v43 gene annotations. Default parameters were used except that MACS3 additionally specified “--keep-dup all --nomodel --extsize 147” for peak calling.
H3K27ac HiChIP data were first processed with nf-core/hic using pairtools processing mode (available from https://github.com/nservant/nf-core-hic/tree/dev) with “--no_digestion --min_cis_dist 1000” and bin sizes ranging from 1 Kb to 1 Mb. The 5 Kb contact maps were then used as input for FitHiChIP v11.0 alongside H3K27ac ChIP-seq consensus peaks from GSE102095 as identified above, running in Peak-to-Peak mode with the following parameters: BINSIZE=5000, LowDistThr=5000, UppDistThr=2000000, UseP2PBackgrnd=1, BiasType=2, MergeInt=1, QVALUE=0.1.
Assembly: hg38
Supplementary files format and content: bigWig of ChIP-seq data scaled by sequencing depth
Supplementary files format and content: cooler file of HiChIP contacts at 5kb resolution, bedpe of significant loops

growth_protocol: 786-O, HKC8 cells were purchased from the American Type Culture Collection (ATCC). The cell lines used in this study were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) high glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin unless specifically indicated. All cells were cultured in a humidified incubator at 37 °C with 5% CO2. All cells were tested negative for mycoplasma by using the mycoplasma detectionkit (Lonza, LT07–218) or mycoplasma elimination reagent-plasmocin (Invivogen, ant-mpt).

extract_protocol:
Immunoprecipitation was performed by incubating chromatin with specific antibodies: STAT3 (CST, 9139), H3K4me1 (Abcam, ab8895), H3K4me3 (Abcam, ab8580), and H3K27ac (Abcam, ab4729), using Protein G magnetic beads at 4°C overnight with rotation. The samples were washed, collected using a magnetic stand, and eluted for cross-link reversal. Proteinase K (Sigma, 3115828001) was used for protein degradation, and RNAse A (Sigma, 70856-3) for RNA degradation. DNA was purified using the Qiagen PCR purification kit (Qiagen, 28106). For HiChIP, Chromatin fragments were incubated with anti-H3K27ac antibody (Cell Signaling Technology, 8173) overnight at 4°C for chromatin immunoprecipitation. The antibody-protein-DNA complex was pulled down with protein A/G-coated magnetic beads. Chromatin ends were repaired and ligated to a biotinylated bridge adapter followed by proximity ligation of adapter containing ends. After proximity ligation, crosslinks were reversed, and the DNA was purified from proteins and converted into a sequencing library.
For ChIP-seq, libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645S) and sequenced at the McDermott Center Next Generation Sequencing Core at UT Southwestern. For HiChIP, the sequencing library was generated using Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of the library. The library was sequenced on Illumina Novaseq platform with paired-end sequencing at 300 million reads per sample.

treatment_protocol: ChIP (Chromatin immunoprecipitation) was performed using SimpleChIP Enzymatic Chromatin IP Kit (CST #9003). Briefly, cells were cross-linked with 1% formaldehyde, followed by treatment with 10X glycine to quench the reaction. Cells were lysed in lysis buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton x-100) and sonicated with a BioRuptor to shear chromatin. The HiChIP experiment was outsourced to Dovetail Genomics (Cantata Bio, LLC) and performed per their established protocol. Briefly, approximately 10 million 786-O cells were collected, and chromatin was subsequently fixed with 3 mM disuccinimidyl glutarate (DSG), followed by another fixation with 1% formaldehyde. The fixed chromatin was digested in situ with micrococcal nuclease (MNase) and then extracted with RIPA lysis buffer.

tag: chip antibody

tag: replicate


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-100
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad

value: BRAF V600E


title: SLE_Control HD A0221045


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-99
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad

value: DMSO or Vemurafenib


title: fecal, GMT, 3-15wk, rep1

description: Library name: GMT3w1


title: SLE_Control HD A0221039

value: HD


title: fecal, SPF, 12wk, rep2

description: Library name: SPF12w2

value: fecal

value: Control


title: 786-O_IgG_ChIP_Rep2

description: Library name: 786-O_IgG_ChIP_Rep2

value: IgG control


title: SLE_Control HD A0221036


title: 786-O_STAT3_ChIP_Rep1

description: Library name: 786-O_STAT3_ChIP_Rep1


title: SLE_Control HD A0221035


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-98
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, SPF, 10wk, rep1

description: Library name: SPF10w1


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-96
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: SLE_Control HD A0221032


