title: brain vasc_Tg350_6 months, rep 3

description: Library name: 20210618_1

value: CADASIL

value: TgN3 R182C


status: Public on Feb 06 2026

type: SRA

city: New York

first: Zihan

last: Xu

state: NY

address: 1230 York Ave

country: USA

institute: The Rockefeller University

description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-53
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities

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

organism: Homo sapiens

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: Lo9_Day0

city: Chicago

first: Joao

last: Barroso-Batista

state: IL

address: The University of Chicago, KCBD room 9220E, 900 E 57th Street

institute: The University of Chicago

description: Library name: NSPF3.RLo9.Day0

library_source: transcriptomic

data_processing:
reads were trimmed with trimmomatic to remove adapter sequence and trailing bases with a quality score less than 20
trimmed reads were mapped to the rhesus macaque genome (RheMac8) using the STAR v2.7.10ac two-pass method
read counts were generated for each gene using HTCseq v2.0 based on overlap of any part of a read with an annotated gene exon
Assembly: RheMac8
Supplementary files format and content: comma-delimited text file includes raw counts for each sample

molecule: total RNA

organism: Macaca mulatta

source_name: blood

extract_protocol:
RNA was extracted using miRNAeasy kit (Qiagen). 300 ng of RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using Illumina Stranded mRNA Prep kit (Illumina) according to the manufacturer’s protocol

tag: tissue

value: blood

value: PBMCs

value: Influenza vaccination


title: brain vasc_Tg350_6 months, rep 2

city: Stockholm

first: Michael

last: Vanlandewijck

address: Blickagången 6

department: Department of Medicine

country: Sweden

institute: Karolinska Institute

description: Library name: 20210616_3

library_source: transcriptomic single cell

data_processing:
The data was sequenced on an Illumina NextSeq2000 sequencer and sequencing output files were demultiplexed and aligned using the Cellranger pipeline (v7.0.0, 10xGenomics). Mapping was done to the mouse mm10 transcriptome where the mouse Clec7a, and human NOTCH3 genes were manually added
Assembly: Mouse mm10
Supplementary files format and content: Tab-separated files and matrix expression files

molecule: polyA RNA

organism: Mus musculus

growth_protocol: All mice were housed in cages of 3-5 mice  with access to ad libitum food and water and kept under a 12h light dark circle at 50% humidity. Animal caretakers inspected animals daily.

extract_protocol:
Cells were extracted using our previously published protocol for microvascular isolation (PMID 37056377). In short, brains were digested into microvascular fragments, which were isolated using magnetic beads coupled to CD31 antibodies. These fragments were then washed and redigested into single cells, which were then loaded onto a 10x Genomics Chromium controller or Chromium iX.
Libraries were contructed according to manufacturers' instructions, using the 10x Genomics NextGEM single cell gene expression kit, v3.2

treatment_protocol: The mice were untreated and were analysed at their respective timepoints

tag: experimental condition

tag: gender

value: female

tag: age

value: 6 months

tag: batch


title: brain vasc_Tg350_6 months, rep 1

description: Library name: 20210616_2

value: male


title: brain vasc_Tg350_9 months, rep 3

description: Library name: 20210420_1

source_name: mouse cerebrovasculature_Tg350_9 months _female

value: 9 months


title: Lo9_Day1

description: Library name: NSPF3.RLo9.Day1


title: brain vasc_Tg350_9 months, rep 2

description: Library name: 20210225_3

source_name: mouse cerebrovasculature_Tg350_9 months _male


title: brain vasc_Tg350_9 months, rep 1

description: Library name: 20210225_2

value: mouse cerebrovasculature_Tg350_9 months _female


title: eCLIP_KPC_shMYC_input

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: eCLIP_KPC_shMYC_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. 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

source_name: PDAC

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

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.

