title: HeLa, H3K27ac Rep2


title: RNA_WW_220511_22-24hmix

status: Public on Apr 13 2023

type: SRA

city: Berlin

first: Emanuel

last: Wyler

address: Robert Roessle Str 10

department: Berlin Institute for Medical Systems Biology

country: Germany

institute: Max Delbrück Center for Molecular Medicine

library_source: transcriptomic

data_processing:
For metagenomics annotation, kaiju (Menzel et al., 2016) was used.
The xHYB data was quantified by aligning raw fastq reads to the reference sequences on which the xHYB panel was built.
Trinity (Haas et al., 2013) and SPAdes (Prjibelski et al., 2020) were used for transcript assembly. Transcript assemblies were then used for detection of novel virus contigs.
Supplementary files format and content: tab-separated values
Supplementary files format and content: fasta files

molecule: total RNA

organism: wastewater metagenome

source_name: wastewater

extract_protocol:
Raw influx was filtered by 2 µm and 0.2 µm. Filtrate was either concentrated using Centricon units (60 ml), or used for Ceres nanotrap based extraction according to the manufacturer's protocol (10 ml) with addition of the ER2 reagent.
RNA was extracted using Trizol/Trizol LS (Thermo Fisher) and the Zymo DirectZol kit (cat# R2050), or with the Zymo Biomics RNA Magbead kit (cat# R2137) using either the RNA isolation protocol from this kit's manual or the RNA/DNA combination isolation protocol from the manual of the Zymo Biomics MagBead DNA/RNA kit (cat# R2135).
DNA was extracted using the Zymo RNA Magbead kit (cat# R2137) with the RNA/DNA combination protocol from the manual of the MagBead DNA/RNA kit (cat# R2135), or with the Zymo Biomics DNA Miniprep Kit (cat# D4300)
RNA sequencing libraries were constructed using the SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara). DNA sequencing libraries were prepared by first applying the ResolveDNA Whole Genome Amplification kit (BioSkryb) and subsequently the Nextera XT DNA Library Preparation Kit (Illumina).
A subset of RNA sequencing libraries was subjected to enrichment using the xHYB adventitious agent panel (Qiagen) where indicated in the sample description.

treatment_protocol: Raw influx from Berlin wastewater treatment plants was provided by municipial water authorities (BWB). Samples were mostly taken as 2 hours composite samples (see time point in sample name), stored at four degrees, and processed within 12 hours.

tag: geographic location

tag: tissue

value: wastewater

tag: processing

value: Concentration

tag: nucleic acid_extraction

value: Trizol/Zymo DirectZol


title: RNA_WW_220511_20-22hmix


title: RNA_WW_220427_22-24hmix


title: 4T1-HA tumors, Micelle IL-12_1

city: Osaka

first: Koji

last: Nagaoka

address: 377-2 Ohno-Higashi, Osaka-Sayama

department: Department of Immunology

country: Japan

institute: Kindai University Faculty of Medicine

data_processing:
Sequence reads were mapped to mm10 using STAR(version 2.7.0f).
Mapped reads were counted by FeatureCounts (version 1.6.4 ).
Genome_build: mm10
Supplementary_files_format_and_content: raw count
Genome_build: mm10
Supplementary_files_format_and_content: csv file including raw counts

organism: Mus musculus

source_name: Tumor tissue

extract_protocol:
Total RNA was isolated from tumor tissues using TRIzol (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and RNeasy spin columns (Qiagen, Venlo, The Netherlands) according to the manufacturer's protocols.
Libraries were prepared using Illumina NEBNext Ultra II RNA Library Prep Kit for Illumina according to the manufacturer's protocols.

tag: strain

value: BALB/c

tag: treatment

value: inoculated with 4T1-HA cells

value: 4T1-HA tumors


title: Tiled-Micro-Capture-C dTAG_11

city: Goettingen

first: Shyam

last: Ramasamy

address: Am Fassberg 11

department: Genome Organization and Regulation

institute: Max Planck Insitute for Multidisciplinary Sciences

description:
dTAG_HMGA2_merged_chimeric.txt
dTAG_ITPRID2_merged_chimeric.txt
dTAG_MTAP_merged_chimeric.txt
dTAG_MYC_merged_chimeric.txt

library_source: genomic

data_processing:
Basecalls were performed using bcl2fastq v2.17 for NextSeq550 output.
MCC pipeline (https://github.com/jojdavies/Micro-Capture-C)
ICE normalization (Imakaev et al. Nature Methods 2012)
Assembly: hg38
Supplementary files format and content: Chimeric junctions files: tab-delimited text files listing the locations of the identified valid junctions
Library strategy: Tiled-Micro-Capture-C

molecule: genomic DNA

organism: Homo sapiens

source_name: Epithelial Tumor Colon

growth_protocol: RPMI-1640, 10% fetal bovine serum, 1x penicillin/streptomycin. Cells were cultured at 37C at 5% CO2.

