title: WT

city: New Haven

first: PABLO

last: FERNANDEZ-TUSSY

state: CT

address: 10 amistad street, 420C

department: Comparative Medicine

institute: Yale University

library_source: transcriptomic single cell

data_processing:
Reads were aligned against mouse genome mm9 using the CellRanger software (10x Genomics).
Gene-barcode matrices were analyzed in R (version 3.0) using Seurat v3. Cells were filtered for more than 500 genes detected in each cell, 20,000 or less total number of molecules detected within a cell and 5% or less mitochondrial gene content.
Significant principal components of variation (PCs) were calculated using JackStraw test, and clusters were calculated with 30 PCs
Differential expression analysis was done using wilcox
Assembly: mmp10
Supplementary files format and content: Cell-gene-barcode matrices

source_name: Hypothalamus

growth_protocol: 6-8 weeks old littermates WT and miR-33AgRPiKO mice were injected with tamoxifen (100mg/Kg every 3 days, after 16h fasting) five times. 2 weeks after last tamoxifen injection, mice were fed a HFD during 1 month.

extract_protocol:
Singlle-cell suspensions were obtained from a pull of 6-8 freshly dissected hypothalamus. Tissue digestion was performed as previously degscribed. riefly, 2 weeks after last tamoxifen injection, 3-month-old miR-33flox/flox/AgRPERT2Cre-Ai14or miR-33wt/wt/AgRPERT2Cre-Ai14 (both male and female) were fed a HFD for 1 month. Then, mice were anesthetized with isofluorane in non-fasted condition. Hippocampi were rapidly dissected and isolated from mice and place into a cold dish with 2 ml HABG buffer. Hippocampi were sliced into small pieces and then transferred to 50 ml tubes with 5 ml of HABG buffer. Then, hippocampi were incubated in water bath with smooth shaking at 30 °C, 8 minutes. Tissue was transferred to a new tube with 15 ml of HABG supplemented with papain (34 U/ml) (Worthington) and incubated at 30 °C, 30 mins, with gentle shaking ( 170 r.p.m.) for enzymatic digestion. Then transfer tissue to a new 15 ml tube with 2 mL of HABG buffer and let 5 minutes at room temperature. Then, we proceeded to mechanical tissue disruption as follows. Hippocampi were triturated with a 9-inches Pasteur pipette with the fire-polished tip  (10 times, 45 seconds, without introducing air bubbles). Tissue pieces were allowed to settle for 1 minute and supernatant was transferred to a clean 15-ml tube. Tissue pieces were resuspended in 2 ml HABG and trituration process was repeated two more times. In parallel, an OptiPrep 1.32 density gradient (Sigma-Aldrich) was prepared as follows: Layer 1 (173 ml OptiPrep + 827 ml medium); Layer 2 (124 ml OptiPrep + 876 ml medium); Layer 3 (99 ml OptiPrep + 901 ml medium); Layer 4 (74 ml OptiPrep + 926 ml medium). Cell suspension from supernatant mixes were carefully placed on top of the OptiPrep density gradient and centrifuged at 8g, 15 minutes, 22 °C, no brake. Top 6 ml containing cell debris were removed and cell fractions 1-4 containing oligodendrocytes, neurons, and microglia were collected. Cell fractions were diluted with 5 ml HABG and centrifuged for 2 minutes at 200g. Cell pellet was resuspended with 1 ml of HABG and filtered through a 40 mm cell strainer. Cells were counted and diluted at 1,000 cells/ ml and immediately processed for single-cell RNA sequencing.
Single cells were encapsulated into droplets and processed following standard manufacturer’s specification, using 10X Genomics GemCode technology. Cells were loaded on a 10x Genomics Chromium controller instrument to generate single-cell Gel Beads in emulsion (GEMS) at the Yale Center for Genome Analysis. Lysis and barcoded reverse transcription of polyadenylated mRNA from single cells were performed inside each GEM followed by complementary DNA (cDNA) generation using the single-Cell 3’ Regent Kits version 2 (10X Genomics). Libraries were sequenced on an Illumina HiSeq 4000 as 2x 100 paired-end reads.

value: Hypothalamus

value: WT

value: High Fat diet 1 month


title: miR-33AgRPiKO

status: Public on Feb 06 2024

type: SRA

country: USA

molecule: total RNA

organism: Mus musculus

tag: strain

tag: tissue

tag: genotype

tag: treatment


title: WT_HS_rep3

city: FuZhou

first: Jing

last: Zhang

state: Fujian Province

address: No.15 Shangxiadian Road, Cangshan District, Fuzhou City, Fujian Province, China

country: China

institute: Fujian Agriculture and Forestry University

library_source: transcriptomic

data_processing:
In total, we yielded paired-end reads of 2x150bp for 24 libraries of RNA  sequencing (RNA-seq), which included wild type and mutation with three biological repeats from immunoprecipitated (IP) and input samples, respectively.
The fastx_clipper script from FASTXtoolkit (http://hannonlab.cshl.edu/fastx_toolkit) was used for adapter sequence trimming of FASTQ files from input and IP samples using following parameters: -l 20 -d 0 -n -v -M 6.
Then above clean reads were aligned to the Arabidopsis reference genome (TAIR10) using TopHat v2.0.11 with following parameters: -a 8 -m 0, which required the min anchor length and maximum number mismatches for junction reads was 8 and 0, respectively.
Reads aligned to multiple genome regions were discarded.
Assembly: TAIR10
Supplementary files format and content: The row data folder contains the raw sequencing data for each sample. Each sample has two FASTQ files with R1 or R2 in the file name
Supplementary files format and content: The row_counts compressed file contains the FPKM file of each sample, including gene, counts and FPKM data

molecule: polyA RNA

organism: Arabidopsis thaliana

source_name: seedling

growth_protocol: Plants were grown in growth chambers or growth room at 22°C under continuous light conditions (24h light ).

extract_protocol:
Conventional RNA extraction method for total RNA, followed by rRNA depletion method for mRNA enrichment
TruSeq Stranded mRNA LT Sample Prep Kit - Set A

treatment_protocol: After one day of blue light treatment, start different temperature (22 or 44°C) treatments

value: seedling

tag: cell line

value: 8-day-old, 44C

tag: ecotype

value: Columbia

value: Wild type

value: heat stress


title: WT_HS_rep1


title: WT_HS_rep2


title: WT_mock_rep1

value: 8-day-old, 22C

value: mock


title: cry1_HS_rep2

value: cry1 mutant


title: WT_mock_rep2


title: cry1_mock_rep2


title: cry1_HS_rep3


title: cry1_mock_rep1


title: Health2

city: Changchun

first: guitian

last: he

institute: jilin university

data_processing:
Single-cell suspension was added to the 10x Chromium chip according to the instructions for the 10X Genomics Chromium Single-Cell 3' kit (V3), with the expectation of capturing 8,000 cells. cDNA amplification and library construction were performed according to standard protocols. Libraries were sequenced by LC-Bio Technology (Hangzhou, China) on an Illumina NovaSeq 6000 sequencing system (double-end sequencing, 150 bp) at a minimum depth of 20,000 reads per cell.
Assembly: mm10
Supplementary files format and content: tab-separated values files and matrix files.

source_name: testis

extract_protocol:
Cell populations were isolated from the testes of mice in the ND and HFD groups. In brief, the albuginea was removed, and the tissue was incubated in dissociation buffer containing collagenase IV (Sigma-Aldrich, St Louis, MO, USA; 1 mg/mL), dispase II (10 mg/mL), and DNase I (DN25, Sigma; 50,000 U/mL) for 30 min at 37°C. Enzymatic digestion was quenched with Dulbecco’s Modified Eagle’s Medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum. Before loading, the cell viability of each sample was evaluated by staining an aliquot with Trypan Blue, and the viability was typically determined to be greater than 80%. The cells were subsequently loaded into each channel of a Chromium Single Cell B Chip (10x Genomics, Pleasanton, CA, USA), and approximately 13,000 cells were recovered from each channel.
A Chromium Single Cell 3ʹ Library and Gel Bead Kit using v3 Chemistry (10X Genomics) was used for single-cell barcoding, synthesis of complementary DNA, and library preparation following the manufacturer’s protocol (Chromium Single Cell 3ʹ Reagent Kits v3 User Guide, Revision B). The libraries were sequenced on an Illumina NovaSeq 6000 system using paired-end sequencing runs.

value: testis

value: Various cell types in the testis

value: ND


title: Fat2

value: HFD


title: Sarc031-NT-CD90

city: Houston

first: Jlenia

last: Guarnerio

state: TX

address: 6767 Bertner Ave

department: Genetics

institute: MD Anderson Cancer Center

data_processing:
Cell Ranger software (10X Genomics, v6 and v7) was used to align, filter, and count reads.
The Seurat (v4) package for R was used to normalize reads and find variable features (SCTransform), perform dimensionality reduction (PCA, UMAP), cluster cells, and to filter for doublets and dead cells. Biological replicate identity (mouse ID) was demultiplexed based on oligo hashtag libraries.
Assembly: mm10
Supplementary files format and content: h5: feature count data as sparse matrix (output from Cell Ranger software)
Supplementary files format and content: csv: original sample ID of each single-cell barcode corresponding to a high-quality cell, and associated cell metadata.

source_name: Cancer-associated fibroblasts enriched from untreated mouse Undifferentiated Pleiomorphic Sarcoma tumors (p53-/-Ccne1+, n = 5), individually hashtagged

growth_protocol: 200,000 mouse Undifferentiated Pleiomorphic Sarcoma cells bearing either p53-/-Ccne1+ or p53-/-Vgll3+ genetics were seeded onto a polyurethane scaffold (DSM Biomedical), which was then implanted subcutaneously into syngeneic Bl6 recipient mice. Tumors were grown for 3 weeks or until tumor size reached 1cm3.

extract_protocol:
Sarcoma tumors were digested into single-cell suspensions using the Mouse Tumor Dissociation Kit and gentleMACS dissociator (Miltenyi) and filtered twice through 70um filters before ACK red blood cell lysis. Cells were washed in FBS-containing medium, stained with antibodies, and enriched by MACS or FACS.
Where applicable, each individual biological replicate (mouse tumors) was stained with a unique hashtag oligonucleotide antibody (Biolegend TotalSeq-B). Biological replicates were then combined in equal proportions into a single pool of cells prior to capture.
For each library, up to 16,000 cells were loaded into one well of a 10X Genomics Chromium chip for NextGEM 3' v3.1 droplet sequencing, at a concentration between 500-1000 cells/uL, targeting capture of up to 10,000 cells per sample.
Each 10X NextGEM 3' v3.1 library was constructed according to manufacturer protocols and sequenced on the NovaSeq X Plus, targeting 20,000 paired-end reads per cell for gene expression libraries, or 500 reads per cell for hashtag libraries.

tag: cell types_enriched_for

value: Cancer-associated fibroblast

tag: Sex

value: Female

tag: enrichment strategy

value: FACS Sorted Live, tumor-dsRed-, CD45-, CD31-, CD90+ cells

tag: hashtag

value: TotalSeq-B Hashtags 1-5


title: Sarc031-NT-CD45

source_name: Immune cells enriched from untreated mouse Undifferentiated Pleiomorphic Sarcoma tumors (p53-/-Ccne1+, n = 5), individually hashtagged

value: Immune cells

value: FACS Sorted Live, tumor-dsRed-, CD45+, CD31-, CD90- cells


title: Sarc031-Glut1i-CD45

source_name: Immune cells enriched from Glut1 inhibitor-treated mouse Undifferentiated Pleiomorphic Sarcoma tumors (p53-/-Ccne1+, n = 5), individually hashtagged


title: Sarc031-Glut1i-CD90

source_name: Cancer-associated fibroblasts enriched from Glut1 inhibitor-treated mouse Undifferentiated Pleiomorphic Sarcoma tumors (p53-/-Ccne1+, n = 5), individually hashtagged


title: Fat1


title: cry1_HS_rep1

value: seedling

value: 8-day-old, 44C


title: Health1


title: AB6946

city: Rehovot

first: Ido

last: Amit

address: 234 Herzl st.

department: Immunology

country: Israel

institute: Weizmann Institute of Science

description:
BiSE PICseq
metadata_q_SPID_BiSE.txt

data_processing:
bcl2fastq/2.15.0.4
Sequences with RMT of low quality (defined as RMT with minimum Phred score of less than 27) were filtered out.
Pool-barcode used i5+i7 barcodes each of them is unique in run, 1 error was allowed.  well-barcode N7 folowed by UMI N8 in read 2.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include mRNA molecule count values for each Sample
Supplementary files format and content: metadata_tumor_early_HISAT_annotated.txt
Supplementary files format and content: metadata_dln_late_annotated.txt
Supplementary files format and content: metadata_tumor_late_annotated.txt
Supplementary files format and content: metadata_q_SPID_BiSE.txt

source_name: dLN

growth_protocol: 8 to 10 weeks-old mice housed at the Weizmann Institute animal facility.