treatment_protocol: Treatment as indicated

value: PDAC

value: KPCshMYC

value: KPC cells

value: Ptf1a-Cre, KRasG12D/+, Tp53R172H/+

value: doxycycline (1 µg/ml, 72 h)


title: Lm8_Day7

description: Library name: NSPF3.RLm8.Day7


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-54
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities

value: A375 cell line


title: Lm8_Day3

description: Library name: NSPF3.RLm8.Day3


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-50
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities

value: Melanoma


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-51
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities

value: BRAF V600E


title: eCLIP_KPC_wtMYC_input

description: Library name: eCLIP_KPC_wtMYC_input


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-52
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities

value: DMSO or Vemurafenib


title: eCLIP_KPC_shMYC_R2

description: Library name: eCLIP_KPC_shMYC_R2


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-49
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities


title: eCLIP_KPC_IgG_input

description: Library name: eCLIP_KPC_IgG_input

value: merge


title: eCLIP_KPC_RBRIIImutMYC_input

description: Library name: eCLIP_KPC_RBRIIImutMYC_input


title: eCLIP_KPC_shMYC_R1

description: Library name: eCLIP_KPC_shMYC_R1


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-48
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities


title: eCLIP_KPC_RBRIIImutMYC_R2

description: Library name: eCLIP_KPC_RBRIIImutMYC_R2


title: Lm8_Day0

description: Library name: NSPF3.RLm8.Day0

value: blood

value: PBMCs


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-47
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities


title: eCLIP_KPC_RBRIIImutMYC_R1

description: Library name: eCLIP_KPC_RBRIIImutMYC_R1


title: eCLIP_KPC_wtMYC_R2

description: Library name: eCLIP_KPC_wtMYC_R2


title: eCLIP_KPC_IgG_merge_R2

description: Library name: eCLIP_KPC_IgG_merge_R2


title: Gq10_Day7

description: Library name: NSPF3.RGq10.Day7

value: Influenza vaccination


title: Gq10_Day3

description: Library name: NSPF3.RGq10.Day3


title: Lm8_Day1

description: Library name: NSPF3.RLm8.Day1


title: Gq10_Day1

description: Library name: NSPF3.RGq10.Day1


title: DRIPc_Rx_KPC_shExosc10_r2

description: Library name: DRIPc_Rx_KPC_shExosc10_r2

data_processing:
Fastq files of DRIPc-Rx samples were mapped separately to either murine GRCm39 or human GRCh38 genome and to the fly dm6 genome using STAR (Dobin et al., 2013). Subsequently, aligned reads were spike-normalized by dividing the number of mapped reads aligned to GRCm39 or GRCh38 by the number of reads mapped to dm6 for each sample and multiplying this ratio with the smallest number of reads aligned to dm6 for any sample. Peaks were called using MACS (Zhang et al., 2008) with default broad peak settings. For metagenes and heatmaps, log2FC of DRIPs over inputs were determined based on bam files using bamCompare and plotted using deeptools (Ramirez et al., 2014).
Assembly: GRCh38, GRCm39 and dm6
Supplementary files format and content: bedgraph files