extract_protocol:
Cells were fixed with 2% formaldehyde for 10 minutes and permeabilised with 0.05% digitonin. Chromatin was digested by MNase and religated. Decrosslinking and DNA extraction was performed. Samples were sonicated using a S220 focused-ultrasonicator (Covaris).
Tiled-Micro-Capture-C (Tiled-MCC) combines Micro-Capture-C (MCC) library preparation with oligonucleotide capture enrichment for deep, targeted sequencing of chromatin interactions within regions of interest. Details are described in the Methods section of the paper.

treatment_protocol: Cells were treated as described in the Methods section of the paper

value: Epithelial Tumor Colon

tag: cell line

tag: cell type

value: human colon cancer cells

tag: genotype

value: dTAG


title: HeLa, H4K16ac Rep2

city: London

first: Pradeepa

middle: M

last: Madapura

state: UK

address: 4 Newark Street, Whitechapel

country: United Kingdom

institute: Queen Mary University of London

data_processing:
150bp paired-end reads for the CUT&Tag-seq were trimmed for adapters using the Trimmomatic tool and aligned to the hg38 genome through local Bowtie2 (version 2.4.5) with these parameters for pair-end mapping: --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700. For analyses, multi-mapped reads were filtered out and only uniquely mapped reads were retained with samtools flag of -q 2 -f 0x200. The bam files were sorted and indexed using the samtools (version 1.9) sort and the samtools index. Merging of multiple replicates was performed using samtools merge.
The sorted bam files were used to generate bed, bedgraph and bigwig formats for individual modifications. Sorted bam files were subjected to bigwig generation via deepTools (version 3.5.1) bamCoverage tool with --binSize 20 –normalize Using BPM or CPM --scaleFactor=1.0 --smoothLength 60 --extendReads 150 --centerReads options. The signal was normalized to IgG through bigwigCompare with option –operation first or subtract. For TDF, sequencing runs involving knockout systems, the corresponding bigwig files were normalized by factoring in the reads mapped to E. coli in WT (icas9) and KO (MSL1/3 KO).
Peak-calling and analyses  The reads were extracted from the bam to bed by the bedtools bamtobed option. Further reads were processed as mentioned in the SEACR (version 1.3) manual to get the bedgraph. These bedgraph files were subjected to peak calling through SEACR with a stringent p-value of ≤1e-6 with the norm and relaxed options.
Assembly: hg38
Supplementary files format and content: bigWig
Library strategy: CUT&TAG

source_name: HeLa

growth_protocol: H9 hPSC line were grown on geltrex coated plates (ThermoFisher Scientific, A1413302) in mTeSR™ Plus medium (StemCell Technologies, #100-0276) supplemented with 100 U/ml penicillin-streptomycin (Gibco, 15140122) and passaged every 3-4 days with ReLeSR™ (StemCell Technologies, 100-0484) according to manufacturer’s protocols. TDF MSL-LOF and WT lines were grown in MEM (Gibco, 11095080) supplemented with 10% FBS (Sigma, F7524), 1X Glutamax (Gibco, 35050061), 1X non-essential amino acid solution (Sigma, M7145) and 100 U/ml penicillin-streptomycin. HEK293T and HeLa cells were grown in DMEM, high glucose (Lonza, BE12-614Q) supplemented with 10% FBS (Sigma, F7524), 1X Glutamax (Gibco, 35050061) and 100 U/ml penicillin-streptomycin. PC3 and LNCaP cells were grown in RPMI medium (Gibco, 21875034) supplemented with 10% FBS (Sigma, F7524) and 100 U/ml penicillin-streptomycin. RWPE1 cells were grown in keratinocyte serum-free medium (Gibco, 10724011) supplemented with 100 U/ml penicillin-streptomycin. K562 cells were grown in Iscove’s Modified Dulbecco’s Medium (Lonza, BE12-722F) supplemented with 10% FBS (Sigma, F7524) and 100 U/ml penicillin-streptomycin. SH-SY5Y cells were grown in DMEM / F12 (1:1) medium (Gibco, 11320033) supplemented with 10% FBS (Sigma, F7524) and 100 U/ml penicillin-streptomycin. All the cell lines were tested for Mycoplasma contamination using EZ-PCR Kit (Geneflow, K1-0210).