extract_protocol:
Mice were sacrificed at various time points by cervical dislocation. Tumor, spleen, and tdLN were collected and put through a process of mechanical and enzymatic digestion. For single-cell suspensions, tumors were chopped into small pieces and suspended in RPMI supplemented with DNase (2mg/ml, Sigma-Aldrich) and collagenase IV (100mg/ml, Worthington). Tumors were homogenized by GentleMacs tissue homogenizer (Miltenyi Biotec) and incubated at 37oC for 30min, with frequent agitation. Following dissociation procedures, cells were washed with cold PBS, filtered through a 70-μm cell strainer, and centrifuged at 300g for 5 min at 4 °C. Spleen and tdLN were mashed through a 100uM cell strainer and washed with ice-cold MACS buffer (PBS supplemented with 0.2mM EDTA, pH 8, and 0.5% BSA). Cells were centrifuged at 300g, 5 min, and 4°C. Spleen was suspended in red blood lysis buffer (Sigma-Aldrich) and DNase (0.33U/ml, Sigma-Aldrich), incubated for 5min at room temperature, washed with cold PBS, passed through a 70μm mesh strainer, centrifuged at 300g, 5 min, 4°C and then resuspended in ice-cold MACS buffer.
Single-cell RNA-seq libraries were prepared as previously described {Jaitin, 2014}. In brief, mRNA from single cells sorted into capture plates were barcoded and converted into cDNA and then pooled using an automated pipeline.
3' end mRNA libraries were prepared for sequencing using MARS-seq (Jaitin et al, Science 2014)

treatment_protocol:
Mice were anesthetized and the indicated inactivated pathogens were administered by intradermal injection into the ear pinna. PBS was injected into the ear pinna of control animals.
mice were treated with 250ug of aPD1 or/and 200ug of a41BB.

value: dLN

tag: cell type


title: WT_mock_rep3

value: Columbia

value: Wild type


title: AB6944

description:
BiSE PICseq
metadata_q_SPID_BiSE.txt


title: AB6349

value: TCRB+


title: AB6945

description:
BiSE PICseq
metadata_q_SPID_BiSE.txt

value: Lin-T+


title: AB6347


title: AB6348

value: TCRB


title: FV_RNAPIIS2P_WT_1h_B2

city: Cambridge

first: Larry

last: Melidis

state: Not US or Canada

address: Robinson Way

department: CRUK-CI

country: United Kingdom

institute: University of Cambridge

library_source: genomic

data_processing:
fq files were trimed with cutadapt version 1.18 to remove adaptors and bases with quality lower than 20 (-q 20) mapped with bwa-mem 0.7.17-r2188 to hg38 genome and the coresponding modified genomes, and only the reads within the whitelist have been retained.
bw files presented here were created using bamCoverage with counts per milion normalisation (CPM)  after extending the paired-end reads and accepting maximum mapped fragment length of 350nt. For the polymerase tracks the tracks were further rescaled, normalised for the E coli reads in the library.
Assembly: hg38 , hg38_MutMYCG4, hg38_SwapKRASG4, hg38_MYCFlip_StrandSwap, mm10
Supplementary files format and content: bigWig
Library strategy: CUT&Tag

molecule: genomic DNA

organism: Homo sapiens

growth_protocol:
Human embryonic kidney (HEK293T), cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, Gibco, 41966-029) growth media supplemented with 10% fetal bovine serum (Gibco, A3840401) and 2 mM L-Glutamine (Gibco, 25030024). Cells were maintained at standard conditions (5% CO2, 37°C) on TC-treated 6-well plates (Corning, 3516) and regularly tested for mycoplasma contamination (RICS, CRUK-CI Core Facility). For passaging, cells were washed with DPBS pH 7.4 (Gibco, 14190094), detached with StemPro Accutase (Gibco, A11105-01) at 37°C for 5mins and resuspended in 10-fold excess of growth media. Wild type J1 (129/Sv/Jae) mouse embryonic stem cell (mESC) cultures were routinely maintained in DMEM high glucose (Sigma, D6546) containing 10% FBS (Gibco, 16141079), 1x GlutaMax-I (Gibco, 35050-038), 1x NEAA (Gibco, 11140-035), 0.1 mM β-mercaptoethanol (Sigma, M3148 diluted to 50 mM in 50 µM EDTA/PBS solution), 1000 U/mL recombinant LIF (PeproTech, murine LIF, 250-02) and 1 µM PD (Sigma, PZ0162-5MG), 3 µM CHIR (Cambridge biosciences, HY-10182A-5MG). Cells were cultured under standard conditions at 37°C in water saturated, CO2 (5%) enriched atmosphere on 0.2% (w/v) gelatine-coated (Sigma, G9391) plates and regularly tested for mycoplasma contamination (RICS, CRUK CI core). Authentication of mESCs was performed by mouse STR analysis (RICS, CRUK CI core). For passaging cells were washed with PBS (Gibco, 14190094), detached with TrypLE (Gibco, 12604-013) at 37°C for 5 min and resuspended in 10-fold excess of mESC growth medium as described.
Human embryonic kidney (HEK293T), cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, Gibco, 41966-029) growth media supplemented with 10% fetal bovine serum (Gibco, A3840401) and 2 mM L-Glutamine (Gibco, 25030024). Cells were maintained at standard conditions (5% CO2, 37°C) on TC-treated 6-well plates (Corning, 3516) and regularly tested for mycoplasma contamination (RICS, CRUK-CI Core Facility). For passaging, cells were washed with DPBS pH 7.4 (Gibco, 14190094), detached with StemPro Accutase (Gibco, A11105-01) at 37°C for 5mins and resuspended in 10-fold excess of growth media. Wild type J1 (129/Sv/Jae) mouse embryonic stem cell (mESC) cultures were routinely maintained in DMEM high glucose (Sigma, D6546) containing 10% FBS (Gibco, 16141079), 1x GlutaMax-I (Gibco, 35050-038), 1x NEAA (Gibco, 11140-035), 0.1 mM β-mercaptoethanol (Sigma, M3148 diluted to 50 mM in 50 µM EDTA/PBS solution), 1000 U/mL recombinant LIF (PeproTech, murine LIF, 250-02) and 1 µM PD (Sigma, PZ0162-5MG), 3 µM CHIR (Cambridge biosciences, HY-10182A-5MG). Cells were cultured under standard conditions at 37°C in water saturated, CO2 (5%) enriched atmosphere on 0.2% (w/v) gelatine-coated (Sigma, G9391) plates and regularly tested for mycoplasma contamination (RICS, CRUK CI core). Authentication of mESCs was performed by mouse STR analysis (RICS, CRUK CI core). For passaging cells were washed with PBS (Gibco, 14190094), detached with TrypLE (Gibco, 12604-013) at 37°C for 5 min and resuspended in 10-fold excess of mESC growth medium as described.

extract_protocol:
CUT&Tag and Chem-map were performed as previously described (Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019),Yu, Z., et al. Nat Biotechnol (2023)).G4 mapping (BG4, SG4, SG4mut-R105A) and R-loop profiling were performed as 3-layer CUT&Tag on DynabeadsTMMyOneTMStreptavidinT1 (Thermo Fisher, cat#65601). SG4 and SG4mutR105A CUT&Tag was performed in extracted nuclei as previously described (Hui W.W.I., et al. Sci Rep (2021)). PDS Chem-map, G4 interactome CUT&Tag experiments (SP1, CNBP, MLL1, MLL4, SIN3A, SMARCA4, RNAPIIS5P, RNAPIIS2P) and histone marks (H3K4me1, H3K4me3, H3K27me) were performed as 2-layer CUT&Tag on ConcanavalinA (ConA)-coated magnetic beads (Bangs Laboratories, BP531).MyOneT1 beads were washed twice with PBS pH 6.8, resuspended in 1xPBS pH 6.8 supplemented with 0.01% Tween-20 and mixed with the biotin-conjugated ConA (Sigma-Aldrich, cat#C5275-5MG) solution at 2.3mg/mL at a 1:0.5 ratio. The mix was then incubated at 22°C at 400 rpm for 30min. Aliquots of 500,000 cells/100mL were added to 10mL of beads, washed twice with 100mL wash buffer and resuspended in 1% BSA Antibody Buffer. Primary antibodies were added (1:50), and the mix was incubated at 4°C overnight at 600 rpm. Cells were then washed twice with 100mL dig-wash buffer (0.05% digitonin, 20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and resuspended in 50mL dig-wash buffer. Secondary antibodies were added 1:50 to the samples and incubated at 25°C for 2h at 600 rpm (2-layer CUT&Tag). For G4-mapping samples, cells were washed three times with 500mL dig-wash buffer and resuspended in 50mL dig-wash buffer and 0.5mL of guinea-pig anti-rabbit antibody (Antibodies online, ABIN101961)was added and incubated with the cells at 25°C for 1h at 600 rpm (3-layer CUT&Tag). Cells were then washed three times with 100mL Dig-300 buffer (0.01% digitonin, 20mM HEPES pH7.5, 300mM KCl, 0.5mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and incubated in 1:250 dilution of pA-Tn5 in 50µL Dig-300 buffer at 25°C for 1h at 600rpm.Cells were then washed three times with 500 µL Dig-300 buffer and the Tn5 tagmentation was activated by incubating in 300mL Dig-300 buffer supplemented with 10 mM MgCl2at 37°C for 1h at 600 rpm. Cells were then washed twice with 500 µL TAPS buffer and resuspended in 0.5 mg/mL Proteinase K, 0.5% SDS in 10 mM Tris-HCl buffer pH 8.0 at 55°C for 1h at 800 rpm. Tagmented DNA was purified with DNA clean & concentrator columns (Zymo Research, D4013).
For Illumina library preparation, genomic DNA extracted by CUT&Tag was used. Libraries were generated as previously described with a universal i5 and a uniquely barcoded i7 primer(Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019)). Libraries were visualized and quantified on a high-sensitivity D1000 tape on a Tape Station (AgilentTechnologies 2200)andwith the NEBNext®Library Quant Kit for Illumina (NEB, cat#7630S), following the manufacturer’s instructions.Libraries were sequenced on an Illumina NextSeq 500 (High Output Kit v2.5, 75 cycles) or NextSeq 2000 instrument(P2 or P3 reagents, 100 cycles). RNA-seq and library preparation Total RNA was extracted from 1.0x106 cells using the RNeasy Plus Mini Kit (Qiagen, cat#74134) as per manufacturer’s instructions. RNA quality was checked via TapeStation (Agilent Technologies 2200, RIN>9) and RNA concentration determined using a Qubit fluorometer (Thermo Fisher, Qubit 4). Libraries were generated from 700ng RNA using the NEBNext®Poly(A) mRNA Magnetic Isolation Module (NEB, cat#7490S) together with the NEBNext®UltraTMII Directional RNA Library Prep Kit for Illumina (NEB, cat#7760S) as per manufacturer’s instructions. Libraries were sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles). For MNase 1.0 x 106 cells were washed in ice-cold PBS and incubated in 1mL lysis buffer (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT, 0.1% NP-40) for 10 mins on ice. Cells were then centrifuged at 1,000 x g for 5 mins at 4 °C and resuspended in lysis buffer without NP-40 (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT). Cells were then incubated at 37 °C for 5 mins supplemented with 2 mM CaCl2 and digested with 10 U MNase at 37 °C for 60 mins. The digestion was stopped with a final concentration of 40 mM EDTA, 0.5% SDS. DNA was incubated with RNase A (Thermo Scientific, cat #EN0531) for 1 h at 37oC. DNA was purified using Phenol-chloroform-isoamyl alcohol (PCl, Thermo Scientific, cat #15593049). Libraries were generated with the NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina (NEB, cat #7760S) as per manufacturer’s instructions. A bead-based size selection of 200 bp was performed to isolate mononucleosomal fragments. Libraries were then quantified with the NEBNext® Library Quant Kit for Illumina (NEB, cat #7630S) and sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles).

treatment_protocol: Cell treatment with transcriptional inhibitors Wild-type HEK293T cells were grown in 6-well plates until 80-90% confluent. To inhibit transcription initiation, cells were treated with triptolide (11973-1mg-CAY, Cayman Chemicals, 10 mM DMSO stock) at a final concentration of 10mM in complete growth medium for different time points (30 min, 1h, 2h). As a vehicle control, cells were treated with DMSO (diluted 1:1000 v/v in complete medium) for the entire duration of the time course (2h). To inhibit transcription elongation, cells were treated with flavopiridol hydrochloride (10009197-5mg-CAY, Cayman Chemicals, 1 mM DMSO stock) at a final concentration of 1mM in complete growth medium for different time points (30 min, 1h, 2h). Similarly, a DMSO control was included for the entire time course (2h). At the end of the treatment course, medium was removed, cells were detached with StemPro™ Accutase™ (Gibco™ A1110501), resuspended in cold medium and kept on ice until ready for CUT&Tag.