extract_protocol:
DRIPc-sequencing was adapted from Sanz and Chédin, 2019. Cells were lysed rotating overnight at 37°C in TE buffer (pH 8) supplemented with 10% SDS and 10 µL proteinase K (10 mg/mL). For spike-normalization 10% Drosophila S2 cells were added according to the cell number. DNA was isolated by phenol/chloroform extraction using MaXtract High Density Tubes (Qiagen) and fragmented with a mixture of restriction enzymes. For this DNA was incubated overnight with 60 U of BsrGI, EcoRI, HindIII, SSpI and Xbal (New England Biolabs) at 37°C. DNA was cleaned up again with phenol/chloroform and fragmentation was validated by agarose gel electrophoresis to ensure successful digestion of the DNA to a size below 1 kb. Subsequently, 200 µg of DNA was treated with 30 U of RNaseT1 (Thermo Fisher Scientific) at 37°C for 30 min to eliminate ssRNA. To avoid potential dsRNA contamination the DNA was treated with 3 U RNaseIII (Thermo Fisher Scientific) for 1 h at 37°C. Samples were then incubated for 6 h at 37°C with or without 60 U of RNaseH1 (New England Biolabs) to ensure specificity of the following immunoprecipitation step. For this DNA in 1 x DRIP binding buffer (10 x DRIP binding buffer: 100 mM Sodium phosphate, 1.4 M NaCl, 0.5 % Triton X-100) was split into two tubes and incubated overnight with 4 µL of S9.6 antibody (New England Biolabs) while rotating. For total input samples, 10 % of DNA was taken before the immunoprecipitation. On the next day wash 40 µL/IP of Protein A and Protein G-coupled Dynabeads (Thermo Fisher Scientific), add to the DNA/antibody mix and incubate for 4 h on a rotating wheel at. 4°C Immunoprecipitated complexes were then washed 3 times each with ice-cold 1 x DRIP binding buffer. Finally, DNA was eluted by adding DRIP elution buffer (50 mM TRIS pH 8,10 mM EDTA, 0.5 % SDS) and roteinase K for 45 min at 55°C while shaking at 900 rpm. DNA was purified with phenol/chloroform extraction and finally eluted in 50 µl TE buffer. Before proceeding to library preparation, 4 µL were used to test enrichment by qPCR.
DNA:RNA hybrids were then treated with DNase (Thermo Fisher Scientific) for 45 min at 37°C. DNase was inactivated by inactivation buffer (Thermo Fisher Scientific) for 5 min at room temperature. After 5 min of centrifugation at 10’000 rpm the supernatant was transferred to a new PCR tube and libraries were generated using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) following manufacturer’s instructions.

value: 24 h doxycycline (1 µg/ml)


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-45
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities


title: Gq10_Day0

description: Library name: NSPF3.RGq10.Day0


title: eCLIP_KPC_IgG_merge_R1

description: Library name: eCLIP_KPC_IgG_merge_R1


title: DRIPc_Rx_KPC_shExosc10_input_r2

description: Library name: DRIPc_Rx_KPC_shExosc10_input_r2


title: Cl13_Day3

description: Library name: NSPF3.RCl13.Day3


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-43
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Old RNA and ATAC-44
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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_KPC_wtMYC_R1

description: Library name: eCLIP_KPC_wtMYC_R1


title: SLE Severe A0221010

type: RNA

city: Durham

first: James

middle: M

last: Ward

state: NC

address: 111 TW Alexander Dr

department: Integrative Bioinformatics

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

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: SLE

value: Severe


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-46
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-42
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: DRIPc_Rx_KPC_EtOH_r2

description: Library name: DRIPc_Rx_KPC_EtOH_r2


title: SLE Moderate A0221579

value: Moderate


title: Cl13_Day1

description: Library name: NSPF3.RCl13.Day1


title: DRIPc_Rx_KPC_EtOH_input_r2

description: Library name: DRIPc_Rx_KPC_EtOH_input_r2

value: PDAC


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-41
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: Cl13_Day7

description: Library name: NSPF3.RCl13.Day7


title: DRIPc_Rx_KPC_RBRIII_MUT_MYC_Input_r2

description: Library name: DRIPc_Rx_KPC_RBRIII_MUT_MYC_Input_r2

value: KPCshMYC


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-40
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Moderate A0221091


title: SLE Moderate A0221062


title: Cl13_Day0

description: Library name: NSPF3.RCl13.Day0


title: Rn9_Day7

description: Library name: NSPF2.RRn9.Day7


title: Rn9_Day3

description: Library name: NSPF2.RRn9.Day3


title: DRIPc_Rx_KPC_RBRIII_MUT_MYC_r2

description: Library name: DRIPc_Rx_KPC_RBRIII_MUT_MYC_r2

value: KPC cells


title: SLE Moderate A0221105


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-38
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: DRIPc_Rx_KPC_WT_MYC_r2