title: 786-O_IgG_ChIP_Rep1

description: Library name: 786-O_IgG_ChIP_Rep1


title: SLE_Control HD A0221031


title: eCLIP_fractionation_nucleoplasm_MYC_R2

description:
Library name: eCLIP_fractionation_nucleoplasm_MYC_R2
+

data_processing:
For seCLIP experiments, the analysis was performed according to the Skipper workflow (Boyle et al 2023). Fastq files of CLIP and input samples were normalized to sequencing depth using seqtk. Per condition, the same input subsampled using different random seeds was provided. For samples spiked with cDNA, reads mapping to the DNA spike-in were identified using STAR (Dobin et al. 2013). Accordingly, fastq files were normalized according to the number of reads mapping to the spike-in in each sample. Multiplexed eCLIPs were demultiplexed using UMItools (Smith et al. 2017) and used without further normalization. Subsequently, samples were trimmed utilizing skewer (Jiang et al. 2014) and extracts unimolecular identifiers (UMIs) using fastp (Chen et al 2018) and aligned to the human genome version GRCh38 for all MYC seCLIP and eCLIP experiments or to the murine genome version GRCm38 for TLR3 seCLIPs using STAR (Dobin et al. 2013). PCR duplicates were removed by using UMItools (Smith et al. 2017). To identify genomic regions with enriched signal in the CLIP over the SMI input, the automated Skipper analysis pipeline was used 46. The GENCODE version 38 gene annotations were obtained from gencodegenes.org. Transcripts were filtered based on total RNA-seq data in K562 cells with a minimum expression level of one transcript per million. A custom parse_gff.R script (Boyle et al 2023) was used to define RNA feature and transcript types (CDS_START ‘Start codon’, CDS_STOP ‘Stop codon’, CDS ‘CDS’ (of protein coding genes), UTR5 ‘5’ UTR’, UTR3 ‘3’ UTR’, EXON_SMALL ‘ncRNA’ (including snoRNA, snRNA, scaRNA, scRNA, miRNA, vault_RNA, rRNA, rRNA_pseudogene, Mt_rRNA, Mt_tRNA, ribozyme, sRNA, misc_RNA), INTRON ‘Intron’, EXON_LNCRNA ‘lncRNA’ (including lncRNA, processed_transcript), EXON_MRNA ‘Other’ (including Immunoglobulin (Ig) variable chain and T-cell receptor (TcR) genes, retaind_intron, non_stop_decay, protein_cidubg_LoF, nonsense_mediated_decay, TEC), PSEUDO ‘Pseudogene’ (including inactivated immunoglobulin genes, pseudogene, polymorphic_pseudogene, translated_pseudogene, translated_unprocessed_pseudogene, pseudogene, transcribed_unprocessed_pseudogene, transcribed_processed_pseudogene, transcribed_unitary_pseudogene, unitary_pseudogene, processed_pseudogene, unprocessed_pseudogene), PRIMIRNA ‘pri-miRNA’, SS3_ADJ ‘3’ splice site adj. (100 bp intronic region downstream of the 3’ splice site), SS3_ADJ ‘5’ splice site adj. (100 bp intronic region upstream of the 5’ splice site), SSB_ADJ ‘Branch point prox. (500 bp intronic region centered around the branch point), SS3_PROX ‘3’ splice site prox. (500 bp intronic region downstream of the 3’ splice site), SS5_PROX ‘5’ splice site prox. (500 bp intronic region upstream of the 5’ splice site), SSB_PROX ‘Branch point prox. (500 bp intronic region centered around the branch point)). Each feature was tiled into evenly spaced windows of maximum 100 nucleotides in length and then tested for enrichment in immunoprecipitated over SMI samples using a beta-binomial distribution that accounts for overdispersion in read counts and GC bias. For the analysis of TLR3 seCLIPs by Skipper, statistical thresholds calculated from the fit of the beta-binomial distribution were derived from the automated Skipper pipeline (only windows passing a 20% false discovery rate in both replicates of each experiment were considered enriched), while for MYC seCLIP experiments the statistical thresholds were customized. Within individual replicates, windows that passed a p value threshold p<0.05 were considered enriched windows. Of these, windows that were identified in both replicates and passed a 20% FDR in at least one of the replicates were designated as reproducibly enriched windows. Windows that were called in more than 17% of all K562 and HepG2 ENCODE eCLIP samples were blacklisted and removed. For calling TLR3 reproducible enriched windows in the TLR3-IPs, reproducible enriched windows identified in IgG and NXL controls were blacklisted additionally. Reproducible windows were fine mapped by centering them around the local maximum of binding enrichment per window and adjusted to a fixed size of 75 nt. For RNA motif identification the HOMER script findMotifsGenome.pl (http://homer.ucsd.edu/homer/) was run with the following options: -preparsedDir <directory> -size given -rna -nofacts -S 20 -len 5,6,7,8,9 -nlen 1 -bg <background windows>. A random RNA background was specified by selecting random 75 nt windows belonging to the same RNA feature group. To visualize similarities in binding characteristics to different RNA transcript types in a t-SNE plot, performed MYC seCLIP data was pooled with ENCODE reference RBP eCLIP data, and plotted together in a two-dimensional space using the Rtsne package (https://lvdmaaten.github.io/tsne/). Several random seeds were tested to confirm a reproducible association of the queried protein with a certain feature type. Enrichment odds ratios (ORs), represented by the enrichment of observed CLIP enrichment over a baseline CLIP enrichment per RNA feature type, were calculated based on the call_enriched_windows.R script (Boyle et al 2023). For metagenes and heatmaps, log2FCs of CLIP/input were determined based on deduplicated bam files using bamCompare and plotted using deeptools (Ramirez et al. 2014) using the –sortUsingSamples and --averageTypeSummaryPlot mean options. Intersections between different sets of reproducible enriched windows were generated and visualized using intervene (Khan et al. 2014). Publicly available ENCODE eCLIP and control datasets were downloaded and log2 ratios of CLIP over SMIs were calculated using bamCompare and plotted over called windows of interest using deeptools (Ramirez et al. 2014).
Assembly: GRCh38 (MYC seCLIPs/eCLIPs), GRCm38 (TLR3 seCLIPs in KPC cells), GRCm39 (MYC eCLIPs in KPC cells)
Supplementary files format and content: bedgraph or bw files