extract_protocol:
CUT&Tag was performed according to the protocol of Kaya-Okur et al (1) with modifications to tissue processing as described below. ~100,000 cells were pelleted by centrifugation for 3 min at 600 x g at room temperature and resuspended in 500μl of ice-cold NE1 buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 mM spermidine, 1% Triton X-100, and 20 % glycerol and cOmplete EDTA free protease inhibitor tablet) and was let to sit for 10 min on ice. Nuclei were pelleted by centrifugation for 4 min at 1300 x g at 4°C, resuspended in 500μl of wash buffer and held on ice until beads were ready. Required amount of BioMag Plus Concanavalin-A-conjugated magnetic beads (ConA beads, Polysciences, Inc) were transferred into binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1mM CaCl2 and 1mM MnCl2), washed once in the same buffer, each time placing them on a magnetic rack to allow the beads to separate from the buffer and resuspended in binding buffer. 10 μl of beads was then added to each tube containing cells and rotated on an end-to-end rotator for 10 minutes. After a quick spin to remove liquid from the cap, tubes were placed on a magnet stand to clear, the liquid was withdrawn and 800μl of antibody buffer containing 1μg of primary antibodies: normal rabbit IgG (Santa Cruz Cat no sc-2027), H3K27ac (Abcam, ab4729), H4K16ac (Abcam, ab109463), H3K122ac (Abcam, ab33309), H3K4me1(Abcam, ab8895), H3K36me3 (Abcam, ab9050)) H3K4me3 (Millipore, 07-473),  H3K27me3 (Abcam, ab192985) and H3K9me3 (Abcam, ab176916)) was added and incubated at 4ºC overnight in a nutator. Secondary antibodies (guinea pig α-rabbit antibody, Antibodies online, ABIN101961) were added 1:100 in Dig-wash buffer (5% digitonin in wash buffer) and 100 µL was squirted in per sample while gently vortexing to allow the solution to dislodge the beads from the sides and incubated for 60 min on a nutator. Unbound antibodies were washed in 1 ml of Dig-wash buffer three times. 100 μl of (1:250 diluted) protein-A-Tn5 loaded with adapters in Dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine with Roche cOmplete EDTA free protease inhibitor) was added to the samples, placed on nutator for 1 hour and washed three times in 1 ml of Dig-300 buffer to remove unbound pA-Tn5. 300 µL Tagmentation buffer (Dig-300 buffer + 5 mM MgCl2) was added while gentle vortexing and samples were incubated at 37ºC for 1 hr on an incubator. Tagmentation was stopped by adding 10 µL 0.5M EDTA, 3 µL 10% SDS and 2.5 µL 20 mg/mL Proteinase K to each sample. Samples were mixed by full speed vortexing for ~2 seconds and incubated for 1 hr at 55ºC to digest proteins. DNA was purified by phenol: chloroform extraction using phase-lock tubes (Quanta Bio) followed by ethanol precipitation.
Libraries were prepared using NEBNext HiFi 2x PCR Master mix (M0541S) with a 72ºC gap-filling step followed by 13 cycles of PCR with 10-second combined annealing and extension for the enrichment of short DNA fragments. Libraries were sequenced in Novaseq 6000 (Novogene) with 150bp paired-end reads.

treatment_protocol: All KO and WT TDF lines were treated with 1μg/ml doxycycline (Sigma) for 4 to 7 days

value: HeLa

value: Adenocarcinoma Epithelial cells

tag: antibody


title: 4T1-HA tumors, Free IL-12, anti-PD-1_3

value: Free IL-12_anti-PD-1


title: Tiled-Micro-Capture-C dTAG_10


title: 4T1-HA tumors, Free IL-12, anti-PD-1_2


title: RNA_WW_220427_20-22hmix


title: HeLa, H4K16ac Rep1


title: RNA_WW_220421_22-24hmix_cer_trizol

value: Ceres nanotrap


title: 4T1-HA tumors, Micelle IL-12_3


title: Tiled-Micro-Capture-C dTAG_9


title: HeLa, IgG Rep1

value: IgG(Santa Cruz Biotechnology, sc-2027)