description: Library name: DRIPc_Rx_KPC_WT_MYC_r2

value: Ptf1a-Cre, KRasG12D/+, Tp53R172H/+


title: DRIPc_Rx_KPC_WT_MYC_Input_r2

description: Library name: DRIPc_Rx_KPC_WT_MYC_Input_r2


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-39
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Moderate A0221033


title: DRIPc_Rx_KPC_shExosc10_r1

description: Library name: DRIPc_Rx_KPC_shExosc10_r1


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-36
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: DRIPc_Rx_KPC_shExosc10_input_r1

description: Library name: DRIPc_Rx_KPC_shExosc10_input_r1


title: Rn9_Day0

description: Library name: NSPF2.RRn9.Day0


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-37
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Moderate A0221004


title: Rn9_Day1

description: Library name: NSPF2.RRn9.Day1


title: SLE Moderate A0221029


title: SLE Moderate A0220993


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-35
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: Ph7_Day7

description: Library name: NSPF2.RPh7.Day7


title: DRIPc_Rx_KPC_EtOH_r1

description: Library name: DRIPc_Rx_KPC_EtOH_r1


title: Ph7_Day3

description: Library name: NSPF2.RPh7.Day3


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-34
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Moderate A0220997


title: Ph7_Day1

description: Library name: NSPF2.RPh7.Day1


title: Ph7_Day0

description: Library name: NSPF2.RPh7.Day0


title: SLE Mild A0221583

value: Mild


title: DRIPc_Rx_KPC_RBRIII_MUT_MYC_r1

description: Library name: DRIPc_Rx_KPC_RBRIII_MUT_MYC_r1


title: SLE Mild A0222017


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-31
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Old RNA and ATAC-33
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: DRIPc_Rx_KPC_RBRIII_MUT_MYC_Input_r1

description: Library name: DRIPc_Rx_KPC_RBRIII_MUT_MYC_Input_r1


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-32
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: Ed8_Day7

description: Library name: NSPF2.REd8.Day7


title: SLE Mild A0221142


title: Ed8_Day3

description: Library name: NSPF2.REd8.Day3


title: Ed8_Day1

description: Library name: NSPF2.REd8.Day1


title: SLE Mild A0221582


title: DRIPc_Rx_KPC_WT_MYC_r1

description: Library name: DRIPc_Rx_KPC_WT_MYC_r1


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-30
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: DRIPc_Rx_KPC_WT_MYC_Input_r1

description: Library name: DRIPc_Rx_KPC_WT_MYC_Input_r1


title: seCLIP_KPC_TLR3_IP_NXL_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) or GRCm38 (TLR3 seCLIPs)
Supplementary files format and content: bedgraph 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.
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), a 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: merge


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-29
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: DRIPc_Rx_KPC_EtOH_input_r1

description: Library name: DRIPc_Rx_KPC_EtOH_input_r1


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-28
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: seCLIP_KPC_TLR3_IP_NXL_r1


title: Bm7_Day7

description: Library name: NSPF2.RBm7.Day7

value: blood


title: seCLIP_KPC_IgG_IP_r2


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-27
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: Ed8_Day0

description: Library name: NSPF2.REd8.Day0

value: PBMCs


title: SLE Mild A0221117


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-26
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: seCLIP_KPC_IgG_IP_r1


title: Bm7_Day1

description: Library name: NSPF2.RBm7.Day1

value: Influenza vaccination


title: Bm7_Day3

description: Library name: NSPF2.RBm7.Day3


title: SLE Mild A0221060


title: SLE Mild A0221050


title: Ai10_Day7

description: Library name: NSPF2.RAi10.Day7


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-24
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Mild A0221158


title: SLE Mild A0221090


title: Bm7_Day0

description: Library name: NSPF2.RBm7.Day0


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-23
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: seCLIP_KPC_TLR3_IP_shMYC_r1

value: doxycycline (1 µg/ml, 72 h)


title: SLE Mild A0221134

value: SLE


title: seCLIP_KPC_TLR3_IP_Ctrl_r2

value: EtOH ( 72 h)


title: SLE Mild A0221034


title: Ai10_Day3

description: Library name: NSPF2.RAi10.Day3


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-25
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Mild A0221057