value: Human embryonic kidney

tag: chip antibody

value: Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb #13499


title: AB6319

source_name: tumor

value: tumor

value: PBS


title: AB6316


title: AB6318


title: AB6346


title: AB6317


title: AB6315


title: cry1_mock_rep3

value: 8-day-old, 22C

value: cry1 mutant

value: mock


title: AB6305


title: AB6301


title: AB6303


title: AB6300


title: AB6308


title: AB6313


title: AB6304


title: AB6309


title: AB6307


title: AB6312


title: AB6310


title: AB6293


title: AB6302


title: AB6292


title: AB6306


title: AB6288


title: AB6350


title: AB6286


title: AB6295


title: AB6296


title: AB6291


title: AB6290


title: AB6297_1


title: AB6287


title: AB6289


title: AB6285


title: AB6299


title: AB6284


title: AB6282


title: AB6283


title: AB6298_1


title: AB6294


title: AB6279


title: AB6277


title: AB6276


title: AB6272


title: AB6273


title: AB6270


title: AB6271


title: AB6274


title: AB6268


title: AB6267


title: AB6263


title: AB6265


title: AB6262


title: AB6264


title: AB6266


title: AB6216


title: AB6217


title: AB6210


title: AB6211


title: AB6209


title: AB6281


title: AB6207


title: AB6280


title: AB6275


title: AB6208


title: AB6205


title: AB6204


title: AB6203


title: AB6269


title: AB6200


title: AB6202


title: AB6199


title: AB6198


title: AB6201


title: sRNA-Cattle-1

city: Lanzhou

first: shaoke

last: guo

state: Gansu

address: West Lake Street

department: Lanzhou institute of husbandry and pharmaceutical sciences

institute: Chinese Academy of Agricultural Sciences

data_processing:
The raw data in fastq format was removed by Trimmomatic software to remove adapters, remove poly-N reads and low-quality reads, and obtain clean reads. Clean reads were aligned to the cattle genome using HISAT2 (version 2.0.5) (version: Bos_taurus.UMD3.1). The mapped reads of each sample were assembled by StringTie (v1.3.4) in a reference-based approach. Cufflinks was used to calculated FPKM (Fragments Per Kilobase of exon Model per million mapped fragments) values of transcripts.
The raw data in fastq format is used to remove joints and low-quality reads by custom perl and python scripts. Bowtie was used to map small RNAs with a length range of 18-35 nt to the reference genome. Mapped small RNA tags were used to looking for known miRNA. miRBase20.0 was used as reference, modified software mirdeep2 and srna-tools-cli were used to obtain the potential miRNA and draw the secondary structures. To remove tags originating from protein-coding genes, repeat sequences, rRNA, tRNA, snRNA, and snoRNA, small RNA tags were mapped to RepeatMasker, Rfam database or those types of datas from the specified species itself. The signature hairpin structure of miRNA precursors can be used to predict new miRNAs, and integrate miREvo and mirdeep2 miRNA prediction software to analyze new miRNAs. miRNA expression levels were estimated by TPM through the criteria。The predicted miRNA target genes were the intersection results of miRanda, PITA and RNAhybrid.
Assembly: Bos_taurus.UMD3.1
Supplementary files format and content: Excle include FPKM/TPM values for each Sample...

organism: Bos taurus

growth_protocol: Four-year-old cattle, yaks and cattle-yaks ( three each ) were selected from the same cooperative in Gannan Tibetan Autonomous Prefecture for castration.

extract_protocol:
Total RNA was extracted from each testicular sample using TRIzol reagent. RNA quality was assessed using Nanodrop-2000 and 1% agarose gel electrophoresis.
RNA-seq：A total amount of 5μg RNA per sample was used as input material for the RNA sample preparations. Firstly, ribosomal RNA was removed by Epicentre Ribozero™ rRNA Removal Kit (Epicentre, USA). Second, the sequencing libraries were generated by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Third, the library quality was assessed on the Agilent Bioanalyzer 2100 system. At last, the clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. And sequencing the library on the Illumina Hiseq 2500 platform and and 125bp.paired -end reads were generated.      miRNA-seq：A total amount of 3 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA high sensitivity chips. After clustering the samples, the library preparations were sequenced on an Illumina Hiseq 2500 platform and 50 bp single-end reads were generated.

treatment_protocol: The testicular tissue was washed with PBS and quickly placed in a freezer tube, immediately stored in liquid nitrogen.

value: testis

value: Four years old


title: AB6197


title: Cattle-3


title: AB6196


title: sRNA-Cattle-2


title: AB6194


title: AB6193


title: Teratoma-derived fibroblasts that were established from DM14 hESCs, rep A

city: jerusalem

first: Rachel

last: Eiges

address: Shmu'el Bait St 12, Jerusalem, 9103102

institute: Shaare Zedek medical center

data_processing:
Bisulfit-Seq
Raw sequence reads were mapped to the human genome (build GRCh37) using Bismark (30). Methylation calls were extracted after duplicate sequences were removed. Data visualization and analysis were performed using custom R and Java scripts. CpG methylation was calculated as the average methylation at each CpG position, and non‐CpG methylation was extracted from the Bismark reports.
Assembly: GRCh37
Supplementary files format and content: Bismark
RNA-Seq
Sequence reads were trimmed for adapter\low quality sequences using Trimmomatics (v0.39).
Trimmed reads were then aligned to GRCh38/v42 using STAR (v2.5.4b) with default parameters.
Count tables were created by processing the aligned BAM files from the previous step using Subread's featurecounts function.
Assembly: GRCh38/v42
Supplementary files format and content: tab-delimited text files wich includes raw counts for each sample

source_name: DM14 TOFs

extract_protocol:
Total RNA was extracted using NucleoSpin RNA Plus kit (Macherey-Nagel, Cat # 740984.50) following the manufacturer's instructions. RNA was quantitated by NanoDrop 200 (ThermoFisher).
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: DM14 TOFs

value: Teratoma-derived fibroblasts

value: 2000 CTG expansion in DMPK


title: Teratoma-derived fibroblasts that were established from DM14 hESCs, rep B


title: Cattle-2


title: AB6192

value: dLN


title: Mutant hESC line with a 2000 CTG expansion in DMPK, undifferentiated, rep C

value: Human embryonic stem cells


title: AB6206


title: AB6191

value: TCRB+


title: Mutant hESC line with a 2000 CTG expansion in DMPK, undifferentiated, rep B


title: Mutant hESC line with a 2000 CTG expansion in DMPK, undifferentiated, rep A


title: AB6190


city: Oxford

first: Jane

last: Mellor

address: South Parks Road

department: Biochemistry

institute: University of Oxford

description:
D8
Native RNA from Rbp3 immunoprecipitated material size selected 500-1000nt and alkali fragmented  before library construction
Trimmed to 3' end

data_processing:
The adapter Sequence ‘ATCTCGTATGCCGTCTTCTGCTTG’ was first trimmed from the raw reads by running cutadapt. Bowtie was utilized to align the reads to the hybrid genome of S.pombe and S.cerevisiae with the following settings: ‘?q ?p 16 ?S ?m 1 ?n 1 ?e 70 ?l 28 ?k 1 ?? best ??phred33?quals’. The uniquely aligned sam files were eventually converted into the bedgraph files by samtools, bamtobed and genomecov. The data is aligned as for the conventional NET-Seq (Churchman & Weissman, 2011; Fischl et al., 2017). For the smallest size fraction (20-50nt), the reads trimming by ‘genomecov’ was performed on either the 3’ ends and the 5’ ends of reads, which produced two sets of bedgraph files 5’ and 3’. Bedgraph files were visualised using IGV (Thorvaldsdottir et al., 2013).
Assembly: S. cerevisiae sacCer3 and S. pombe (ASM294v2) spike ins
Supplementary files format and content: Bedgraphs

organism: Saccharomyces cerevisiae

source_name: S. cerevisiae with S.pombe spike in

growth_protocol: Yeast were grown in YPD to OD600>0.6 using standard protocols as in Fischl et al., 2017; http://dx.doi.org/10.1016/j.molcel.2017.01.006 and Uzun et al., 2021;  http://dx.doi.org/10.1016/j.celrep.2021.109755

extract_protocol:
Size selected NET-Seq was developed to map new transcription units and their putative transcription start sites (TSS) on the S.cerevisiae genome. The step up to ligation of linker-1 were performed as for the conventional NET-Seq protocol (Fischl et al., 2017; http://dx.doi.org/10.1016/j.molcel.2017.01.006); Uzun et al., 2021;  http://dx.doi.org/10.1016/j.celrep.2021.109755). For size selected NET-Seq, 6ug of RNA (>3ug) was used and split into six non-stick RNase-free tubes for the ligation reaction, just as for conventional NET-Seq. After 3 hours incubation at 37°C, the reaction was quenched by adding 0.7ul of 0.5M EDTA to each tube. The ligation product was purified and concentrated by isopropanol precipitation. Specifically, 0.56ml of RNA solution and 0.75ml of isopropanol were added to each tube. After vortexing, the mixture was incubated at ~20°C for 2 hours, followed by centrifugation at 4°C, 20,000g for 30mins. Next, the supernatant was removed without disturbing the RNA pellet. The pellet was then washed once with 0.75ml of 80% ice?cold ethanol. After brief centrifugation, residual ethanol was carefully removed by pipetting. The pellet was air-dried at room temperature for 10mins. The RNA pellets were resuspended in 15ul of 10mM Tris.Cl (pH 7.0), mixed with 15ul of 2X TBU denaturing loading buffer, denatured at 80°C for 3 mins, and 10ul loaded onto 3 wells of an ice-cold 3.5% TBU polyacrylamide gel (10mls 4.8g Urea, 1.17ml 30% acrylamide solution, 50uL 10% APS, 1 mL 10X UltraPure TBE buffer, MilliQ water to 10mls, 10uL TEMED poured between glass plates cleaned with 1% SDS and washed thoroughly with MilliQ water to remove RNase contamination. The gels were left for one hour to set and then pre?run for 15mins. Before loading, a pipette with a clean tip was used to flush the wells with 1XTBE buffer several times. Two empty wells were left between the ladders (heated denatured 10bp, 50bp, 100bp and 1kb DNA ladders) and the sample. The samples were subject to electrophoresis using ice-cold 1xTBE running buffer, at 80V for 80mins. The gel was stained for 5mins in 50ml of 1xTBE buffer containing 5ul of SYBR Gold nucleic acid gel stain (S11494, ThermoFisher Scientific), followed by destaining in 1xTBE buffer for another 5mins. The gel was frozen at ~20°C for 15mins, prior to cutting into size ranges, normally 20-50nts (samples A), 50-170nts (samples B), 170-500 (samples C) and 500-1000nts (samples D). Sample E are fractions >1000nt but did not yield much beyond rRNA sequences. The RNA was recovered from mashed gel pieces using electroelution in 1 x TBE at 80V with the electrophoresis tank sitting in wet ice. Fluorescence was monitored every 10mins with the transilluminator, with 10mins being sufficient for the smallest fraction and up to 2hrs for the longest RNA.  After electroelution, RNA from each size fraction was dissolved in 600ul of 10mM Tris.Cl (pH 7.0). To purify and concentrate the RNA, 2ul of Glycoblue, 50ul of 3M sodium acetate (pH 5.5) and 0.75ml of isopropanol were added. The RNA pellet of the smallest fraction was dissolved in 10ul of 10mM Tris.Cl (pH 7.0). All the other size fractions were dissolved in 20ul of the same buffer. The RNA from each fraction, except the smallest, is subject to alkaline fragmentation and library construction, with S.pombe NET-Seq spike-in, following the conventional NET-Seq protocol (Fischl et al., 2017; http://dx.doi.org/10.1016/j.molcel.2017.01.006); Uzun et al., 2021;  http://dx.doi.org/10.1016/j.celrep.2021.109755), except that up to 2 more PCR cycles may be needed. The sequencing of NET-Seq libraries was performed on the Illumina NEXTSeq 500 Sequencer with the high throughput flow cell, single end reads and 50 cycles. NET-Seq normally requires 100 million reads to obtain good coverage on each S.pombe spiked S.cerevisiae sample.
NET-Seq nascent RNA sequencing libraries based on protocols in Fischl et al., 2017; http://dx.doi.org/10.1016/j.molcel.2017.01.006; Uzun et al., 2021;  http://dx.doi.org/10.1016/j.celrep.2021.109755 in