title: Tiled-Micro-Capture-C dTAG_8


title: RNA_WW_220421_20-22hmix_cer_trizol


title: 4T1-HA tumors, Free IL-12_3

value: Free IL-12


title: Tiled-Micro-Capture-C dTAG_7


title: HEK, H3K27ac Rep2

value: Human Embryonic Kidney

value: H3K27ac(Abcam, ab4729)


title: RNA_WW_220421_22-24hmix

value: wastewater


title: HEK, H3K27ac Rep1


title: RNA_WW_220421_20-22hmix

value: Concentration


title: 4T1-HA tumors, Free IL-12_2


title: 4T1-HA tumors, Free IL-12_1


title: 4T1-HA tumors, anti-PD-1_4


title: RNA_WW_220413_20-22hmix

value: Trizol/Zymo DirectZol


title: HEK, H4K16ac Rep2


title: Tiled-Micro-Capture-C dTAG_4


title: Tiled-Micro-Capture-C dTAG_5


title: RNA_WW_220413_22-24hmix


title: RNA_WW_220404_20-22hmix_captu_elu_conc

value: captuvir concentrated eluate


title: 4T1-HA tumors, Free IL-12, anti-PD-1_1

value: BALB/c

value: inoculated with 4T1-HA cells


title: TDF, MSL1KO H4K16ac Rep2

source_name: TDF

value: TDF

value: Transformed Dermal Fibroblasts


title: Tiled-Micro-Capture-C dTAG_3

value: Epithelial Tumor Colon

value: human colon cancer cells

value: dTAG


title: HEK, H4K16ac Rep1


title: Tiled-Micro-Capture-C dTAG_2


title: RNA_WW_220404_20-22hmix_captu_elu

value: captuvir eluate


title: 4T1-HA tumors, anti-PD-1_2

value: 4T1-HA tumors


title: 4T1-HA tumors, Free IL-12_4


title: 4T1-HA tumors, anti-PD-1_3


title: TDF, MSL1KO H4K16ac Rep1


title: 4T1-HA tumors, anti-PD-1_1


title: Tiled-Micro-Capture-C dTAG_1


title: RNA_WW_220404_20-22hmix_captu_ft

value: captuvir flowthrough


title: Tiled-Micro-Capture-C DMSO_14

description:
DMSO_HMGA2_merged_chimeric.txt
DMSO_ITPRID2_merged_chimeric.txt
DMSO_MTAP_merged_chimeric.txt
DMSO_MYC_merged_chimeric.txt

value: DMSO


title: RNA_WW_220404_20-22hmix_befo_captu

value: before captuvir


title: TDF, icas9 H4K16ac Rep3


title: 4T1-HA tumors, PBS_5

value: PBS


title: TDF, icas9 H4K16ac Rep2


title: HEK, IgG Rep1

value: Human Embryonic Kidney

value: IgG(Santa Cruz Biotechnology, sc-2027)


title: 4T1-HA tumors, PBS_4


title: Tiled-Micro-Capture-C DMSO_13


title: TDF, MSL1KO Igg


title: RNA_WW_220404_20-22hmix

value: wastewater

value: Concentration

value: Trizol/Zymo DirectZol


title: TDF, MSL3KO H3K9me3


title: Tiled-Micro-Capture-C DMSO_11


title: TDF, MSL3KO H3K27ac

value: H3K27ac(Abcam, ab4729)


title: 4T1-HA tumors, PBS_3

value: BALB/c

value: inoculated with 4T1-HA cells


title: Tiled-Micro-Capture-C DMSO_12

value: Epithelial Tumor Colon


title: RNA_WW_220204_22-24hmix


title: 4T1-HA tumors, PBS_1


title: 4T1-HA tumors, PBS_2


title: RNA_WW_220119_22-24hmix


title: Tiled-Micro-Capture-C DMSO_10

value: human colon cancer cells


title: Tiled-Micro-Capture-C DMSO_9


title: ISC-ATAC-2

city: Beijing

first: Ye-guang

last: Chen

address: Tsinghua Road, Medical Building

department: School of life science

country: China

institute: Tsinghua University

data_processing:
HISAT2 was used to align the sequences to the mouse genome and generate bam files
bamCoverage was used to generate bigwig files from bam files
MACS2 (v2.2.5) was used for peak calling and to generate bed files from aligned reads
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files

source_name: Intestinal stem cells from tissue

growth_protocol: The cells were obtained from wild type mice.