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-22
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: seCLIP_KPC_TLR3_IP_Ctrl_r1


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-21
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: seCLIP_KPC_TLR3_IP_shMYC_r2


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-20
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: Ai10_Day0

description: Library name: NSPF2.RAi10.Day0


title: Ai10_Day1

description: Library name: NSPF2.RAi10.Day1


title: seCLIP_KPC_Input_IgG


title: SLE Mild A0221027


title: SLE Mild A0221022


title: Sm8_Day7

description: Library name: NSPF1.RSm8.Day7


title: SLE Mild A0221028


title: seCLIP_KPC_Input_NXL


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-19
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Mild A0221012


title: Sm8_Day3

description: Library name: NSPF1.RSm8.Day3


title: seCLIP_KPC_Input_shMYC


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-18
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: seCLIP_KPC_Input_Ctrl


title: mRNA_KPC_shMYC_r3

data_processing:
Base calling was performed with Illumina’s BCL Convert Software, implemented in the Illumina Dragen software (v3.7.4 or v3.10.4) and high-quality PF-clusters (according to the chasity filter) were selected for further analyses. Overall sequencing quality was determined with FastQC (v0.11.9).
STAR (version: 2.7.10a) (Dobin et al. 2013) was used to align the reads against the GRCm39 genome and quantify transcripts in the quant mode. The quantified transcripts were used as input for the R package DESeq2 (Love et al. 2014) for differential expression analysis.
Assembly: GRCm39
Supplementary files format and content: bedgraph files for each replicate (r1, r2, r3), and one count table including all conditions and replicates

extract_protocol:
After treatment, cells were washed once with PBS and lysed with RLT buffer (QIAGEN) supplemented with beta-mercaptoethanol. Total RNA was extracted with RNeasy mini columns (QIAGEN) including an on-column DNase I digestion step according to manufacturer’s instructions. RNA concentration and RQN values were determined with a Fragment Analyzer (Agilent).
RNA samples with RQN > 9 were used for mRNA isolation with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). cDNA libraries were then prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer’s instructions. Libraries were amplified for 11 PCR cycles and size selected using SPRI selected beads (Beckman Coulter).

value: 48 h doxycycline (1 µg/ml)


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-16
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Mild A0221014


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-17
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: Rz6_Day7

description: Library name: NSPF1.RRz6.Day7


title: Sm8_Day0

description: Library name: NSPF1.RSm8.Day0


title: SLE Mild A0221006


title: SLE Mild A0221002


title: SLE Mild A0220995


title: SLE Mild A0221000


title: Sm8_Day1

description: Library name: NSPF1.RSm8.Day1


title: Rz6_Day0

description: Library name: NSPF1.RRz6.Day0


title: Rz6_Day1

description: Library name: NSPF1.RRz6.Day1


title: mRNA_KPC_MYC_WT_r2

value: PDAC

value: KPCshMYC + MYC WT


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-15
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Old RNA and ATAC-13
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: mRNA_KPC_MYC_WT_r3

value: KPC cells


title: mRNA_KPC_shMYC_r1

value: KPCshMYC

value: Ptf1a-Cre, KRasG12D/+, Tp53R172H/+


title: Rz6_Day3

description: Library name: NSPF1.RRz6.Day3


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-14
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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


title: mRNA_KPC_MYC_WT_r1


title: mRNA_KPC_shMYC_r2


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-11
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: mRNA_KPC_MYC_MUT_III_r3