tag: spike in

value: yeast cell

value: BY4741  RPB3-3xFLAG

tag: size selected


description:
E8
Native RNA from Rbp3 immunoprecipitated material size selected >1000nt and alkali fragmented before library construction
Trimmed to 3' end


description:
C6
Native RNA from Rbp3 immunoprecipitated material size selected 170-500nt and alkali fragmented before library construction
Trimmed to 3' end


description:
B5
Native RNA from Rbp3 immunoprecipitated material size selected 50-170nt and alkali fragmented before library construction
Trimmed to 3' end


title: AB6189


title: Human immortalized myoblasts with a 2600 CTG expansion in DMPK, rep C

value: Immortalized myoblasts

value: 2600 CTG expansion in DMPK


description:
A6
Native RNA from Rbp3 immunoprecipitated material size selected 20-50nt


title: AB6188


title: Human immortalized myoblasts with a 2600 CTG expansion in DMPK, rep B


title: Human immortalized myoblasts with a 2600 CTG expansion in DMPK, rep A


title: Teratoma-derived fibroblasts that were established from DM14 hESCs, rep C


title: AB6187


title: DM14_DNMT3B_KO_clone_19

extract_protocol:
Genomic DNA (2 µg) was modified by bisulfite treatment (EZ DNA methylation kit, Zymo Research) and amplified by FastStart DNA polymerase (Roche). PCR was carried out using 5’ primer: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGGTTGTGGGTTAGTGTT-3’, and 3’ primer: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCAACAACCTACAACTATTAT-3’ (DMPK-specific sequences are underlined; adaptors for library preparation are at the 5’ end of each primer). Amplified products were quality controlled for size using the D1000 ScreenTape kit; Agilent Technologies) and for concentration using the Qubit® DNA HS Assay kit (catalog #32854; Invitrogen). Subsequently, the PCR products were bead purified with Agencourt Ampure XP beads (Beckman Coulter) according to the manufacturer’s protocol.
Next, amplicons were subjected to a second PrimMax Takara PCR reaction (1st purified PCR DNA (7.5 μl), 2.5 μl N7XX primer (nextera barcode 1), 2.5 ul S5XX primer (nextera barcode 2), 12.5 μl 2x Primstar ReadyMix). PCR program: 98°C for 1 minute followed by 8 cycles of 98°C for 10 seconds, 55°C for 10 seconds, 72°C for 30 seconds, then 72°C for 5 minutes and finally held at 10°C. The 2nd PCR was bead purified, quality controlled using the D1000 ScreenTape kit and the Qubit® DNA HS Assay kit, and all resultant libraries were normalized and pooled at a 10 nM concentration. Denaturation and dilution of the sequencing pool were performed according to the standard Illumina protocol.

value: 2000 CTG expansion in DMPK+knockout of DNMT3B


title: AB6186


title: DM14_DNMT3B_KO_clone_3


title: Cattle-1


title: TMF

city: Grenoble

first: Jérémy

last: LUCAS

address: 17 avenue des Martyrs

country: France

institute: CNRS

description: Recombiant proteins were produced by TnT in vitro transcription translation system from Promega

data_processing:
Alignement to genome is done by Bowtie2
poor quality and multimapped reads are filtered out using samtools
peak-calling is done by MACS2 with parameters (-q 0.001 --call-summits)
Peaks called where Control read coverage is too high are removed from analysis
replicates are treated by MSPC to create consensus peaks
Assembly: Consensus peaks are reworked around maximum of peaks.
Supplementary files format and content: bed format, contains consensus peaks locations of replicates (one per sample, input/control excluded)
Supplementary files format and content: bedgraph format, contains genomic signal values
Library strategy: ampDAP-seq

source_name: 2-weeks old seedlings

growth_protocol: 2-weeks old seedlings of Arabidopsis thaliana

extract_protocol:
Genomic DNA was extracted by chloroform isoamyl alcohol dna extraction protocol
Libraries were contructed using Truseq Illumina kits
Illumina TruSeq adaptors with single end index

value: 2-weeks old seedlings


title: LOS2


title: HeLa, GFP-NLS-TAF2d1142-1171 expression, rep2, GFP, greenCUT&RUN, -Ca2+ control

city: Freiburg im Breisgau

first: Tanja

last: Bhuiyan

address: Albertstrasse 25

department: Institute of Experimental and Clinical Pharmacology and Toxicology

country: Germany

institute: University of Freiburg

data_processing:
Reads were processed using bcl2fastq version: 2.20.0.422. for Novaseq output
CUT&RUN reads were trimmed using Trim-galore v0.6.4
Alignment to human and drosophila genomes was carried out using bowtie2 v2.3.5.1 according to Nizamuddin et al., 2021. The total number of aligned reads, representing both human and drosophila spike-in, was calculated using the Flagstat program of samtools (version 1.10).
Spike-in normalized genome coverage (BigWig) files were created with DeepTools bamCoverage v3.5.1.0.0 using a blacklist region file for hg38, bin size 50 bases and 1x normalization (RPCG). The scaling factor was calculated 10⁶/spike-in reads for CUT&RUN and 10⁵/spike-in reads for greenCUT&RUN
Peaks were identified using HOMER with default parameters, except for the inclusion of –C 0. Normalization of reads during peak calling was conducted using spike-in according to Nizamuddin et al., 2021 #69. Peaks in the experiment were called against control samples.
To merge peaks, bedtools intersect intervals (v 2.30.0+galaxy1) was used with 1 bp required overlap.
Assembly: hg38 (human) and BDGP5 (drosophila)
Supplementary files format and content: bigWig, bed

source_name: HeLa

growth_protocol: DMEM, 10% FBS, 1 mM sodium pyruvate, cells were cultured at 37 °C with 5% CO2

extract_protocol:
Standard CUT&RUN according to Skene et al., 2018
NEB Next Ultra II library preparation kit (NEB #E7645L) without size selection according to manufacturer’s instructions with modifications: we increased the end prep duration to 60 min at 50 °C instead of 30 min at 65 °C and used only 10 s annealing and extension time instead of 75 s, and 15 PCR cycles during the amplification step

treatment_protocol: 1 ug/mL doxycycline overnight (17-22 h)

value: HeLa

value: 1 ug/mL doxycycline

tag: cut&run antibody

value: GFP-nanobody-MNase


title: LOS1


title: HeLa, GFP-NLS-TAF2d1142-1171 expression, rep2, GFP, greenCUT&RUN


title: G1


title: DM14_DNMT3B_KO_clone_1


title: DM14_DKO_clone_I4

value: 2000 CTG expansion in DMPK+double Ko in DNMT3B+DNMT3A


title: LSH3


title: AB6183


title: DM14_DKO_clone_I3


title: LSH2


title: HeLa, GFP-NLS-TAF2 expression, GFP, greenCUT&RUN, -Ca2+ control


title: AB6181


title: DM14_DKO_clone_3K


title: LSH1


title: AB6182


title: DM14_DKO_clone_3G


title: AB6185


title: HeLa, GFP-NLS-TAF2dIDR expression, TBP, CUT&RUN

description: use same control file as for HeLa, GFP-NLS-TAF2dIDR expression, TAF1, CUT&RUN, IgG control

value: TBP (Abcam #ab28175)


title: Input


title: DM14_DKO_clone_3C


title: HeLa, GFP-NLS-TAF2dIDR expression, TAF7, CUT&RUN


title: AB6179


title: AB6184


title: AB6180


title: HeLa, GFP-NLS-TAF2dIDR expression, TAF1, CUT&RUN, IgG control

description: control file to be used also for GFP-NLS-TAF2dIDR expression, TAF1 and TAF7 CUT&RUN


title: DM14 hESCs-control


title: AB6178


title: Pat50

city: Lund

first: Martin

last: Lauss

address: Barngatan 2B

department: Dept of Oncology

country: Sweden

institute: Lund University

data_processing:
RNA sequencing data of bulk tumor samples were aligned using HISAT2 version 2.1.0  and reference hg38.analysisSet_gencode27_snp150, and further processed using StringTie to obtain FPKM values.
Transcripts with the same gene name were added up. The data were reduced to protein-coding genes.
The data were transformed as log2(data+1) and quantile-normalized using limma. Six samples that did not undergo ICB treatment were removed.
Principal Component (PC) PC2 was associated with a batch variable and was removed from the data using R package swamp.
Assembly: hg38
Supplementary files format and content: tab-delimited text file that includes FPKM values, which have undergone log-transformation, quantile-normalization and removal of PC2.

source_name: melanoma

extract_protocol:
Tumor DNA and RNA was extracted using AllPrep DNA/RNA Mini Kit (Qiagen)
RNA libraries were prepared for sequencing using standard Illumina protocols

value: melanoma

value: anti-PD1 resistant

tag: gender

value: F

tag: Stage

value: unknown

tag: previous. brafi

value: no_During, day 7

value: TRUE

tag: groups

value: anti-PD1 resistant, BRAFi day7


title: HeLa, GFP-TAF2 expression, TBP, CUT&RUN

description: use same control file as for HeLa, GFP-TAF2 expression, TAF1, CUT&RUN, IgG control


title: Pat49

tag: tissue

value: M


title: HCC827ASCL1, persister, rep2

first: Bomiao

last: Hu

address: 310 Cedar Street

data_processing:
RNA-seq raw data were trimmed of adaptor sequences using Trim Galore and mapped to the human reference genome (hg38) using HISAT2.
The sequencing reads were annotated according to GENCODE v27 model and gene expression levels were quantified using StringTie
Differentially expressed genes were identified using DESeq2.
Assembly: hg38
Supplementary files format and content: excel file including normalized gene counts for all the cell lines and experimental conditions

growth_protocol: RPMI 1640 supplemented with 10% tet system-certified FBS, 1μg/ml doxycycline, 1mM sodium pyruvate, 10mM HEPES, 100U/ml penicillin and 100μg/ml streptomycin.

extract_protocol:
Total RNA was extracted from cell lines using AllPrep DNA/RNA mini kit (Qiagen, 80204).
RNA-seq libraries were constructed using KAPA mRNA HyperPrep Kit (Kapa Biosystems, KR1352).

treatment_protocol: vehicle or 100nM osimertinib

value: human NSCLC cell line

tag: transfection

value: inducible ASCL1 transfected

tag: treatment

value: osimertinib for 10 days


title: HCC827ASCL1, persister, rep1

tag: cell line


title: HeLa, GFP-NLS-TAF2dIDR expression, TAF1, CUT&RUN

value: TAF1 (cTAF1, 177-4, in-house)


title: HeLa, GFP-NLS-TAF2 expression, GFP, greenCUT&RUN

value: HeLa


title: HeLa, GFP-TAF2 expression, TAF7, CUT&RUN

value: 1 ug/mL doxycycline


title: HCC827ASCL1, osimertinib, rep2

value: osimertinib for 3 days


title: Pat47

value: skin


title: HeLa, GFP-TAF2 expression, TAF1, CUT&RUN, IgG control

description: control file to be used also for GFP-TAF2 expression, TAF1 and TAF7 CUT&RUN


title: AB6174

value: dLN

value: TCRB+


title: HCC827ASCL1, osimertinib, rep3


title: AB6175


title: AB6176


title: HCC827ASCL1, persister, rep3


title: HCC827ASCL1, osimertinib, rep1


title: HeLa, GFP-TAF2 expression, TAF1, CUT&RUN


title: Pat46

value: melanoma

value: anti-PD1 resistant


title: sRNA-Cattle-3

value: testis

value: Four years old


title: AB6173


title: HeLa, RNAPII, CUT&RUN, IgG control


title: Pat45

value: F

value: FALSE


title: HCC827ASCL1, vehicle, rep2

value: vehicle treated


title: AB6177


title: HeLa, RNAPII, CUT&RUN

value: RNA pol II (8WG16, Santa Cruz sc-56767))


title: Pat44

value: IIIb

value: TRUE


title: AB6171

value: PBS


title: Pat43

value: M

value: no_During, day 7

value: anti-PD1 resistant, BRAFi day7


title: Pat48

value: skin


title: HeLa, TBP, CUT&RUN

description: use same control file as for HeLa, TAF1, CUT&RUN, IgG control

value: HeLa

value: TBP (Abcam #ab28175)