extract_protocol:
Lgr5-high ISCs were sorted from mice and re-suspended in 6 μL of lysis buffer (10mM Tris-HCl, pH 7.4, 10Mm NaCl, 3 mM MgCl2, 0.5% NP-40) for 10 min. Then 4 μL of 5X TTBL, 5 mL of TTE mix V5 and 4 μL of ddH2O were added and incubated at 37 oC for 30 min. Next, 40 ng of carrier RNA (EpiTect Fast DNA Bisulphite Kit, 59824), 78μl of Tris-EDTA buffer and phenol–chloroform was added to purify the DNA.
The library was constructed with TruePrep DNA library Prep Kit V2 for Illumina (Vazyme, TD 202 and TD502). Subsequently, the library was purified with AMPuro XP beads (Beckman, A63881) and sequenced with Illumina Novaseq 6000.

treatment_protocol: no more treatment

tag: strategy

value: ATAC-seq

value: wild type

value: Intestinal stem cells from tissue


title: TDF, MSL3KO H4K16ac

value: TDF

value: Transformed Dermal Fibroblasts


title: TDF, MSL3KO_Igg


title: ISC-ATAC-1


title: Tiled-Micro-Capture-C DMSO_8

value: DMSO


title: RNA_WW_220404_22-24hmix


title: TDF, icas9_K9me3


title: Tiled-Micro-Capture-C DMSO_7


title: TDF, icas9 H3K27ac


title: RNA_WW_220113_22-24hmix


title: RNA_WW_220113_20-22hmix


title: Tiled-Micro-Capture-C DMSO_6


title: Fosb-chip-IP-2

data_processing:
HISAT2 was used to align the sequences to the mouse genome and generate bam files
Deeptools bamCoverage (CPM normalized and extended reads) was used to generate bigwig files from bam files
MACS2 was used for peak calling and to generate bed files from aligned reads
HOMER annotatePeaks.pl was used to annotate the peaks.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig

source_name: Intestinal organoid cells

growth_protocol: Organoids were embedded in Matrigel and seeded on 48- or 24- well plate. After Matrigel polymerization, the ENR medium (Advanced DMEM/F12 supplemented with Penicillin/Streptomycin, GlutaMAX, N2, B27, and N-acetylcysteine containing 50 ng/mL EGF, 100 ng/mL Noggin, and 500 ng/mL R-spondin1) was added and refreshed every 2 days.

extract_protocol:
Organoids were harvested at day 6 after 4-OHT treatment and alive cells were sorted by FACS. After crosslink with 1% formaldehyde and stopped with glycine, the cells were re-suspended in SDS cell lysis buffer (0.3% SDS, 50mM Tris-HCl, pH 8.0, 20mM EDTA, 1X EDTA-free protease inhibitor cocktail). The crosslinked DNA was sheared to lengths between 100 bp and 2500 bp with sonication, and diluted in DB buffer (16.7Mm Tris-HCl, pH 8.0, 0.01% SDS, 1.2mM EDTA, 1.1% Triton X-100, 1XEDTA-free protease inhibitor cocktail). After pre-cleared with 30 μL of 1:1 protein A/G Dyna beads (Life Technologies, 10001D/10003D), the samples were incubated with anti-Fosb antibody (Abcam, ab184938) overnight at 4 oC, and washed 5 times with 1 mL of RIPA buffer (50mM Hepes, pH 8.0, 1% NP-40, 0.7% DOC, 0.5M LiCl, 1X EDTA-free protease inhibitor cocktail) and once with 1mL TE buffer. The beads were eluted in 100 μL of Elution buffer (10 mM Tris-HCl, pH 8.0, 1% SDS, 1 mM EDTA) for 30 min at 65 oC. The Protein-DNA complexes were reversed crosslink at 65 oC overnight. After incubation at 55 oC for 1 hour to digest proteins with 0.4mg/mL Proteinase K (AMRESCO, 0706-100mg) and another 1 hour at 37 oC to digest RNA with 0.4mg/mL RNase A (Thermo Scientific, EN0531), DNA were purified by phenol chloroform and ethanol-precipitated.
Purified DNA was subjected to Tru-seq library construction using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S). The library were purified and size-selected with AMPure XP beads (Beckman, A63881) and sequenced as paired-end with Illumina Novaseq 6000.