extract_protocol:
seCLIP (single-end enhanced cross-linking and immunoprecipitation) sequencing was performed as described in Van Nostrand et al. and Blue et al. All seCLIP experiments were conducted in duplicates with one pooled size-matched inputs per condition. Per IP sample, 30-40 million cells were crosslinked with UV-C light (254 nm, 2 min, 400 mJ/ cm2). Not UV-crosslinked cells were used as negative control. Cells were lysed in seCLIP lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% (vol/vol) Igepal CA-630, 0.1% (vol/vol) SDS and 0.5% (wt/vol) sodium deoxycholate) supplemented with a protease inhibitor cocktail (Sigma-Aldrich) and 440 U/ml murine RNase inhibitor (NEB), and 10% of crosslinked murine NIH 3T3 cells were added to each sample as spike-in. Samples were sonicated using the M220 Focused-ultrasonicator (Covaris) for 3 min. Next, samples were treated with 40 U RNase I (Thermo Fisher Scientific) and 10 U Turbo DNase for 5 min at 37  °C shaking at 1200 rpm. After centrifugation at 15,000 g for 10 min at 4 °C, supernatants were added to pre-coupled protein A/G Dynabeads (62.5 µl each per IP) to the antibody of interest (10 µg per sample) and incubated rotating at 4 °C overnight. 2% v/v of the lysate (20 µl) including beads were taken as SMInput, both for the RNA gel and for the diagnostic western blot. The remaining lysate was magnetically separated and washed 2 times with high-salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1% (vol/vol) Igepal CA-630, 1 mM EDTA, 0.1% (vol/vol) SDS and 0.5% (wt/vol) sodium deoxycholate), followed by a transition wash which was conducted by adding 500 µl of high-salt wash buffer, mixing and then adding 500 µl of wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% (vol/vol) Tween-20 and 5 mM NaCl). Three more washes with wash buffer were conducted, before the immunoprecipitated RNA was dephosphorylated on-beads using the FastAP and PNK enzymes. Therefore, 1X FastAP buffer (Thermo Fisher Scientific), 80 U murine RNase inhibitor (NEB), 4 U Turbo DNase (NEB) and 3 U FastAP enzyme (Thermo Fisher Scientific) were added in a total volume of 50 µl and incubated for 20 min at 37 °C shaking at 1,200 rpm. Without removing the phosphorylation mix, 150 µl PNK master mix (1x PNK7 buffer (700 mM Tris-HCl pH 7, 100 mM MgCl2), 40 U PNK4 enzyme) were added and incubated for another 20 min at 37 °C (shaking at 1,200 rpm). Samples were then washed with 200 µl seCLIP high-salt wash buffer once, followed by a transition wash and three further washings with seCLIP wash buffer. 10% of the IP samples were saved for biotin-labeling to visualize the RNA cross-linked to the protein of interest. To the remaining RNA, InvRiL19 RNA adapters (IDT) were ligated by incubating the samples with 25 µl 3’RNA linker mix (1.2x RNA ligase buffer (no DTT), 1.2 µM ATP, 3.6% DMSO, 0.024% v/v Tween-20, 18% w/v PEG 8000, 0.8 U murine RNase inhibitor (NEB), 72 U T4 RNA ligase high-concentration enzyme) rotating at RT for 75 min. Beads were then washed as follows: 1x wash buffer, transition wash to high-salt wash buffer, 1x high-salt wash buffer, transition wash to wash buffer, 3x wash buffer. After the final wash, 20% per IP sample was kept for the diagnostic western blot. To all IP and SMInput samples, NuPAGE LDS buffer (Thermo Fisher Scientific) containing DTT (98 mM, freshly prepared) was added before samples were incubated at 70 °C for 10 min. After magnetically separating the beads from the lysates, RNA-protein complexes were separated by gel electrophoresis using 8% Bis-Tris gels, and transferred onto nitrocellulose or methanol-activated PVDF membranes for the RNA gels and western blots, respectively. The diagnostic western blot was used as a guide to cut out the region of the membrane starting from the observed size of the protein of interest and extending until the maximal expected size of the RNA-protein complexes (but at least ~75 kDa larger). Membrane pieces were then sliced into ~2 mm x 2 mm squares and transferred into a fresh tube. To digest the protein, membranes were incubated with 24 U proteinase K (Roth) in PKS buffer for 20 min at 37 °C followed by an incubation at 50 °C for another 20 min. RNA was extracted using the RNA Clean and Concentrator-5 kit (Zymo) following the manufacturer’s instructions, and RNA was eluted in 10 µl H2O. Next, SMInput samples were dephosphorylated by incubating each SMInput RNA sample with 20 µl of dephosphorylation master mix (1x FastAP buffer (Thermo Fisher Scientific), 40 U murine RNase inhibitor (NEB), 2 U FastAP enzyme (Thermo Fisher Scientific) incubating at 37 °C for 20 min shaking at 1,200 rpm, followed by another 20 min incubation at 37 °C (1,200 rpm) in PNK master mix (1.05x PNK7 buffer, 5.26 mM DTT, 2 U Turbo DNase (NEB), 40 U T4 PNK enzyme). RNA was purified using the Clean & Concentrator 5 kit (Zymo) as described before. To append the SMInput RNA with 3’ RNA adapters, 1.5 µl 100% DMSO and 0.5 µl InvRil19 adapter were added per sample and incubated at 65 °C for 2 min. 13.5 µl of SMInput RNA ligation master mix (0.976x RNA ligase buffer (NEB), 0.976 mM ATP, 2.93% DMSO, 0.02% v/v Tween-20, 14.63 w/v PEG 8000, 12 U murine RNase inhibitor, 36 U T4 RNA ligase high-concentration enzyme) were added and samples were incubated at RT for 60 min on a rotating wheel. To purify the RNA, 15 µl MyOne Silane Dynabeads per sample were washed ones with RLT buffer (Qiagen), resuspended in RLTW buffer (RLT + 0.025% v/v Tween-20) and added to the RNA. An equal volume of 100% EtOH was added and samples were incubated at RT for 10 min, before samples were washed three times with 80% v/v EtOH. After the final wash, beads were tried and subsequently eluted in 9.5 µl of TT elution buffer (10 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 0.01% (vol/vol) Tween-20). All IP and SMInput samples were next reverse transcribed using the SuperScript III enzyme. For that, 1 µl of 5 µM InvAR17 RT primer  and 1 µl of 10 mM dNTPs were added per RNA sample and incubated for 2 min at 65 °C. 10 µl of reverse transcription master mix (1x first strand buffer (Invitrogen), 5 mM DTT, 8 U murine RNase inhibitor (NEB), 120 U Superscript III enzyme (Invitrogen)) were added to each sample and incubated at 55 °C for 20 min. 2.5 µl ExoSAP-IT (Thermo Fisher Scientific) were used to remove unincorporated dNTPs and RT primers (37 °C, 15 min) before 1 µl of 0.5 M EDTA was added. To remove RNA stands, 3 µl of 1 M sodium hydroxide were added and incubated at 70 °C for 10 min. 3 µl of 1 M hydrochloric acid were added to neutralize the sodium hydroxide. RNAs were again purified using MyOne Silane beads as described earlier. After drying, 2.5 µl of 5’ adapter master mix (4.66 µM InvRand3Tr3 adapter and 7.77% DMSO in TT elution buffer) were directly added to the beads and incubated at 70 °C for 2 min before 7.8 µl of ligation master mix (0.097% RNA ligase buffer (NEB), 1.94 mM DTT, 0.97 mM ATP, 0.019% v/v Tween-20, 17.48% PEG 8000, 30 U T4 RNA ligase high-concentration enzyme (NEB), 5’ deadenylase enzyme (NEB)) and incubated at RT overnight. RNA was subsequently purified using MyONE silane beads as described earlier. To estimate the number of cycles required for the final PCR, qPCRs were conducted. For multiplexing of eCLIPs, each RNA adapters containing unique barcodes (RNA_A01, RNA_B06, RNA_C01, RNA_D08, RNA_X1A, RNA_X1B) based on 44 were ligated to the RNA after immunoprecipitation. At least one third of each sample was mixed and further processed as a pool. SMI were separately run through the gel and barcoded RNA adapters were ligated after RNA isolation from the nitrocellulose membranes. As for the IP samples, inputs were merged to a pool and processed together. For total input samples, 20 µl of lysate were taken before the immunoprecipitation. Using the eCLIP adapter strategy 44, the in-line barcode on read 1 was later used to demultiplex the samples bioinformatically. Samples labeled with * contain 10% murine spike-in (NIH 3T3) that has not been used for further analysis. For fractionation eCLIPs (+) cells were cross-linked as described above. Cells were then fractionated as described in Nojima et al. Briefly, cells were resuspended in ice-cold HLB+N buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2 and 0.5% (vol/vol) NP-40) and incubated for 5 min on ice. The lysates were then resuspended in 1 ml ice-cold HLB-NS buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2, 0.5% (vol/vol) NP-40 and 10% (wt/vol) sucrose) and centrifuged at 420 g for 5 min. The cytosolic fraction in the supernatant was removed and the nuclear pellet was resuspended in ice-cold NUN1 buffer (20 mM Tris-HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, and 50% (vol/vol) glycerol), followed by the addition of ice-cold NUN2+E buffer (20 mM HEPES-KOH (pH 7.6), 300 mM NaCl, 0.2 mM EDTA, 7.5 mM MgCl2, 1% (vol/vol) NP-40, 1 M urea, and 3% Empigen (Sigma, Cat. No. 30326)). After incubation for 5 min on ice, samples were centrifuged at 300 g for 1 min at 4°C and the nucleoplasmic supernatant was separated from the chromatin pellet. The chromatin pellet was washed twice with ice-cold PBS. Immunoprecipitations were performed in seCLIP Lysis Buffer. All subsequent steps were performed as described for eCLIP multiplex sequencing.
cDNA libraries were amplified using a three-step, six cycle amplification followed by a two-step amplification bringing each sample to its calculated number of cycles. The final libraries were either purified using an agarose gel-based strategy cutting out gel pieces corresponding to 175-300 bp in size and purifying the DNA using the Qiagen MiniElute gel extraction kit following the manufacturer’s instructions. Alternatively, samples were cleaned using a two-sided selection with SPRI beads (0.7x right-sided, followed by a 1x left-sided selection). The quality and quantity of the libraries were assessed using the Fragment Analyzer (Agilent). To account for different cycle number used during final PCR amplification (e.g. for IgG controls), DNA spike-in containing adaptors for the Illumina index primers used in the final PCR was generated and equal amounts were added to the immunoprecipitation samples prior to the final PCR for indicated experiments (#), resulting in different relative ratios of CLIP:DNA spike-in in the final libraries as observed upon measurement using the fragment analyzer.