title: HCC827ASCL1, vehicle, rep1

value: human NSCLC cell line

value: inducible ASCL1 transfected


title: AB6170

value: dLN

value: TCRB+


title: Pat42

value: melanoma

value: anti-PD1 resistant

value: F


title: HeLa, TAF7, CUT&RUN


title: HCC827EV, osimertinib, rep2

value: empty vector transfected

value: osimertinib for 3 days


title: HCC827EV, osimertinib, rep3


title: AB6169


title: Pat41

value: TRUE


title: AB13955

description: metadata_tumor_early_HISAT_annotated.txt

value: tumor


title: HCC827EV, osimertinib, rep1


title: Pat40

value: M


title: HCC827ASCL1, vehicle, rep3

value: vehicle treated


title: AB6172


title: HCC827EV, vehicle, rep2


title: HCC827EV, vehicle, rep3


title: HeLa, TAF1, CUT&RUN

value: TAF1 (cTAF1, 177-4, in-house)


title: HeLa, TAF1, CUT&RUN, IgG control

description: control file to be used also for HeLa, TAF1 and TAF7 CUT&RUN


title: Pat37

value: skin

value: no_During, day 7

value: FALSE

value: anti-PD1 resistant, BRAFi day7


title: AB13954


title: HCC827EV, vehicle, rep1

value: human NSCLC cell line


title: AB13953


title: HeLa, GFP-NLS-TAF2dIDR, dox, rep3

data_processing:
Reads were processed using bcl2fastq version: 2.20.0.422. for Novaseq output
RNA-seq pre-processing was performed on the FRT (control), GFP-NLS-TAF2∆IDR, and GFP–TAF2 FASTQ samples to produce a joined count matrix.
Downstream differential gene analysis then contrasted each sample condition against the other to produce differentially expressed (DE) genes. The pre-processing utilized the Rsubread v2.12.0
Raw FASTQ reads were aligned to the Homo Sapiens (hg38) reference genome, with the resulting BAM files then being quantified at the exon feature level to produce raw feature tables which were then joined into a single count matrix. The downstream analysis was run via the R package RNA-seq Helper based on DESeq2 v1.40.2.
Using the "run_deseq" function, quality control was run on the matrix; filtering out any genes with less than 30 total counts across all samples, as well as any that did not appear in more than 4 samples.
DESeq2 normalization, along with a variance-stabilizing transform (VST) to correct for unwanted variation on the data was also performed via the "pca_and_matrices" function
Assembly: hg38
Supplementary files format and content: tab-delimited count matrices

extract_protocol:
Total RNA was isolated using an RNA isolation kit (RNeasy Mini Kit, Qiagen #74106). 8 ug of RNA were treated with DNaseI and purified according to the manufacturer’s protocol (TURBO DNA-free kit, Thermo Fisher Scientific #AM1907)
Libraries were prepared with 100 ng RNA starting material using the Illumina Standard mRNA Prep kit (Illumina, 20040534). Oligo(dT) magnetic beads purify and capture polyadenylated RNAs. PolyA+ RNAs were fragmented and copied into first strand cDNA using reverse transcriptase and random primers. In a second strand cDNA synthesis step, dUTP replaces dTTP to achieve strand specificity. After adapter ligation, the resulting products were purified and amplified using unique dual index primers for multiplexed sequencing.

treatment_protocol: 1 ug/mL doxycycline for 17.5 h

value: HeLa

value: 1 ug/mL doxycycline

tag: genotype


title: AB13952


title: AB13951


title: PC9ASCL1, osimertinib, rep3

value: inducible ASCL1 transfected


title: Pat36

value: melanoma

value: anti-CTLA4 resistant

value: IIIb

value: mucosa

value: mucosal


title: PC9ASCL1, osimertinib, rep2

value: osimertinib for 3 days


title: HeLa, GFP-NLS-TAF2dIDR, dox, rep2


title: Pat35

value: anti-PD1 resistant

value: F

value: TRUE


title: HeLa, TAF2, CUT&RUN, IgG control


title: AB13950

value: tumor


title: HeLa, GFP-NLS-TAF2dIDR, dox, rep1


title: Pat34

value: M

value: skin


title: HeLa, TAF2, CUT&RUN


title: Pat33

value: unknown


title: Pat39


title: HeLa, GFP-TAF2, dox, rep3


title: AB13942

value: TCRB+


title: AB13943


title: Pat32


title: PC9ASCL1, osimertinib, rep1

value: human NSCLC cell line


title: PC9ASCL1, vehicle, rep3

value: vehicle treated


title: HeLa, GFP-TAF2, dox, rep2

value: HeLa

value: 1 ug/mL doxycycline


title: Pat31

value: melanoma

value: anti-CTLA4 resistant

value: no_During, day 7


title: AB13940


title: HeLa, GFP-TAF2, dox, rep1


title: PC9ASCL1, vehicle, rep1

value: inducible ASCL1 transfected


title: AB13941


title: HeLa, control, dox, rep3

value: control


title: Pat29

value: anti-PD1 resistant

value: anti-PD1 resistant, BRAFi day7


title: HeLa, control, dox, rep2


title: PC9EV, osimertinib, rep3

value: empty vector transfected

value: osimertinib for 3 days


title: AB13938

value: tumor


title: PC9EV, osimertinib, rep2


title: Pat28

value: F

value: skin

value: FALSE


title: HeLa, control, dox, rep1


title: Pat27

value: unknown

value: TRUE


title: PC9EV, osimertinib, rep1

value: human NSCLC cell line


title: Human Uterine Leiomyosarcoma, C10/C12

data_processing:
Cell Ranger software (10X Genomics, v7) was used to align, filter, and count probe reads.
The Seurat (v4) package for R was used to normalize reads and find variable features (SCTransform), perform dimensionality reduction (PCA, UMAP), and cluster cells.
Assembly: GRCh38
Supplementary files format and content: h5: feature count data as sparse matrix (output from Cell Ranger Multi pipeline)
Supplementary files format and content: csv: original sample ID of each single-cell barcode corresponding to a high-quality cell, and associated cell metadata.

source_name: Uterine Leiomyosarcoma tumor

extract_protocol:
Formalin-fixed paraffin-embedded block was sectioned at 50um thickness, digested to single-cell suspension with the Qiagen FFPEasy kit, and filtered with 30um cell strainers.
Cell suspension was hybridized to the 10X Genomics Fixed RNA Profiling human transcriptome probeset.
Sample was tagged with Human Probe Barcodes BC003 and BC004 (10X p/n 2000497 and 2000498) prior to pooling.
After probe and barcode hybridization, suspension was partitioned into single cells according to manufacturer instructions.
Library was constructed according to manufacturer protocols.

value: Uterine Leiomyosarcoma tumor

value: Tumor, Immune, Cancer-associated fibroblast

tag: cancer subtype

value: Uterine Leiomyosarcoma


title: PC9EV, vehicle, rep3

value: vehicle treated


title: Human Uterine Leiomyosarcoma, A32

extract_protocol:
Formalin-fixed paraffin-embedded block was sectioned at 50um thickness, digested to single-cell suspension with the Qiagen FFPEasy kit, and filtered with 30um cell strainers.
Cell suspension was hybridized to the 10X Genomics Fixed RNA Profiling human transcriptome probeset.
Sample was tagged with Probe Barcode BC001 (10X p/n 2000495).
After probe and barcode hybridization, suspension was partitioned into single cells according to manufacturer instructions.
Library was constructed according to manufacturer protocols.


title: AB13937


title: PC9ASCL1, vehicle, rep2

value: inducible ASCL1 transfected


title: AB13936

value: TCRB+


title: AB13935


title: LP9 E+/-KTS 2

city: New Orleans

first: Sean

middle: Bong

last: Lee

state: LA

address: 1700 Tulane Ave

department: Pathology

institute: Tulane University School of Medicine

data_processing:
Gene counts were generated by alignment to human transcriptome GRCh38 using STAR in RSEM.
Differential gene expression analysis was performed using DESeq2
Assembly: GRCh38
Supplementary files format and content: txt file with count data and TPM from RSEM

growth_protocol: Cells were grown in DMEM/F12 with 10% FBS, 1% glutamine, and 1% Penstrep.

extract_protocol:
RNAs were prepared with RNeasy Plus kit (Qiagen, Hilden, Germany)
libraries were created from 500ng of total RNA using the Illumina TruSeq Stranded Total RNA LT Sample Prep kit (San Diego, CA)

treatment_protocol: Cells were treated with or without 1 ug/mL Doxycycline for 4 days

value: mesothelial cells

value: E+/-KTS


title: Pat25

value: melanoma

value: anti-CTLA4 resistant

value: M


title: PC9EV, vehicle, rep2

value: empty vector transfected


title: Pat30

value: anti-PD1 resistant

value: no_During, day 7

value: anti-PD1 resistant, BRAFi day7


title: Pat23

value: F

value: skin

value: TRUE


title: PC9EV, vehicle, rep1

value: human NSCLC cell line


title: AB13934

value: tumor


title: AB13933


title: LP9 E-KTS 2

value: E-KTS


title: Pat22


title: LP9 E+/-KTS 1


title: AB13939


title: Pat21


title: LP9 E-KTS 1


title: AB13925


title: Pat20


title: LP9 E+KTS 2

value: E+KTS


title: Pat26


title: Pat19


title: LP9 E+KTS 1


title: AB13924

value: TCRB+


title: Pat18

value: mucosa

value: mucosal


title: AB13923


title: YU-006 PDX, residual, rep3

data_processing:
Sequencing reads were trimmed of adaptor sequences using Trim Galore.
Reads were mapped to a combined human (hg38) and mouse (mm10) genome using STAR to separate the components of the two species and to a combined transcriptome (GENCODE v24 for human, vM10 for mouse) to add gene annotation.
The uniquely mapped reads were counted per annotation using featureCounts.
The counting matrix went through DESeq2 package to identify the genes that are differentially expressed between different conditions.
Assembly: Hybrid human (hg38) and mouse (mm10) genome
Supplementary files format and content: csv files including RPKM values for each gene in different PDXs

source_name: Patient-derived xenograft

growth_protocol: Dissociated PDX tissues were implanted subcutaneously into NSG mice. Treatment was started as the tumors grew to appropriate size.

extract_protocol:
Total RNA was extracted from tissue powder using AllPrep DNA/RNA mini kit (Qiagen, 80204).
RNA-seq libraries were constructed using KAPA mRNA HyperPrep Kit (Kapa Biosystems, KR1352).

treatment_protocol: Mice were treated with with osimertinib at 25mg/kg for five consecutive days each week. The treatment lasted for 6 weeks before drug tolerant residual tumor was excised.

value: Patient-derived xenograft

value: Osimertinib for 6 weeks


title: AB13922


title: LP9 GFP 1

value: mesothelial cells

value: GFP


title: LP9 GFP 2


title: YU-006 PDX, residual, rep2


title: AB13921

value: PBS


title: YU-006 PDX, residual, rep1


title: AB13920


title: Pat16

value: melanoma

value: anti-CTLA4 resistant


title: Pat15

value: F


title: AB13932


title: YU-006 PDX, untreated, rep2

value: Untreated


title: Pat14

value: M

value: skin

value: TRUE


title: LP9 ctrl 2

value: ctrl


title: Pat17


title: AB13918

value: tumor


title: YU-006 PDX, untreated, rep1


title: Pat13


title: LP9 ctrl 1


title: AB13917


title: YU-003 PDX, residual, rep3


title: AB13919


title: Pat11


title: SK-DSRCT2 shWT1 -dox 2

value: DSRCT

value: -dox


title: SK-DSRCT2 shWT1 +dox 1

value: +dox


title: AB13916


title: YU-003 PDX, residual, rep2

value: Patient-derived xenograft


title: Pat9


title: SK-DSRCT2 shWT1 -dox 1


title: AB13654


title: YU-003 PDX, residual, rep1

value: Osimertinib for 6 weeks


title: BOD-DSRCT shWT1 +dox 2

source_name: BOD-DSRCT

value: BOD-DSRCT


title: Pat8


title: SK-DSRCT2 shWT1 +dox 2


title: YU-003 PDX, untreated, rep2


title: AB13653

value: TCRB+


title: Pat6

value: unknown


title: AB13652

value: PBS


title: Pat5

value: melanoma

value: anti-CTLA4 resistant


title: BOD-DSRCT shWT1 +dox 1


title: BOD-DSRCT shWT1 -dox 2


title: AB13651


title: YU-003 PDX, untreated, rep1

value: Untreated


title: BOD-DSRCT shWT1 -dox 1


title: lsh1 lsh3 lsh4 B8

city: Cassina Rizzardi

first: Veronica

middle: Maria

last: Beretta

state: Co

address: Via A. Vivaldi 36

country: Italy

institute: Università degli Studi di Milano

data_processing:
The raw data were cleaned with fastq-mcf.
cleaned and trimmed reads were mapped on the Arabidopsis thaliana genome assembly TAIR10 with annotation Araport10 using RSEM
DEG analysis was conducted with DESeq2 in R.
Assembly: Arabidopsis thaliana genome assembly TAIR10 with annotation Araport11
Supplementary files format and content: FPKM_Replicates.xlsx excel file that contains the FPKM values of each of the replicates
Supplementary files format and content: complete_table.xlsx excel file that contains the expression values, log2 fold changes lsh1 lsh3 lsh4 vs WT, pvalue and padj