treatment_protocol: Organoids derived from Lgr5-EGFP-IRES-CreERT2; Villin-CreERT2 mice were infected with Fosb-OE retrovirus, retro-Fosb-OE organoids were selected out under puromycin treatment. retro-Fosb-OE organoids were treated with 4-OHT to induce Fosb overexpression.

value: Retro-Fosb-OE organoids

value: Treated with 4-OHT for 2 days

value: ChIP-Seq

tag: chip antibody

value: Fosb antibody (Abcam, ab184938)

value: Intestinal organoid cells


title: Fosb-chip-input-2


title: Fosb-chip-IP-1


title: RNA_WW_220105_22-24hmix

value: wastewater

value: Concentration

value: Trizol/Zymo DirectZol


title: TDF, icas9 Igg

value: IgG(Santa Cruz Biotechnology, sc-2027)


title: RNA_WW_220105_20-22hmix


title: Fosb-chip-input-1


title: Tiled-Micro-Capture-C DMSO_5

value: Epithelial Tumor Colon


title: RNA_WW_211223_22-24hmix


title: RNA_WW_220119_20-22hmix


title: Tiled-Micro-Capture-C DMSO_3

value: human colon cancer cells


title: H9, H3K9me3 Rep2

value: Human embryonic stem cells


title: M3-KO-m6A-input-2

data_processing:
Library strategy: MeRIP-seq
Reads were aligned to the mouse genome in Ensembl (release 95) with HISAT2
m6A peaks in each sample were identified using exomePeak (v2.17.0) R/Bioconductor package
Deeptools bamCoverage (CPM normalized and extended reads) was used to generate bigwig files from bam files
Genome_build: mm10
Supplementary_files_format_and_content: bigwig

extract_protocol:
Intestinal crypts were isolated and digested with Tryple. Lgr5-high cells were sorted by flow cytometry. Total RNA was extracted with RNeasy Mini Kit (QIAGEN, 74104). mRNA was purified using Dynabeads mRNA Purification kit (Invitrogen, 61006). Then, mRNA was fragmented into ~100-nucleotide-long fragments by RNA Fragmentation Reagents (Ambion, AM8740) at 94 ℃ for 45s. After that, mRNA were immunoprecipitation with m6A antibody (NEB, NO.E1610S) to enrich the m6A-marked mRNA followed by RNA extraction by RNA clean&concentrator-5 (ZYMO RESEARCH, 1013).
The cDNA libraries were generated using SMARTER Stranded Total RNA-seq kit v2 (Takara, 634411), ) and sequenced on an Illumina Novaseq 6000 platform.

treatment_protocol: Mettl3fl/fl mice and Mettl3fl/fl; Lgr5-EGFP-IRES-CreERT2; Villin-CreERT2 mice were injected with 20mg/ml tamoxifen for four days.

value: MeRIP-Seq

tag: merip antibody

value: Treated with Tamoxifen for 4 days


title: H9, H3K9me3 Rep1


title: RNA_WW_211208_22-24hmix


title: M3-KO-m6A-IP-1

value: m6A antibody (NEB, NO.E1610S)

value: Intestinal stem cells from tissue


title: RNA_WW_211223_20-22hmix


title: Tiled-Micro-Capture-C DMSO_1

value: DMSO


title: RNA_WW_220204_20-22hmix


title: H9, H3K122ac Rep2


title: Tiled-Micro-Capture-C dTAG_6

value: dTAG


title: TDF, icas9 H4K16ac Rep1

value: TDF

value: Transformed Dermal Fibroblasts


title: Tiled-Micro-Capture-C DMSO_2


title: Micro-Capture-C dTAG_12

description:
ANGPT2_dTAG_HCT-116_merged.wig
DCBLD2_dTAG_HCT-116_merged.wig
DDIT4_dTAG_HCT-116_merged.wig
EREG_dTAG_HCT-116_merged.wig
ERRFI1_dTAG_HCT-116_merged.wig
HMGA2_dTAG_HCT-116_merged.wig
HS3ST1_dTAG_HCT-116_merged.wig
IRS1_dTAG_HCT-116_merged.wig
ITPRID2_dTAG_HCT-116_merged.wig
KITLG_dTAG_HCT-116_merged.wig
KRR1_dTAG_HCT-116_merged.wig
KRT19_dTAG_HCT-116_merged.wig
KRT80_dTAG_HCT-116_merged.wig
MTAP_dTAG_HCT-116_merged.wig
MYC_dTAG_HCT-116_merged.wig
PCDH7_dTAG_HCT-116_merged.wig
PHLDA1_dTAG_HCT-116_merged.wig
SPRY4_dTAG_HCT-116_merged.wig
TRIB1_dTAG_HCT-116_merged.wig
VTI1A_dTAG_HCT-116_merged.wig