value: KPCshMYC + MYC RBRIIIMUT


title: SLE Inactive A0221156

value: Inactive


title: SLE Mild A0220992

value: Mild


title: Qv5_Day1

description: Library name: NSPF1.RQv5.Day1

value: blood


title: Qv5_Day3

description: Library name: NSPF1.RQv5.Day3

value: PBMCs


title: mRNA_KPC_MYC_MUT_III_r2


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-10
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: mRNA_KPC_MYC_MUT_III_r1


title: SLE Mild A0220988

value: SLE


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-09
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: Qv5_Day0

description: Library name: NSPF1.RQv5.Day0

value: Influenza vaccination


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-12
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: Qv5_Day7

description: Library name: NSPF1.RQv5.Day7


title: SLE Inactive A0221130


title: SLE Inactive A0221149


title: SLE Inactive A0221109


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-08
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: RT_M4

city: Pamplona

first: Juan

last: Dubrot

state: (None)

address: Avda. Pío XII 55

country: Spain

institute: Cima-Universidad de Navarra; Cancer Center Clinica Universidad de Navarra (CCUN)

data_processing:
The quality of the samples was verified using FastQC v0.11.8 software.
FASTQ files were mapped to GRCm39 mouse assembly using STAR v2.7.9a with default parameters.
featureCounts v1.6.0 software and Gencode vM32 release were used to obtain read counts at gene level considering only those genes aligned to exonic regions
Obtained gene counts were normalized using edgeR and voom function of limma.
Assembly: GRCm39
Supplementary files format and content: tab-delimited text files include gene counts obtained with featureCounts for each sample.

source_name: Tumor

extract_protocol:
RNA was extracted from cell pellets using the Qiagen RNeasy Mini kit according to the manufacturer’s instructions. RNA was subjected to quantity and quality control using Qubit HS RNA Assay Kit (Thermo Fisher Scientific) and 4200 Tapestation with High Sensitivity RNA ScreenTape (Agilent Technologies). All RNA samples were high-quality, with RIN values higher than 8.
Library preparation was performed using the Illumina Stranded mRNA Prep Ligation kit (Illumina) following the manufacturer’s protocol. All sequencing libraries were constructed from 50 ng of total RNA according to the manufacturer's instruction. Briefly, the protocol selects and purifies poly(A) containing RNA molecules using magnetic beads coated with poly(T) oligos. Poly(A)-RNAs are fragmented and reverse transcribed into first cDNA strand using random primers. The second cDNA strand is synthesized in the presence of dUTP to ensure strand specificity. Resulting cDNA fragments are purified with AMPure XP beads (Beckman Coulter), adenylated at 3′ ends and then ligated with uniquely indexed sequencing adapters. Ligated fragments are purified and PCR amplified to obtain the final libraries. The quality and quantity of the libraries were verified using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and 4200 Tapestation with High Sensitivity D1000 ScreenTape (Agilent Technologies).

treatment_protocol: Single-cell suspensions from B16 tumors taken 5 days after treatment were stained with antibodies against CD45 and CD31 and Zombie Nir staining. CD45- CD31- cells were sorted in a MoFlo Astrios-EQ cell sorter (Beckman Coulter).

value: Tumor

value: RT (8Gy) + AAV-iLuc


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-07
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Inactive A0221077


title: RT_L3


title: Ie6_Day3

description: Library name: NSPF1.RIe6.Day3


title: Ie6_Day1

description: Library name: NSPF1.RIe6.Day1


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-06
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Inactive A0221049


title: Ie6_Day0

description: Library name: NSPF1.RIe6.Day0


title: RT_K2


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-05
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: RT_J1