source_name: reproductive meristems enriched in IM, FM and first stages of flower development until stage 8

growth_protocol: Plants  grown in a controlled environment at 20–22 °C under long day conditions (16 h light/8 h dark)

extract_protocol:
RNA was extracted from the samples using the RNeasy Plant Mini Kit
Sequencing libraries were prepared by NovoGene with poly-A enrichment

value: meristematic cells

value: lsh1 lsh3 lsh4 mutant

value: pants sown in growth chamber under LD conditions

tag: strain


title: lsh1 lsh3 lsh4 B7

value: reproductive meristems enriched in IM, FM and first stages of flower development until stage 8


title: SP1_WT_Bio3

extract_protocol:
CUT&Tag and Chem-map were performed as previously described (Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019),Yu, Z., et al. Nat Biotechnol (2023)).G4 mapping (BG4, SG4) and R-loop profiling were performed as 3-layer CUT&Tag on DynabeadsTMMyOneTMStreptavidinT1 (Thermo Fisher, cat#65601). PDS Chem-map, G4 interactome CUT&Tag experiments (SP1, CNBP, MLL1, MLL4, SIN3A, SMARCA4, RNAPIIS5P, RNAPIIS2P) and histone marks (H3K4me1, H3K4me3, H3K27me) were performed as 2-layer CUT&Tag on ConcanavalinA (ConA)-coated magnetic beads (Bangs Laboratories, BP531). MyOneT1 beads were washed twice with PBS pH 6.8, resuspended in 1xPBS pH 6.8 supplemented with 0.01% Tween-20 and mixed with the biotin-conjugated ConA (Sigma-Aldrich, cat#C5275-5MG) solution at 2.3mg/mL at a 1:0.5 ratio. The mix was then incubated at 22°C at 400 rpm for 30min. Aliquots of 500,000 cells/100mL were added to 10mL of beads, washed twice with 100mL wash buffer and resuspended in 1% BSA Antibody Buffer. Primary antibodies were added (1:50), and the mix was incubated at 4°C overnight at 600 rpm. Cells were then washed twice with 100mL dig-wash buffer (0.05% digitonin, 20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and resuspended in 50mL dig-wash buffer. Secondary antibodies were added 1:50 to the samples and incubated at 25°C for 2h at 600 rpm (2-layer CUT&Tag). For G4-mapping samples, cells were washed three times with 500mL dig-wash buffer and resuspended in 50mL dig-wash buffer and 0.5mL of guinea-pig anti-rabbit antibody (Antibodies online, ABIN101961)was added and incubated with the cells at 25°C for 1h at 600 rpm (3-layer CUT&Tag). Cells were then washed three times with 100mL Dig-300 buffer (0.01% digitonin, 20mM HEPES pH7.5, 300mM KCl, 0.5mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and incubated in 1:250 dilution of pA-Tn5 in 50µL Dig-300 buffer at 25°C for 1h at 600rpm.Cells were then washed three times with 500 µL Dig-300 buffer and the Tn5 tagmentation was activated by incubating in 300mL Dig-300 buffer supplemented with 10 mM MgCl2at 37°C for 1h at 600 rpm. Cells were then washed twice with 500 µL TAPS buffer and resuspended in 0.5 mg/mL Proteinase K, 0.5% SDS in 10 mM Tris-HCl buffer pH 8.0 at 55°C for 1h at 800 rpm. Tagmented DNA was purified with DNA clean & concentrator columns (Zymo Research, D4013).
For Illumina library preparation, genomic DNA extracted by CUT&Tag was used. Libraries were generated as previously described with a universal i5 and a uniquely barcoded i7 primer(Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019)). Libraries were visualized and quantified on a high-sensitivity D1000 tape on a Tape Station (AgilentTechnologies 2200)andwith the NEBNext®Library Quant Kit for Illumina (NEB, cat#7630S), following the manufacturer’s instructions.Libraries were sequenced on an Illumina NextSeq 500 (High Output Kit v2.5, 75 cycles) or NextSeq 2000 instrument(P2 or P3 reagents, 100 cycles). RNA-seq and library preparation Total RNA was extracted from 1.0x106 cells using the RNeasy Plus Mini Kit (Qiagen, cat#74134) as per manufacturer’s instructions. RNA quality was checked via TapeStation (Agilent Technologies 2200, RIN>9) and RNA concentration determined using a Qubit fluorometer (Thermo Fisher, Qubit 4). Libraries were generated from 700ng RNA using the NEBNext®Poly(A) mRNA Magnetic Isolation Module (NEB, cat#7490S) together with the NEBNext®UltraTMII Directional RNA Library Prep Kit for Illumina (NEB, cat#7760S) as per manufacturer’s instructions. Libraries were sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles). For MNase 1.0 x 106 cells were washed in ice-cold PBS and incubated in 1mL lysis buffer (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT, 0.1% NP-40) for 10 mins on ice. Cells were then centrifuged at 1,000 x g for 5 mins at 4 °C and resuspended in lysis buffer without NP-40 (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT). Cells were then incubated at 37 °C for 5 mins supplemented with 2 mM CaCl2 and digested with 10 U MNase at 37 °C for 60 mins. The digestion was stopped with a final concentration of 40 mM EDTA, 0.5% SDS. DNA was incubated with RNase A (Thermo Scientific, cat #EN0531) for 1 h at 37oC. DNA was purified using Phenol-chloroform-isoamyl alcohol (PCl, Thermo Scientific, cat #15593049). Libraries were generated with the NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina (NEB, cat #7760S) as per manufacturer’s instructions. A bead-based size selection of 200 bp was performed to isolate mononucleosomal fragments. Libraries were then quantified with the NEBNext® Library Quant Kit for Illumina (NEB, cat #7630S) and sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles).

value: Human embryonic kidney

value: anti-SP1, Protein Tech, 21962-1-AP


title: BER-DSRCT shWT1 +dox 1

source_name: BER-DSRCT

value: BER-DSRCT

value: DSRCT


title: SP1_WT_Bio2


title: AB13649

extract_protocol:
Mice were sacrificed at various time points by cervical dislocation. Tumor, spleen, and tdLN were collected and put through a process of mechanical and enzymatic digestion. For single-cell suspensions, tumors were chopped into small pieces and suspended in RPMI supplemented with DNase (2mg/ml, Sigma-Aldrich) and collagenase IV (100mg/ml, Worthington). Tumors were homogenized by GentleMacs tissue homogenizer (Miltenyi Biotec) and incubated at 37oC for 30min, with frequent agitation. Following dissociation procedures, cells were washed with cold PBS, filtered through a 70-μm cell strainer, and centrifuged at 300g for 5 min at 4 °C. Spleen and tdLN were mashed through a 100uM cell strainer and washed with ice-cold MACS buffer (PBS supplemented with 0.2mM EDTA, pH 8, and 0.5% BSA). Cells were centrifuged at 300g, 5 min, and 4°C. Spleen was suspended in red blood lysis buffer (Sigma-Aldrich) and DNase (0.33U/ml, Sigma-Aldrich), incubated for 5min at room temperature, washed with cold PBS, passed through a 70μm mesh strainer, centrifuged at 300g, 5 min, 4°C and then resuspended in ice-cold MACS buffer.
Single-cell RNA-seq libraries were prepared as previously described {Jaitin, 2014}. In brief, mRNA from single cells sorted into capture plates were barcoded and converted into cDNA and then pooled using an automated pipeline.
3' end mRNA libraries were prepared for sequencing using MARS-seq (Jaitin et al, Science 2014)

value: tumor


title: lsh1 lsh3 lsh4 B6


title: AB13648


title: SP1_SWP_Bio3


title: SP1_WT_Bio1


title: AB13647


title: wild type B4

value: wild type


title: BER-DSRCT shWT1 -dox 2

value: -dox


title: BER-DSRCT shWT1 -dox 1


title: lsh1 lsh3 lsh4 B5


title: SP1_SWP_Bio2


title: JN-DSRCT-1 shWT1 +dox 2

value: +dox


title: AB13646


title: SP1_SWP_Bio1


title: AB13645


title: JN-DSRCT-1 shWT1 +dox 1


title: wild type B3


title: AB13644


title: AB13650


title: SP1_MUT_Bio3


title: wild type B1


title: SP1_MUT_Bio2


title: JN-DSRCT-1 shWT1 -dox 1


title: JN-DSRCT-1 shWT1 -dox 2


title: Pat_ICBnaive2

description: 10x Genomics

data_processing:
The data was processed using Cell Ranger software v6.1.2.
The h5 files were processed and merged using Seurat 4.0.1. The data were reduced to protein-coding genes. Further, translational genes (RPS/RPL in gene names), mitochondrial genes (MT- in gene names), and genes with <= 4 maximum counts were removed. Cell with less than 500 expressed genes were discarded.
Normalization of the data was performed using SCTransform, counts that were zero before normalization were returned to zero, and data was transformed by log2(data+1).
Assembly: hg38
Supplementary files format and content: tab-delimited text file that includes raw counts for each cell
Supplementary files format and content: tab-delimited text file that includes normalized, log-transformed values for each cell

extract_protocol:
Finely chopped cryopreserved material was gently thawed and dissociated using Dri Tumor & Tissue Dissociation Reagent (BD Horizon) according to manufacturer’s protocol, with digestion incubation times up to 1 hour. Dead cells were removed using Dead Cell Removal Kit (Miltenyi Biotech).
The single cell suspension was processed using Chromium Single Cell 3’ Kit with Dual Index Kit TT Set A sample barcodes (10x Genomics) according to manufacturer’s recommendations. Libraries were sequenced on NovaSeq6000 (Illumina) with read length settings 28-10-10-90, according to the 10x Genomics User Guide.

value: ICB naive


title: LED1000_3

city: Chengdu

first: Shuhuai

last: Meng

address: Huaxi

institute: Huaxi

data_processing:
After sequencing on an Illumina Novaseq 6000 platform, raw reads were processed, mapped, and analyzed. Differential expression analysis and enrichment analyses were performed, including GO, KEGG pathway, Reactome, and WikiPathways, using R (v 3.2.0). Column, chord, and bubble plots were generated, and GSEA software was utilized for gene set enrichment analysis.
Assembly: Gene Set Enrichment Analysis (GSEA) was performed using GSEA software[ Subramanian A , Tamayo P , Mootha V K , et al. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(43) :P.15545-15550.]-[ Mootha, V., Lindgren, C., Eriksson, KF. et al. PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34, 267–273 (2003).]. The analysis was used a predefined gene set, and the genes were ranked according to the degree of differential expression in the two types of samples. Then it is tested whether the predefined gene set was enriched at the top or bottom of the ranking list.
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

organism: Rattus norvegicus

source_name: Retina

extract_protocol:
After 6 months of exposure to light, extract rat retina tissues from different light groups, including Control, Halogen 200, Halogen 1000, Blue 200, Blue 1000, White 200, White 1000, a total of seven groups, each with three samples, for RNA transcriptome sequencing
The libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instruction

value: Retina

value: Sprague Dawley rat

value: 8 months


title: LED1000_2


title: AB13643


title: SP1_MUT_Bio1


title: Pat_ICBnaive1

description: 10x Genomics

value: melanoma


title: RNAseq_WT_Bio3

data_processing:
fq files were trimed with cutadapt version 1.18 to remove adaptors and bases with quality lower than 20 (-q 20) mapped with bwa-mem 0.7.17-r2188 to hg38 genome and the coresponding modified genomes, and only the reads within the whitelist have been retained.
bw files presented here were created using bamCoverage with counts per milion normalisation (CPM)  after extending the paired-end reads and accepting maximum mapped fragment length of 350nt. For the polymerase tracks the tracks were further rescaled, normalised for the E coli reads in the library.
Assembly: hg38 , hg38_MutMYCG4, hg38_SwapKRASG4, hg38_MYCFlip_StrandSwap, mm10
Supplementary files format and content: bigWig