data_processing:
Basecalls were performed using bcl2fastq v2.17 for NextSeq550 output.
MCC pipeline (https://github.com/jojdavies/Micro-Capture-C)
Assembly: hg38
Supplementary files format and content: Normalised wiggle files
Library strategy: Micro-Capture-C (MCC)

extract_protocol:
Cells were fixed with 2% formaldehyde for 10 minutes and permeabilised with 0.05% digitonin. Chromatin was digested by MNase and religated. Decrosslinking and DNA extraction was performed. Samples were sonicated using a S220 focused-ultrasonicator (Covaris).
Micro-Capture-C libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Libraries were amplified using 6 cycles on the thermocycler. Capture enrichment was done with IDT biotinylated DNA oligonucleotides and washes were performed using Roche KAPA HyperCapture Reagent Kit Post amplification libraries were purified using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity NGS Analysis Kit.


title: WT-m6A-IP-2

value: Wild type

value: Untreated


title: RNA_WW_211208_20-22hmix

value: wastewater


title: RNA_WW_211124_22-24hmix

value: Concentration


title: M3-KO-m6A-input-1


title: Tiled-Micro-Capture-C DMSO_4


title: H9, IgG Rep3


title: RNA_WW_211124_20-22hmix

value: Trizol/Zymo DirectZol


title: Micro-Capture-C dTAG_11


title: M3-KO-m6A-IP-2


title: RNA_WW_211117_22-24hmix


title: H9, IgG Rep2

value: IgG(Santa Cruz Biotechnology, sc-2027)


title: Micro-Capture-C dTAG_10

value: Epithelial Tumor Colon


title: H9, IgG Rep1


title: WT-m6A-IP-1


title: H9, H3K122ac Rep1


title: WT-m6A-input-1


title: Micro-Capture-C dTAG_9

value: human colon cancer cells


title: H9, H4K16ac Rep3

value: Human embryonic stem cells


title: Micro-Capture-C dTAG_8


title: RNA_WW_211117_20-22hmix


title: Micro-Capture-C dTAG_7


title: RNA_WW_211028_20-22hmix


title: H9, H4K16ac Rep2


title: RNA_WW_211028_22-24hmix


title: H9, H4K12ac Rep3


title: Micro-Capture-C dTAG_6


title: RNA_WW_210929_22-24hmix


title: H9, H4K16ac Rep1


title: WT-m6A-input-2

value: MeRIP-Seq


title: Micro-Capture-C dTAG_5


title: Micro-Capture-C dTAG_4


title: WT-scRNA-seq

data_processing:
Raw reads were aligned to the GRCm38/mm10 mouse genome, and Cell Ranger (v3.1.0) was used to estimate unique molecular identifiers
Raw aligned features were loaded and processed using the Seurat package (v4.0.2)
Low-quality cells were filtered if they expressed no more than 200 genes or with more than 20% of mitochondrial genes
Genome_build: mm10
Supplementary_files_format_and_content: text files

source_name: Intestinal epithelium

extract_protocol:
Intestinal crypts were isolated and digested with collagenⅠ and Tryple, alive cells were sorted by flow cytometry. Single cell suspensions were captured using the 10X Genomics Chromium Single cell 3’ Solution according to the manufacturers protocol. Briefly, a single-cell suspension was mixed with RT-PCR master mix and loaded together with Single Cell 3’ Gel beads and partitioning Oil into a Single Cell 3’ Chip. About 8,000 cells were loaded into each reaction. RNA transcripts from single cell were uniquely barcoded and reverse-transcribed.
ScRNA-seq libraries were generated using the Chromium Single Cell 3’ Reagent Ki V3 (10X Genomics). The libraries were sequenced as paired-end with Illumina Novaseq 6000.