value: MYC-Tet-On

value: doxycycline (1 µg/ml, 24 h)


title: fecal, SPF, 10wk, rep2

description: Library name: SPF10w2


title: fecal, SPF, 8wk, rep3

description: Library name: SPF8w3


description:
Library name: eCLIP_fractionation_nucleoplasm_MYC_input
+


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-94
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, SPF, 8wk, rep2

description: Library name: SPF8w2


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-93
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: eCLIP_fractionation_nucleoplasm_MYC_R1

description:
Library name: eCLIP_fractionation_nucleoplasm_MYC_R1
+


title: SLE_Control HD A0221030


description:
Library name: eCLIP_fractionation_nucleoplasm_IgG_input
+


title: SLE_Control HD A0221040


title: SLE_Control HD A0221018


title: fecal, SPF, 6wk, rep2

description: Library name: SPF6w2


title: fecal, SPF, 12wk, rep1

description: Library name: SPF12w1


title: eCLIP_fractionation_nucleoplasm_IgG_R2

description:
Library name: eCLIP_fractionation_nucleoplasm_IgG_R2
+


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-102
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-97
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-91
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-92
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, SPF, 8wk, rep1

description: Library name: SPF8w1


title: BcBKO,  rep3

city: Hangzhou

first: Zhuoying

last: Yang

address: Yuhangtang Road

country: China

institute: Zhejiang University

description: Library name: Sample 6

data_processing:
These reads were trimmed to 38-bp paired-end reads using the fastx trimmer tool.
Broad peaks were identified using the MACS2 (v.2.1.1.20160309) package with specific parameters (using the parameter of (--nomodel --shift −100 --extsize 200 -B --broad)
Assembly: GRCm38
Supplementary files format and content: bigwig

source_name: islet

extract_protocol:
Islets were obtained from six mice of each genotype and immediately digested into single cells.  For each transposase (TD501; Vazyme) reaction, 50,000 nuclei were used, followed by barcoding and sequencing library amplification.
DNA sequencing of islets was carried out using the HiSeq 4000 instrument, with 150-bp paired-end reads.