extract_protocol:
Total RNA was extracted from 1.0x106 cells using the RNeasy Plus Mini Kit (Qiagen, cat#74134) as per manufacturer’s instructions. RNA quality was checked via TapeStation (Agilent Technologies 2200, RIN>9) and RNA concentration determined using a Qubit fluorometer (Thermo Fisher, Qubit 4). Libraries were generated from 700ng RNA using the NEBNext®Poly(A) mRNA Magnetic Isolation Module (NEB, cat#7490S) together with the NEBNext®UltraTMII Directional RNA Library Prep Kit for Illumina (NEB, cat#7760S) as per manufacturer’s instructions. Libraries were sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles).
For Illumina library preparation, genomic DNA extracted by CUT&Tag was used. Libraries were generated as previously described with a universal i5 and a uniquely barcoded i7 primer(Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019)). Libraries were visualized and quantified on a high-sensitivity D1000 tape on a Tape Station (AgilentTechnologies 2200)andwith the NEBNext®Library Quant Kit for Illumina (NEB, cat#7630S), following the manufacturer’s instructions.Libraries were sequenced on an Illumina NextSeq 500 (High Output Kit v2.5, 75 cycles) or NextSeq 2000 instrument(P2 or P3 reagents, 100 cycles). RNA-seq and library preparation Total RNA was extracted from 1.0x106 cells using the RNeasy Plus Mini Kit (Qiagen, cat#74134) as per manufacturer’s instructions. RNA quality was checked via TapeStation (Agilent Technologies 2200, RIN>9) and RNA concentration determined using a Qubit fluorometer (Thermo Fisher, Qubit 4). Libraries were generated from 700ng RNA using the NEBNext®Poly(A) mRNA Magnetic Isolation Module (NEB, cat#7490S) together with the NEBNext®UltraTMII Directional RNA Library Prep Kit for Illumina (NEB, cat#7760S) as per manufacturer’s instructions. Libraries were sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles). For MNase 1.0 x 106 cells were washed in ice-cold PBS and incubated in 1mL lysis buffer (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT, 0.1% NP-40) for 10 mins on ice. Cells were then centrifuged at 1,000 x g for 5 mins at 4 °C and resuspended in lysis buffer without NP-40 (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT). Cells were then incubated at 37 °C for 5 mins supplemented with 2 mM CaCl2 and digested with 10 U MNase at 37 °C for 60 mins. The digestion was stopped with a final concentration of 40 mM EDTA, 0.5% SDS. DNA was incubated with RNase A (Thermo Scientific, cat #EN0531) for 1 h at 37oC. DNA was purified using Phenol-chloroform-isoamyl alcohol (PCl, Thermo Scientific, cat #15593049). Libraries were generated with the NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina (NEB, cat #7760S) as per manufacturer’s instructions. A bead-based size selection of 200 bp was performed to isolate mononucleosomal fragments. Libraries were then quantified with the NEBNext® Library Quant Kit for Illumina (NEB, cat #7630S) and sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles).


title: LED1000_1


title: BER-DSRCT shWT1 +dox 2


title: AB13638

data_processing:
bcl2fastq/2.15.0.4
Sequences with RMT of low quality (defined as RMT with minimum Phred score of less than 27) were filtered out.
Pool-barcode used i5+i7 barcodes each of them is unique in run, 1 error was allowed.  well-barcode N7 folowed by UMI N8 in read 2.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include mRNA molecule count values for each Sample
Supplementary files format and content: metadata_tumor_early_HISAT_annotated.txt
Supplementary files format and content: metadata_dln_late_annotated.txt
Supplementary files format and content: metadata_tumor_late_annotated.txt
Supplementary files format and content: metadata_q_SPID_BiSE.txt


title: LED200_3


title: RNAseq_WT_Bio2


title: wild type B2

value: meristematic cells


title: RNAseq_WT_Bio1


title: LED200_2


title: Pat5

description: 10x Genomics

value: anti-CTLA4 resistant


title: AB13636


title: LED200_1


title: RNAseq_SWP_Bio3


title: Human Myxofibrosarcoma

data_processing:
Cell Ranger software (10X Genomics, v6 and v7) was used to align, filter, and count reads.
The Seurat (v4) package for R was used to normalize reads and find variable features (SCTransform), perform dimensionality reduction (PCA, UMAP), cluster cells, and filter for doublets and dead cells.
Assembly: GRCh38
Supplementary files format and content: h5: Hierarchical data file
Supplementary files format and content: csv: original sample ID of each single-cell barcode corresponding to a high-quality cell, and associated cell metadata.

source_name: Myxofibrosarcoma tumor

extract_protocol:
Sarcoma tumors were digested into single-cell suspensions using the Tumor Dissociation Kit and gentleMACS dissociator (Miltenyi) and filtered twice through 70um filters before ACK red blood cell lysis. Cells were washed in FBS-containing medium.
For each library, 16,000 cells were loaded into one well of a 10X Genomics Chromium chip for NextGEM 3' v3.1 droplet sequencing, at a concentration between 600-1400 cells/uL, thus targeting capture of 10,000 cells per sample.
Each 10X NextGEM 3' v3.1 library was constructed according to manufacturer protocols and sequenced on the NovaSeq 6000, targeting 20,000 paired-end reads per cell for gene expression libraries, or 500 reads per cell for hashtag libraries.

value: Myxofibrosarcoma tumor

value: Tumor, Immune, Cancer-associated fibroblast

value: Myxofibrosarcoma


title: RNAseq_SWP_Bio2

value: Human embryonic kidney


title: AB13635

value: TCRB+


title: Human Uterine Leiomyosarcoma

value: Uterine Leiomyosarcoma tumor

value: Uterine Leiomyosarcoma


title: Hal1000_3


title: AB13640

value: tumor


title: RNAseq_SWP_Bio1


title: AB13634


title: RNAseq_MUT_Bio3


title: AB13639


title: HeLa - Chip4 Subpool3 RNA Rep 3

city: Los Angeles

first: Xinshu

middle: Grace

last: Xiao

state: CA

address: 610 Charles E. Young Drive South, Room 2000E

department: Integrative Biology and Physiology

institute: UCLA

data_processing:
Read sequences and adaptors were extracted using UMItools (v 0.5.5). The library sequences were then aligned to the designed reference sequences using Botwtie2 (version 2.3.5), allowing up to 1 mismatch per alignment. We only retained perfectly mapped reads or reads with 1 mismatch at positions other than the designed SNPs. PCR duplicates were removed by retaining only one read with the same UMI that mapped to the same reference sequence. The UMIs were then counted in the DNA- and RNA-seq libraries, respectively. The counts were quantile-normalized across the 3 biological replicates.
Assembly: hg19
Supplementary files format and content: Tab-delimited text files contain UMI counts for each Sample

source_name: Uterus; Cervix

growth_protocol: HeLa cells were maintained in DMEM (Gibco, Cat# 11995065) with 10% FBS (Gibco, Cat# 26140079) and antibiotic-antimycotic reagent (Gibco, Cat# 15240062) at 37 °C with 5% CO2 supply.

extract_protocol:
24 hours after electroporation, HeLa cells were lysed using TRIzol (Thermo Fisher Scientific, Cat# 15596026). Each 500 μl TRIzol-lysed solution was mixed with 100 μl chloroform (Fisher Chemical, Cat# C298-500) to allow phase separation. The upper aqueous phase was transferred and mixed with equal volume ethanol (200 proof, Fisher BioReagents). The mixture was loaded to the column supplied by the Direct-zol RNA Miniprep Plus kit (Zymo Research, Cat# R2072) to isolate total RNA following the manufacturer’s protocol. PolyA selection was carried out to isolate mRNA from total RNA using Dynabeads™ Oligo(dT)25 (Thermo Fisher Scientific, Cat# 61002).
custom DNA-Seq and RNA-Seq:
The UMI-containing libraries were generated from the plasmid library (DNA) before electroporation, as well as mRNA isolated after electroporation. The mRNA was reverse transcribed into cDNA using the SuperScript™ IV Reverse Transcriptase (Thermo Fisher Scientific, Cat# 18090010) and a gene-specific reverse transcription (RT) primer (MPP3) that contains a 15-mer unique molecular identifier (UMI), which was synthesized as -NNNNNNNNNNNNNNN- (See Supplementary Protocol for primer sequences). After RT, mRNA was removed via RNase H treatment. Both cDNA and plasmid DNA underwent two rounds of PCR. The first-round (3 cycles) utilized custom primers (MPP2 & MPP3) to add UMIs to the cDNA or plasmid DNA samples. The low-cycle number was utilized to minimize PCR amplification bias. The first-round PCR products were cleaned up using the DNA Clean & Concentrator kit (Zymo Research, Cat# D4004). Next, a second-round PCR was performed using the purified first-round PCR products and primers (MPP2 & MPP4), which added sample indexes and Illumina sequencing adaptors (P5/7). To avoid over-amplification, a pilot reaction for the second-step PCR was performed using the PowerUp™ SYBR® Green Master Mix (Thermo Fisher Scientific, Cat# A25742) and ran on a qPCR thermal cycler. An amplification curve was obtained for each sample to determine the cycle number before the plateau. This cycle number (or a smaller value) was then used in the second-round PCR (See Supplementary Protocol). All PCR steps for sequencing library generation were performed using the Q5 polymerase (NEB, Cat# M0492L). PCR reactions for the same sample were pooled and purified using the DNA Clean & Concentrator kit (Zymo Research, Cat# D4004). Purified PCR products were resolved on a 2% agarose gel and the band at the expected library size (377bp) was cut out and purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Cat# D4002). UMI-containing libraries made from DNA/RNA were mixed and sequenced using custom sequencing primers on Illumina Hiseq3000 PE150 or Novaseq SP PE150 with 15% PhiX spike-in.

treatment_protocol: HeLa cells were transfected with MapUTR plasmid library via electroporation and incubated in growth media at 37 °C for 24 hours before extraction.

value: Uterus; Cervix

value: HeLa

value: epithelial cell

value: transiently transfected with MapUTR plasmid library


title: Hal1000_2

value: Retina

value: Sprague Dawley rat


title: Pat42

description: 10x Genomics

value: melanoma

value: anti-PD1 resistant, with prior anti-CTLA4 progression


title: AB13633


title: AB13632


title: Hal200_3

value: 8 months


title: RNAseq_MUT_Bio1


title: AB13637


title: RNAseq_MUT_Bio2


title: AB13631


title: Hal200_2


title: AB13624


title: RNAPIIS5P_WT_Bio3

data_processing:
fq files were trimed with cutadapt version 1.18 to remove adaptors and bases with quality lower than 20 (-q 20) mapped with bwa-mem 0.7.17-r2188 to hg38 genome and the coresponding modified genomes, and only the reads within the whitelist have been retained.
bw files presented here were created using bamCoverage with counts per milion normalisation (CPM)  after extending the paired-end reads and accepting maximum mapped fragment length of 350nt. For the polymerase tracks the tracks were further rescaled, normalised for the E coli reads in the library.
Assembly: hg38 , hg38_MutMYCG4, hg38_SwapKRASG4, hg38_MYCFlip_StrandSwap, mm10
Supplementary files format and content: bigWig
Library strategy: CUT&Tag