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-03
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: Bi10_Day7

description: Library name: NSPF1.RBi10.Day7


title: Bi10_Day3

description: Library name: NSPF1.RBi10.Day3


title: RT_IL12_WT_F3

value: RT (8Gy) + AAV-iIL12


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-04
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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: RT_IL12_WT_E2


title: Ie6_Day7

description: Library name: NSPF1.RIe6.Day7


title: SLE Inactive A0221043


title: Bi10_Day1

description: Library name: NSPF1.RBi10.Day1


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-02
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 Inactive A0221023


title: SLE_Control HD A0221192

value: HD


title: RT_IL12_WT_D1


description:
Library name: PerturbFate pooled screen Tri-omics Old RNA and ATAC-01
PerturbFate Tri-omics single-cell library containing Pre-existing RNA and ATAC modalities
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 A0221189


title: SLE_Control HD A0221187


title: Bi10_Day0

description: Library name: NSPF1.RBi10.Day0


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-128
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: sc189_3

description:
10x Genomics
R1 file contains a cell barcode of 16 nt followed by a UMI sequence of 12 nt

data_processing:
bcl2fastq v2.20.0 was used for sample demultiplexing.
Alignment, filtering, barcode counting, and UMI counting was performed with Cell Ranger v7.0.0 and 'refdata-gex-mm10-2020-A' reference.
Assembly: mm10
Supplementary files format and content: tab separated files and matrix with gene counts per cell and gene annotation.

extract_protocol:
Single-cell suspensions from tumors taken 6 days after treatment were stained with Zombie Nir and CD45. Viable CD45+ cells were sorted in a MoFlo Astrios-EQ cell sorter (Beckman Coulter). Droplet-based isolation of single cells was performed with the Chromium Controller (10x Genomics).
3′ sequencing libraries was performed per the manufacturer’s instructions (10x Genomics). Briefly, 17000 to 20000 cells were loaded at a concentration of 1000 cells/µL on a Chromium X instrument (10X Genomics) to capture single cells in gel bead-in-emulsions (GEMs). In this step, each cell was encapsulated with primers containing a fixed Illumina Read 1 sequence, a cell-identifying 16 nt 10X barcode, a 12 nt Unique Molecular Identifier (UMI) and a poly-dT sequence. Upon cell lysis, reverse transcription yielded full-length, barcoded cDNA. This cDNA was then released from the GEMs, PCR-amplified and purified with magnetic beads (SPRIselect, Beckman Coulter). Enzymatic Fragmentation and Size Selection was used to optimize cDNA size prior to library construction. Fragmented cDNA was then end-repaired, A-tailed and ligated to Illumina adaptors. A final PCR-amplification with barcoded primers allowed sample indexing. Library quality control and quantification was performed using Qubit 3.0 Fluorometer (Life Technologies) and Agilent’s 4200 TapeStation System (Agilent), respectively. Sequencing was performed in a NextSeq2000 (Illumina) using paired-end 91-base pair reads.

value: Immune cells (CD45+)


title: sc189_4


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-127
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-126
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: sc189_2


title: SLE_Control HD A0221182


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-125
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: WT KE Diet S10

city: La Jolla

first: Leigh-Ana

last: Rossitto

state: CA

address: 9500 Gilman Drive

department: Neurosciences

institute: UC San Diego

description:
Library name: Sample10
10x Genomics

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

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

value: Hippocampus

value: WT

tag: Sex

value: male

value: .Diet


title: WT CTL Diet S11

description:
Library name: Sample11
10x Genomics


title: SLE_Control HD A0222021


title: hTau CTL Diet S9

description:
Library name: Sample9
10x Genomics

value: hTau


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-124
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: WT KE Diet S12

description:
Library name: Sample12
10x Genomics


title: sc189_1


title: SLE_Control HD A0221169


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-123
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: luc_4


title: WT KE Diet S8

description:
Library name: Sample8
10x Genomics


title: SLE_Control HD A0221168


title: SLE_Control HD A0221180


title: luc_3


title: luc_2


title: luc_1


title: SLE_Control HD A0221178


title: SLE_Control HD A0221163


description:
Library name: PerturbFate pooled screen Tri-omics New RNA-122
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: WT CTL Diet S7

description:
Library name: Sample7
10x Genomics


title: SLE_Control HD A0221162


title: hTau CTL Diet S5

description:
Library name: Sample5
10x Genomics


title: hTau KE Diet S6

description:
Library name: Sample6
10x Genomics