value: islet

value: BcBKO


title: fecal, SPF, 6wk, rep1

description: Library name: SPF6w1


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-90
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: eCLIP_fractionation_nucleoplasm_IgG_R1

description:
Library name: eCLIP_fractionation_nucleoplasm_IgG_R1
+

value: Bone


title: BcBKO,  rep2

description: Library name: Sample 5


title: fecal, SPF, 4wk, rep2

description: Library name: SPF4w2


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-89
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-88
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: flox/flox,  rep3

description: Library name: Sample 3

value: flox/flox


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-87
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: flox/flox,  rep2

description: Library name: Sample 2


title: fecal, SPF, 2wk, rep2

description: Library name: SPF2w3


title: eCLIP_fractionation_chromatin_MYC_R2

description:
Library name: eCLIP_fractionation_chromatin_MYC_R2
+

value: Osteosarcoma cell line


title: fecal, SPF, 4wk, rep1

description: Library name: SPF4w1


title: eCLIP_fractionation_chromatin_IgG_input

description:
Library name: eCLIP_fractionation_chromatin_IgG_input
+


title: fecal, SPF, 2wk, rep1

description: Library name: SPF2w2


title: SLE_Control HD A0221011


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-86
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: flox/flox,  rep1

description: Library name: Sample 1


title: SLE_Control HD A0221024


title: eCLIP_fractionation_chromatin_MYC_input

description:
Library name: eCLIP_fractionation_chromatin_MYC_input
+


title: SLE_Control HD A0221044


title: eCLIP_fractionation_chromatin_IgG_R2

description:
Library name: eCLIP_fractionation_chromatin_IgG_R2
+


title: BcBKO,  rep1

description: Library name: Sample 4


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-85
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: eCLIP_fractionation_chromatin_MYC_R1

description:
Library name: eCLIP_fractionation_chromatin_MYC_R1
+


title: eCLIP_fractionation_chromatin_IgG_R1

description:
Library name: eCLIP_fractionation_chromatin_IgG_R1
+


title: fecal, LPD, 12wk, rep1

description: Library name: LDP12w1

value: Low-dose penicillin


title: 8505c thyroid cancer cells, veh, replicate 3

city: Detroit

first: Moray

last: Campbell

state: MI

address: 4100 John R

institute: Barbara Ann Karmanos Cancer Institute, Detroit , Michigan, US

description: Library name: 8505c-UT.7.BAM

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using FASTQC and Trimmomatic
Trimmed sequence reads were mapped to hg38 using RSubread
Read count extraction and normalization were performed featureCounts and an edgeR workflow in R
Assembly: hg38
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

source_name: Thyroid

extract_protocol:
Total RNA was isolated via TRIzol® reagent
Sequencing libraries prepared with the TruSeq Stranded Total RNA kit (Illumina Inc), from 1ug total RNA

value: Thyroid

value: Thyroid Cancer Cell Line

value: WT

value: veh


title: 8505c thyroid cancer cells, veh, replicate 1

description: Library name: 8505c-UT.1.BAM


title: fecal, LPD, 10wk, rep2

description: Library name: LDP10w2


title: 8505c thyroid cancer cells, veh, replicate 2

description: Library name: 8505c-UT.5.BAM


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-84
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad

value: A375 cell line

value: Melanoma

value: BRAF V600E

value: DMSO or Vemurafenib


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-83
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, LPD, 12wk, rep2

description: Library name: LDP12w2

value: fecal


description: Library name: eCLIP_U2OSMYCtetOn_CDK9i_MYC_NVP_2_input

value: MYC-Tet-On

value: doxycycline (1 µg/ml, 24 h), NVP-2 (200 nM, 1 h)


title: eCLIP_U2OSMYCtetOn_CDK9i_MYC_NVP_2_R2

description: Library name: eCLIP_U2OSMYCtetOn_CDK9i_MYC_NVP_2_R2


title: eCLIP_U2OSMYCtetOn_CDK9i_MYC_NVP_2_R1

description: Library name: eCLIP_U2OSMYCtetOn_CDK9i_MYC_NVP_2_R1


title: fecal, LPD, 8wk, rep3

description: Library name: LDP8w3


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-82
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: 8505c thyroid cancer cells, CUET 250 nM, 24h, replicate 2

description: Library name: 8505c-CUET.6.BAM

value: CUET 250 nM, 24 hours


title: 8505c thyroid cancer cells, CUET 250 nM, 24h, replicate 3

description: Library name: 8505c-CUET.8.BAM


title: fecal, LPD, 10wk, rep1

description: Library name: LDP10w1


title: 8505c thyroid cancer cells, CUET 250 nM, 24h, replicate 1

description: Library name: 8505c-CUET.4.BAM


title: fecal, LPD, 8wk, rep2

description: Library name: LDP8w2


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-80
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-81
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: eCLIP_U2OSMYCtetOn_CDK9i_IgG_pool_input

description: Library name: eCLIP_U2OSMYCtetOn_CDK9i_IgG_pool_input

value: merge


title: fecal, LPD, 6wk, rep2

description: Library name: LDP6w2


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-79
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: eCLIP_U2OSMYCtetOn_CDK9i_IgG_pool_R1

description: Library name: eCLIP_U2OSMYCtetOn_CDK9i_IgG_pool_R1

value: Bone


title: fecal, LPD, 8wk, rep1

description: Library name: LDP8w1


title: eCLIP_U2OSMYCtetOn_CDK9i_IgG_pool_R2

description: Library name: eCLIP_U2OSMYCtetOn_CDK9i_IgG_pool_R2


title: kdrl:mCherry+ endothelial cells at 30 hours post fertilization crip2-/-;crip3-/-

city: Trieste

first: UDAY

last: RANGASWAMY

state: Trieste

address: via bonomea 265

department: NEUROSCIENCE

country: Italy

institute: SISSA

description: Library name: KA002

data_processing:
The read alignment and gene counting were made using the Cell Ranger software v6.1.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: GRCz11/danRer11
Supplementary files format and content: Tab-separated values files and matrix files

molecule: polyA RNA

organism: Danio rerio

source_name: kdrl:mCherry+ endothelial cells

extract_protocol:
kdrl:mCherry+ cells were isolated from transgenic fluorescent zebrafish embryos using FACS Aria.
Sorted Cell populations were loaded on a Chromium Single Cell Controller (10X Genomics) to generate single-cell gel beads in emulsion (GEMs) by using  Single Cell 3' Reagents Kits v3.1 (10X Genomics). Captured cells were lysed and the released RNA was barcoded through reverse transcription in individual GEMs (Zheng et al., 2017). ScRNA-Seq libraries were prepared using Single Cell 3' library Gel Bead Kit v3.1 (10X Genomics) following the manufacture's instructions.