extract_protocol:
CUT&Tag and Chem-map were performed as previously described (Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019),Yu, Z., et al. Nat Biotechnol (2023)).G4 mapping (BG4, SG4, SG4mut-R105A) and R-loop profiling were performed as 3-layer CUT&Tag on DynabeadsTMMyOneTMStreptavidinT1 (Thermo Fisher, cat#65601). SG4 and SG4mutR105A CUT&Tag was performed in extracted nuclei as previously described (Hui W.W.I., et al. Sci Rep (2021)). PDS Chem-map, G4 interactome CUT&Tag experiments (SP1, CNBP, MLL1, MLL4, SIN3A, SMARCA4, RNAPIIS5P, RNAPIIS2P) and histone marks (H3K4me1, H3K4me3, H3K27me) were performed as 2-layer CUT&Tag on ConcanavalinA (ConA)-coated magnetic beads (Bangs Laboratories, BP531).MyOneT1 beads were washed twice with PBS pH 6.8, resuspended in 1xPBS pH 6.8 supplemented with 0.01% Tween-20 and mixed with the biotin-conjugated ConA (Sigma-Aldrich, cat#C5275-5MG) solution at 2.3mg/mL at a 1:0.5 ratio. The mix was then incubated at 22°C at 400 rpm for 30min. Aliquots of 500,000 cells/100mL were added to 10mL of beads, washed twice with 100mL wash buffer and resuspended in 1% BSA Antibody Buffer. Primary antibodies were added (1:50), and the mix was incubated at 4°C overnight at 600 rpm. Cells were then washed twice with 100mL dig-wash buffer (0.05% digitonin, 20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and resuspended in 50mL dig-wash buffer. Secondary antibodies were added 1:50 to the samples and incubated at 25°C for 2h at 600 rpm (2-layer CUT&Tag). For G4-mapping samples, cells were washed three times with 500mL dig-wash buffer and resuspended in 50mL dig-wash buffer and 0.5mL of guinea-pig anti-rabbit antibody (Antibodies online, ABIN101961)was added and incubated with the cells at 25°C for 1h at 600 rpm (3-layer CUT&Tag). Cells were then washed three times with 100mL Dig-300 buffer (0.01% digitonin, 20mM HEPES pH7.5, 300mM KCl, 0.5mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and incubated in 1:250 dilution of pA-Tn5 in 50µL Dig-300 buffer at 25°C for 1h at 600rpm.Cells were then washed three times with 500 µL Dig-300 buffer and the Tn5 tagmentation was activated by incubating in 300mL Dig-300 buffer supplemented with 10 mM MgCl2at 37°C for 1h at 600 rpm. Cells were then washed twice with 500 µL TAPS buffer and resuspended in 0.5 mg/mL Proteinase K, 0.5% SDS in 10 mM Tris-HCl buffer pH 8.0 at 55°C for 1h at 800 rpm. Tagmented DNA was purified with DNA clean & concentrator columns (Zymo Research, D4013).
For Illumina library preparation, genomic DNA extracted by CUT&Tag was used. Libraries were generated as previously described with a universal i5 and a uniquely barcoded i7 primer(Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019)). Libraries were visualized and quantified on a high-sensitivity D1000 tape on a Tape Station (AgilentTechnologies 2200)andwith the NEBNext®Library Quant Kit for Illumina (NEB, cat#7630S), following the manufacturer’s instructions.Libraries were sequenced on an Illumina NextSeq 500 (High Output Kit v2.5, 75 cycles) or NextSeq 2000 instrument(P2 or P3 reagents, 100 cycles). RNA-seq and library preparation Total RNA was extracted from 1.0x106 cells using the RNeasy Plus Mini Kit (Qiagen, cat#74134) as per manufacturer’s instructions. RNA quality was checked via TapeStation (Agilent Technologies 2200, RIN>9) and RNA concentration determined using a Qubit fluorometer (Thermo Fisher, Qubit 4). Libraries were generated from 700ng RNA using the NEBNext®Poly(A) mRNA Magnetic Isolation Module (NEB, cat#7490S) together with the NEBNext®UltraTMII Directional RNA Library Prep Kit for Illumina (NEB, cat#7760S) as per manufacturer’s instructions. Libraries were sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles). For MNase 1.0 x 106 cells were washed in ice-cold PBS and incubated in 1mL lysis buffer (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT, 0.1% NP-40) for 10 mins on ice. Cells were then centrifuged at 1,000 x g for 5 mins at 4 °C and resuspended in lysis buffer without NP-40 (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT). Cells were then incubated at 37 °C for 5 mins supplemented with 2 mM CaCl2 and digested with 10 U MNase at 37 °C for 60 mins. The digestion was stopped with a final concentration of 40 mM EDTA, 0.5% SDS. DNA was incubated with RNase A (Thermo Scientific, cat #EN0531) for 1 h at 37oC. DNA was purified using Phenol-chloroform-isoamyl alcohol (PCl, Thermo Scientific, cat #15593049). Libraries were generated with the NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina (NEB, cat #7760S) as per manufacturer’s instructions. A bead-based size selection of 200 bp was performed to isolate mononucleosomal fragments. Libraries were then quantified with the NEBNext® Library Quant Kit for Illumina (NEB, cat #7630S) and sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles).

value: Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb #13523


title: AB13623


title: HeLa - Chip4 Subpool2 RNA Rep 3


title: Hal200_1


title: HeLa - Chip4 Subpool2 RNA Rep 2


title: RNAPIIS5P_WT_Bio2


title: con_3

value: control


title: Hal1000_1


title: con_2


title: RNAPIIS5P_WT_Bio1


title: HeLa - Chip4 Subpool2 RNA Rep 1 (mixed with another sample)


title: AB13622


title: RNAPIIS5P_MUT_Bio3


title: HeLa - Chip4 Subpool3 RNA Rep 2 (mixed with another sample)


title: con_1


title: HeLa - Chip4 Subpool1 RNA Rep 3


title: RNAPIIS5P_MUT_Bio2


title: HeLa - Chip4 Subpool1 RNA Rep 2


title: AB13620


title: HeLa - Chip4 Subpool3 RNA Rep 1 (mixed with another sample)


title: HeLa - Chip4 Subpool1 RNA Rep 1

tag: molecule subtype

value: Pre-transfection plasmid DNA


title: RNAPIIS5P_MUT_Bio1

tag: cell type

value: Human embryonic kidney


title: AB13619


title: HeLa - Chip4 Subpool3 Pretransfection DNA Rep 3

library_source: other

molecule: other


title: Blue1000_2


title: AB13618

value: PBS


title: RNAPIIS2P_WT_Bio3

tag: chip antibody

value: Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb #13499


title: HeLa - Chip4 Subpool3 Pretransfection DNA Rep 2


title: RNAPIIS2P_WT_Bio2


title: AB13617

value: TCRB+


title: Blue200_3


title: HeLa - Chip4 Subpool3 Pretransfection DNA Rep 1


title: RNAPIIS2P_WT_Bio1


title: Blue200_2


title: AB13621

value: tumor


title: RNAPIIS2P_MUT_Bio3


title: AB13616


title: HeLa - Chip4 Subpool2 Pretransfection DNA Rep 3


title: HeLa - Chip4 Subpool2 Pretransfection DNA Rep 2


title: Blue1000_3


title: LS174T cells, siKRAS, replicate2

city: Porto

first: Flávia

middle: Oliveira

last: Martins

address: Rua Alfredo Allen, 208

country: Portugal

data_processing:
The quality of reads was evaluated using FASTQC
Adapters were trimmed and reads of poor quality or aligning to rRNA sequences were filtered using Trimmomatic
he cleaned reads were aligned to the human genome (hg38) using STAR
Counts for each condition were estimated from mapped reads using RSEM
Assembly: hg38
Supplementary files format and content: xlxs file includes raw counts for each sample

source_name: cell line

growth_protocol: LS174T cells were maintained in DMEM supplmeneted with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS) in humidified atmosphere with 5% CO2 at 37ºC

extract_protocol:
RNA was harvested using TRIzol Reagent
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads in Illumina platform

treatment_protocol: Prior to 3D cultures, CRC cell lines were seeded in 2D conditions in six-well plates, cultured for approximately 16 hours and then transfected using Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific, USA) in reduced-serum Opti-MEM medium (Gibco, Thermo Fisher Scientific, USA) according to the manufacturer’s guidelines. Gene silencing was performed using ON-TARGETplus SMARTpool small interfering RNA specific for KRAS (siKRAS; L-005069-00-0010) from Dharmacon (USA) at a final concentration of 10 nM. A non-targeting (siControl) siRNA (D-001810-01-50; ON-TARGETplus Non-targeting siRNA #1) was used as negative control at the same concentration as the siRNA targeting KRAS. Twenty-four hours post-transfection, cells were harvested with 0.05% Trypsin-EDTA (Gibco, Thermo Fisher Scientific, USA) for 5 min at 37ºC, resuspended in completed medium, and seeded in 3D CoSeedis

value: cell line

value: colorectal cancer

value: siKRAS


title: RNAPIIS2P_MUT_Bio1


title: RNAPIIS2P_MUT_Bio2


title: AB13614


title: HeLa - Chip4 Subpool2 Pretransfection DNA Rep 1


title: AB13613


title: LS174T cells, siKRAS, replicate1


title: PDSChemMap_WT_Input

data_processing:
fq files were trimed with cutadapt version 1.18 to remove adaptors and bases with quality lower than 20 (-q 20) mapped with bwa-mem 0.7.17-r2188 to hg38 genome and the coresponding modified genomes, and only the reads within the whitelist have been retained.
bw files presented here were created using bamCoverage with counts per milion normalisation (CPM)  after extending the paired-end reads and accepting maximum mapped fragment length of 350nt. For the polymerase tracks the tracks were further rescaled, normalised for the E coli reads in the library.
Assembly: hg38 , hg38_MutMYCG4, hg38_SwapKRASG4, hg38_MYCFlip_StrandSwap, mm10
Supplementary files format and content: bigWig
Library strategy: Chemmap

value: btPDS, CHEMMAP


title: LS174T cells, siControl, replicate2

value: siControl


title: AB13573


title: HeLa - Chip4 Subpool1 Pretransfection DNA Rep 2


title: HeLa - Chip4 Subpool1 Pretransfection DNA Rep 3


title: PDSChemMap_WT_Bio3.Tech2


title: AB13572


title: Blue1000_1

value: Retina

value: Sprague Dawley rat


title: LS174T cells, siControl, replicate1


title: PDSChemMap_WT_Bio3.Tech1


title: HeLa - Chip4 Subpool1 Pretransfection DNA Rep 1


title: Blue200_1


title: AB13615


title: HCT116 cells, siKRAS, replicate2

growth_protocol: HCT116 cells were maintained in RPMI supplmeneted with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS) in humidified atmosphere with 5% CO2 at 37ºC


title: AB13570


title: HCT116 cells, siKRAS, replicate1


title: HeLa - Chip3 Subpool1 RNA Rep 1

growth_protocol: HEK293/HeLa cells were maintained in DMEM (Gibco, Cat# 11995065) with 10% FBS (Gibco, Cat# 26140079) and antibiotic-antimycotic reagent (Gibco, Cat# 15240062) at 37 °C with 5% CO2 supply.

extract_protocol:
24 hours after electroporation, HEK293/HeLa cells were lysed using TRIzol (Thermo Fisher Scientific, Cat# 15596026). Each 500 μl TRIzol-lysed solution was mixed with 100 μl chloroform (Fisher Chemical, Cat# C298-500) to allow phase separation. The upper aqueous phase was transferred and mixed with equal volume ethanol (200 proof, Fisher BioReagents). The mixture was loaded to the column supplied by the Direct-zol RNA Miniprep Plus kit (Zymo Research, Cat# R2072) to isolate total RNA following the manufacturer’s protocol. PolyA selection was carried out to isolate mRNA from total RNA using Dynabeads™ Oligo(dT)25 (Thermo Fisher Scientific, Cat# 61002).
custom DNA-Seq and RNA-Seq
The UMI-containing libraries were generated from the plasmid library (DNA) before electroporation, as well as mRNA isolated after electroporation. The mRNA was reverse transcribed into cDNA using the SuperScript™ IV Reverse Transcriptase (Thermo Fisher Scientific, Cat# 18090010) and a gene-specific reverse transcription (RT) primer (MPP3) that contains a 15-mer unique molecular identifier (UMI), which was synthesized as -NNNNNNNNNNNNNNN- (See Supplementary Protocol for primer sequences). After RT, mRNA was removed via RNase H treatment. Both cDNA and plasmid DNA underwent two rounds of PCR. The first-round (3 cycles) utilized custom primers (MPP2 & MPP3) to add UMIs to the cDNA or plasmid DNA samples. The low-cycle number was utilized to minimize PCR amplification bias. The first-round PCR products were cleaned up using the DNA Clean & Concentrator kit (Zymo Research, Cat# D4004). Next, a second-round PCR was performed using the purified first-round PCR products and primers (MPP2 & MPP4), which added sample indexes and Illumina sequencing adaptors (P5/7). To avoid over-amplification, a pilot reaction for the second-step PCR was performed using the PowerUp™ SYBR® Green Master Mix (Thermo Fisher Scientific, Cat# A25742) and ran on a qPCR thermal cycler. An amplification curve was obtained for each sample to determine the cycle number before the plateau. This cycle number (or a smaller value) was then used in the second-round PCR (See Supplementary Protocol). All PCR steps for sequencing library generation were performed using the Q5 polymerase (NEB, Cat# M0492L). PCR reactions for the same sample were pooled and purified using the DNA Clean & Concentrator kit (Zymo Research, Cat# D4004). Purified PCR products were resolved on a 2% agarose gel and the band at the expected library size (377bp) was cut out and purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Cat# D4002). UMI-containing libraries made from DNA/RNA were mixed and sequenced using custom sequencing primers on Illumina Hiseq3000 PE150 or Novaseq SP PE150 with 15% PhiX spike-in.

treatment_protocol: HEK293/HeLa cells were transfected with MapUTR plasmid library via electroporation and incubated in growth media at 37 °C for 24 hours before extraction.

value: Uterus; Cervix

value: HeLa

value: epithelial cell


title: PDSChemMap_WT_Bio2.Tech2


title: HCT116 cells, siControl, replicate2


title: AB13569


title: PDSChemMap_WT_Bio2.Tech1


title: AB13568


title: PDSChemMap_WT_Bio1.Tech2


title: HeLa - Chip3 Subpool1 RNA Rep 2


title: PDSChemMap_WT_Bio1.Tech1


title: HCT116 cells, siControl, replicate1


title: HeLa - Chip2 Subpool2 RNA Rep 3 / Chip2 Subpool4 RNA Rep 3


title: PDSChemMap_SWP_Input

value: bt,CHEMMAP