value: kdrl:mCherry+ endothelial cells

tag: developmental stage

value: 30 hours post fertilization

tag: transgenic line


title: kdrl:mCherry+ endothelial cells at 30 hours post fertilization wild-type

description: Library name: KA001

value: wild-type


title: eCLIP_U2OSMYCtetOn_CDK9i_MYC_DMSO_input

description: Library name: eCLIP_U2OSMYCtetOn_CDK9i_MYC_DMSO_input

value: Osteosarcoma cell line

value: doxycycline (1 µg/ml, 24 h), DMSO


title: fecal, LPD, 6wk, rep1

description: Library name: LDP6w1

value: Low-dose penicillin


title: eCLIP_U2OSMYCtetOn_CDK9i_MYC_DMSO_R2

description: Library name: eCLIP_U2OSMYCtetOn_CDK9i_MYC_DMSO_R2


title: Mouse embryos, E4.5, KI

city: Barcelona

first: Agustín

middle: Sánchez

last: Belmonte

state: Barcelona

address: Nazaret 115 -117

department: Systems Oncology Program

country: Spain

institute: Vall d ́Hebron Institute of Oncology (VHIO)

description: embryos were collected in the indicated stage and dissagregated for transcriptomic analysis

data_processing:
Quality control was performed using fastqc (version 0.11.9)
We performed read alignment to the mm10 version of GRCh38 mouse reference genome. Quantification and initial quality control were performed using STARsolo of STAR (version 2.7.10a) and STARsolo filtered count matrices were used for downstream analysis.
We integrated the filtered count matrices from STARsolo and analysed them with scanpy v. 1.8.2 following their recommended standard practices (Wolf et al, 2018).
Assembly: GRCh38
Supplementary files format and content: raw matrix of counts, features and barcodes (STAR)
Supplementary files format and content: spliced, unspliced and ambigous matrix from velocyto (STAR)
Supplementary files format and content: filtered barcodes, cells filtered from raw barcodes (STAR)

source_name: Mouse embryos

growth_protocol: Embryos were directly isolated from the female donors prior to transcriptomic analysis

extract_protocol:
E3.5 blastocysts, zona pellucida was removed passing the embryos by mouth pipetting through three consecutive drops of Tyrode’s acidic solution pH 2.5 (Sigma T1788). After careful examination under the microscope for disintegration of the pellucida, tyrode’s solution was immediately neutralized with a double volume of PBS pH 7.4 (Gibco #10010023), and embryos were placed into 20 μL of dissociation buffer composed of TrypLE™ Express Enzyme (Gibco # 12604013) / Accutase (Gibco # A1110501) in a 1:1 ratio, in a low binding 200 μL microcentrifuge tube. To avoid attachment of the embryos to the pulled crystal capillaries, capillaries were coated with 1% BSA in PBS beforehand. Blastocysts were incubated for 20 minutes in dissociation buffer at 37ºC, pipetted up and down gently 60 times with 20 μL low binding pipette tips, incubated 10 additional minutes at 37ºC and pipetted up and down again gently 40 times to disaggregate the last ICM cell clumps. After dissociation, the resulting cell suspension was neutralized with 40uL of 1% cell culture grade BSA (Sigma # A8806). In the case of 8-cell and morula stage embryos, pellucida was removed as abovementioned and embryos were placed in 20 μL of Accutase (Gibco # A1110501) for 20 min at 37ºC. After incubation, embryos were pipetted up and down 50 times until complete disaggregation and neutralized by the addition of 40 μL of 1% cell culture grade BSA (Sigma # A8806) to avoid the formation of clumps after dissociation.  Then, cell suspensions were immediately placed on ice until processing for scRNA-seq
Cell samples were loaded onto a 10x Chromium Single Cell controller chip B (10x Genomics) following manufacturer’s intructions (Chromium Single Cell 3’GEM, Library & Gel Bead Kit v3, ref. PN-1000075). Intended targeted cell recovery of ∼1000 - 10000 cells depending on the embryonic stage. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, cDNA amplification and library construction were all performed as recommended by the manufacturer.

treatment_protocol: In the KI condition, female donors were fed with doxycicline diet from the presence of plug to the isolation stage (E3.5 / E4.5)

value: Mouse embryos

value: E4.5 blastocyst cells

value: miR-203 KI

value: DOX


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-78
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad

value: A375 cell line

value: Melanoma

value: BRAF V600E

value: DMSO or Vemurafenib


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-76
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: fecal, LPD, 4wk, rep2

description: Library name: LDP4w2

value: fecal


title: fecal, LPD, 4wk, rep1

description: Library name: LDP4w1


title: Mouse embryos, E4.5, KO

value: miR-203 KO


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-95
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: eCLIP_U2OSMYCtetOn_CDK9i_MYC_DMSO_R1

description: Library name: eCLIP_U2OSMYCtetOn_CDK9i_MYC_DMSO_R1

value: Bone

value: MYC-Tet-On


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-77
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-75
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: ChIP_Rx_U2OSMYCtetOn_RNAPIIS2_PlaB_R2

description: Library name: ChIP_Rx_U2OSMYCtetOn_RNAPIIS2_PlaB_R2

data_processing:
ChIP-Rx samples were mapped separately to the human GRCh38 genome and to the murine GRCm39 genome using Bowtie2 with -N 1 parameters. Subsequently, aligned reads were spike-normalized by dividing the number of mapped reads aligned to GRCh38 by the number of reads mapped to GRCm39 for each sample and multiplying this ratio with the smallest number of reads aligned to GRCm39 for any sample.
GRCh38
bedgraph files for each condition and replicate.

value: doxycycline (1 µg/ml, 24 h); PlaB (1 µM, 4 h)


title: Mouse embryos, E4.5, WT

value: miR-203 WT


title: fecal, LPD, 2wk, rep2

description: Library name: LDP2w3


title: fecal, LPD, 2wk, rep1

description: Library name: LDP2w2


title: ChIP_Rx_U2OSMYCtetOn_RNAPIIS2_DMSO_R2

description: Library name: ChIP_Rx_U2OSMYCtetOn_RNAPIIS2_DMSO_R2

value: Osteosarcoma cell line

value: doxycycline (1 µg/ml, 24 h); DMSO


title: Mouse embryos, E3.5, KO

value: E3.5 blastocyst cells


title: ChIP_Rx_U2OSMYCtetOn_RNAPIIS2_CBL_R2

description: Library name: ChIP_Rx_U2OSMYCtetOn_RNAPIIS2_CBL_R2

value: doxycycline (1 µg/ml, 24 h); CBL0137 (5 µM, 2 h)


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-73
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: Mouse embryos, E3.5, WT

value: Mouse embryos


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-74
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad


title: GEM-X Flex Gene Expression, pool of 4 samples (Pool 4)

city: San Francisco

first: Ayushi

last: Agrawal

address: 1650 Owens St

institute: Gladstone Institutes

description:
Library name: GEM_Well_4
10X Genomics GEM-X Flex gene expression single-nucleus RNA-sequencing

data_processing:
The demultiplexing, barcoded processing, and gene counting were performed using the 10x Genomics Cell Ranger v9.0.1 multi pipeline (https://www.10xgenomics.com/support/software/cell-ranger/latest). FASTQ files were aligned to the 10x Genomics pre-built GRCm39 reference (refdata-gex-GRCm39-2024-A) using the Chromium mouse transcriptome probe set v1.1.1. The create-bam option was set to false to skip BAM file generation. Sample demultiplexing was performed for each GEM well based on feature barcode oligos defined in the corresponding configuration files. The per-well config files (GEM_Well_1_config.csv, GEM_Well_2_config.csv, GEM_Well_3_config.csv, GEM_Well_4_config.csv) are provided in the processed data files. Filtered gene expression count matrices were generated and used for downstream analysis using the Seurat R package (https://satijalab.org/seurat/).
Assembly: Mouse GRCm39 (10x Genomics pre-built reference refdata-gex-GRCm39-2024-A was used and is available at https://www.10xgenomics.com/support/software/cell-ranger/downloads#reference-downloads)
Supplementary files format and content: Filtered feature-barcode count matrix in the Hierarchical Data Format (HDF5 or H5) (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/h5_matrices)
Supplementary files format and content: Quality control summary metrics from Cell Ranger multi in comma-separated values (CSV) format
Supplementary files format and content: Configuration file in CSV format used for sample demultiplexing within a given GEM well

source_name: Brain

growth_protocol: Mice were housed at room temperature and humidity (approximately 20 °C and 50% humidity) with a 12h:12h light: dark cycle. Mice were fed a chow diet (PicoLab 5058).  All experiments in animals were approved by the Institutional Animal Care and Use Committee at UCSF/Gladstone Institutes. Mice were genotyped by polymerase chain reaction (PCR) followed by MwoI restrcition digestion from a tail clipping.

extract_protocol:
Nuclei were isolated from frozen mouse hemi brains using Dounce homogenization (10 strokes of Pestle A, 20 strokes of Pestle B) in 1 mL of cold homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, 1 mM DTT, 0.5 mM spermidine, 0.15 mM spermine, 1% Triton) containing RNase inhibitor in a 2 mL Dounce, followed by preparation of an Iodixanol gradient (25%, 30%, 40% Iodixanol layers) and density gradient centrifugation in a swinging bucket centrifuge. Nuclei appeared as a band between the 30% and 40% Iodixanol layers after centrifugation. Nuclei were washed, counted, and centrifuged at 350 rcf. After removing the supernatant the nuclei pellet was resuspended in Fixation Buffer B containing 4% Formaldehyde from the 10x Genomics GEM-X Flex Sample Preparation Kit v2. Nuclei samples were fixed overnight at 4 degrees C before addition of Additive C reagent and being quenched with Quenching Buffer B provided with the Sample Preparation Kit.
GEM-X Flex Gene Expression libraries were prepared using the 10x Genomics User Guide CG000787 (GEM-X Flex Reagent Kit for Multiplexed Samples). 300,000 fixed nuclei per mouse sample was used for the GEM-X Flex probe hybridization step. Probe hybridization was conducted using the GEM-X Flex Mouse Transcriptome Probe Kit, 16 Samples. After overnight probe hybridization 4 separate pools containing 4 samples each were generated for a total of 16 samples. Each pool was run across 1 GEM (Gel Bead in Emulsion) reaction for a total of 4 GEM reactions. After GEM generation and barcoding, GEMs were recovered and the resulting material underwent Pre-Amplification and SPRI bead cleanup. For each pool of 4 samples, we generated 1 GEM-X Flex Gene Expression library for a total of 4 libraries. During the final library construction PCR step each library was uniquely indexed using the 10x Genomics Dual Index Plate TS Set A. After the final library construction PCR and SPRI bead size selection, the final GEM-X Flex Gene Expression libraries underwent BioAnalyzer QC (Agilent High Sensitivity DNA Kit). All libraries contain the standard Illumina paired-end constructs that start and end with P5 and P7 sequences. Read 1 encodes the 16 bp GEM barcode and the 12 bp UMI. Read 2 encodes the ligated probe insert, a constant sequence, and the 8 bp Probe Barcode sequence that identifies the mouse probe set used in the hybridization step.

treatment_protocol: At p0, mice were randomly assigned treatment of daily 3uL/g intraperitoneal with either phosphate buffered saline vehicle or 750 mg/kg Nicotinamide Riboside for 5 days until collection.  Before collection, mice were perfused with PBS, and the left hemi-brains (excluding the olfactory bulb) were collected from both Control and NAXD KO mice (n = 4 per genotype per treatment group).

value: Brain


title: ChIP_Rx_U2OSMYCtetOn_RNAPII_DMSO_R2

description: Library name: ChIP_Rx_U2OSMYCtetOn_RNAPII_DMSO_R2


title: Mouse embryos, E3.5, KI

value: miR-203 KI

value: DOX


title: ChIP_Rx_U2OSMYCtetOn_RNAPII_PlaB_R2

description: Library name: ChIP_Rx_U2OSMYCtetOn_RNAPII_PlaB_R2


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-71
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad

value: A375 cell line

value: Melanoma


title: Mouse embryos, E2.5, KO, H3K27ac

description: embryos were collected in the indicated stage and dissagregated for epigenetic analysis

data_processing:
Quality control was performed using fastqc.
Paired-end reads were aligned against mm10 genome using Bowtie2 (version 2.4.2) with the following parameters “-local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700”. Then we excluded reads that fail in the alignment using samtools (version 1.11) and duplicated reads with GATK (version 4.1.9).
Bam files were converted to bigwig using bamCoverage from deeptools (version 3.5.1).
Assembly: GRCh38
Supplementary files format and content: bigwig

growth_protocol: Embryos were directly isolated from the female donors prior to epigenetic analysis

extract_protocol:
E2.5 blastocysts, zona pellucida was removed passing the embryos by mouth pipetting through three consecutive drops of Tyrode’s acidic solution pH 2.5 (Sigma T1788). After careful examination under the microscope for disintegration of the pellucida, tyrode’s solution was immediately neutralized with a double volume of PBS pH 7.4 (Gibco #10010023), and embryos were placed into 20 μL of dissociation buffer composed of TrypLE™ Express Enzyme (Gibco # 12604013) / Accutase (Gibco # A1110501) in a 1:1 ratio, in a low binding 200 μL microcentrifuge tube. To avoid attachment of the embryos to the pulled crystal capillaries, capillaries were coated with 1% BSA in PBS beforehand. Blastocysts were incubated for 20 minutes in dissociation buffer at 37ºC, pipetted up and down gently 60 times with 20 μL low binding pipette tips, incubated 10 additional minutes at 37ºC and pipetted up and down again gently 40 times to disaggregate the last ICM cell clumps. After dissociation, the resulting cell suspension was neutralized with 40uL of 1% cell culture grade BSA (Sigma # A8806). In the case of 8-cell and morula stage embryos, pellucida was removed as abovementioned and embryos were placed in 20 μL of Accutase (Gibco # A1110501) for 20 min at 37ºC. After incubation, embryos were pipetted up and down 50 times until complete disaggregation and neutralized by the addition of 40 μL of 1% cell culture grade BSA (Sigma # A8806) to avoid the formation of clumps after dissociation.  Then, cell suspensions were immediately placed on ice until processing for scRNA-seq
DNA samples were processed through subsequent enzymatic treatments for end-repair, dA-tailing, and ligation to adapters with "NEBNext Ultra II DNA Library Prep Kit for Illumina" (NEB #E7645). Adapter-ligated libraries were completed by a PCR of 21 cycles of amplification, with 63º T(ann/ext) for 10s, and extracted with a (single) double-sided SPRI size selection.
Ultra-low input CUT&RUN

treatment_protocol: In the KI condition, female donors were fed with doxycicline diet from the presence of plug to the isolation stage (E2.5)

value: Blastocyst cells

value: miR203 KO


title: GEM-X Flex Gene Expression, pool of 4 samples (Pool 2)

description:
Library name: GEM_Well_2
10X Genomics GEM-X Flex gene expression single-nucleus RNA-sequencing


title: ChIP_Rx_U2OSMYCtetOn_RNAPII_CBL_R2

description: Library name: ChIP_Rx_U2OSMYCtetOn_RNAPII_CBL_R2


title: GEM-X Flex Gene Expression, pool of 4 samples (Pool 3)

description:
Library name: GEM_Well_3
10X Genomics GEM-X Flex gene expression single-nucleus RNA-sequencing


title: Mouse embryos, E2.5, WT, H3K27ac

value: miR203 WT


title: Mouse embryos, E2.5, KI, H3K27ac

value: miR203 KI


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-70
PerturbFate Tri-omics single-cell library containing Nascent RNA modality
Tri_omics_RNA_cell_metadata.csv
LabeledRNA_gene_count_matrix.RDS
UnlabeledRNA_gene_count_matrix.RDS
Tri_omics_sgRNA_count_matrix.RDS
Tri_omics_ATAC_cell_metadata.csv
Tri_omics_ATAC_peak_count_matrix.h5ad

value: BRAF V600E

value: DMSO or Vemurafenib


title: ChIP_Rx_U2OSMYCtetOn_RNAPIIS2_PlaB_R1

description: Library name: ChIP_Rx_U2OSMYCtetOn_RNAPIIS2_PlaB_R1

value: Bone

value: MYC-Tet-On

value: doxycycline (1 µg/ml, 24 h); PlaB (1 µM, 4 h)


description: Library name: ChIP_Rx_U2OSMYCtetOn_RNAPII_input_pool_R2

value: Osteosarcoma cell line

value: merged of conditions


title: Mouse embryos, E2.5, KI, H3K9ac

value: Mouse embryos