value: scRNA-seq

value: Intestinal epithelium


title: H9, H4K12ac Rep2


title: RNA_WW_211013_20-22hmix


title: FTW-mXENs-sep rep2

city: Kunming

first: Mq

last: Peng

state: Yunnan

address: No. 727, Jingming South Road, Chenggong District

institute: Kunming University of Science and Technology

data_processing:
hisat2 compiler was run on raw fastq files
FeatureCounts was run on the aligned fastqs
Stringtie was run on the aligned fastqs
Concatenated file was processed in Deseq2 (including normalization, estimation of dispersion effects, and differential gene expression)
Genome_build: GRCh38/hg38; GRCm38/mm10; Mmul_10/rheMac10
Supplementary_files_format_and_content: Counts, TPMs

source_name: blastocyst

growth_protocol: All the cells were cultured in FTW condition (FGF2, ActivinA and Chir99021)

extract_protocol:
RNA extraction was performed using a RNeasy Mini Kit (QIAGEN) using DNase treatment (QIAGEN). RNA was analyzed using a 2100 Bioanalyzer (Aglient Technologies).
The RNA Library was generated using TruePrep DNA Library Prep Kit V2 for Illumina (TD501, Vazyme), then the library was adapted for sequencing on an Illmina NovaSeq 6000 platform (sequenced by Annoroad).

treatment_protocol: The separately cultured cells were dissociated with TrypLE Express and centrifgue for RNA extraction. The co-cultured cells were were dissociated with TrypLE Express, sorted by using FACS with different fluorescence (GFP or mKO) and centrifuged for RNA extraction.

value: Separately cultured cells

tag: tissue type

value: Extra-embryonic

value: Mouse XENs


title: Micro-Capture-C dTAG_3

value: dTAG


title: RNA_WW_210916_22-24hmix


title: H9, H4K12ac Rep1


title: RNA_WW_210929_20-22hmix


title: KO-scRNA-seq

value: Treated with Tamoxifen for 4 days


title: FTW-mXENs-Dif rep1

value: differentiated Mouse XENs


title: H9, H3K4me3 Rep3

value: H3K4me3(Millipore, 07-473)


title: Micro-Capture-C dTAG_2


title: H9, H3K4me3 Rep2


title: RNA_WW_210901_22-24h

value: wastewater


title: RNA_WW_210916_20-22hmix

value: Concentration


title: FTW-mES cells-sep rep2

value: Embryonic

value: mouse ES cells


title: Micro-Capture-C DMSO_14

description:
ANGPT2_DMSO_HCT-116_merged.wig
DCBLD2_DMSO_HCT-116_merged.wig
DDIT4_DMSO_HCT-116_merged.wig
EREG_DMSO_HCT-116_merged.wig
ERRFI1_DMSO_HCT-116_merged.wig
HMGA2_DMSO_HCT-116_merged.wig
HS3ST1_DMSO_HCT-116_merged.wig
IRS1_DMSO_HCT-116_merged.wig
ITPRID2_DMSO_HCT-116_merged.wig
KITLG_DMSO_HCT-116_merged.wig
KRR1_DMSO_HCT-116_merged.wig
KRT19_DMSO_HCT-116_merged.wig
KRT80_DMSO_HCT-116_merged.wig
MTAP_DMSO_HCT-116_merged.wig
MYC_DMSO_HCT-116_merged.wig
PCDH7_DMSO_HCT-116_merged.wig
PHLDA1_DMSO_HCT-116_merged.wig
SPRY4_DMSO_HCT-116_merged.wig
TRIB1_DMSO_HCT-116_merged.wig
VTI1A_DMSO_HCT-116_merged.wig

value: Epithelial Tumor Colon

value: DMSO


title: FTW-mXENs-sep rep1


title: RNA_WW_210901_20-22h

value: Trizol/Zymo DirectZol


title: H9, H3K4me1 Rep3

value: Human embryonic stem cells

value: H3K4me1(Abcam, ab8895)


title: Micro-Capture-C DMSO_13

value: human colon cancer cells


title: H9, H3K4me1 Rep1


title: RNA_WW_210819_22-24h


title: FTW-mXENs-Dif rep2

value: Separately cultured cells


title: FTW-mES cells-sep rep1


title: H9, H3K36me3 Rep1

value: H3K36me3(Abcam, ab9050)


title: FTW-mES cells-Co rep2

value: Co-cultured cells


title: Micro-Capture-C DMSO_11


title: RNA_WW_210804_20-22h


title: H9, H3K4me3 Rep1


title: FTW-mES cells-Co rep1


title: Micro-Capture-C DMSO_10


title: Micro-Capture-C dTAG_1

value: dTAG


title: RNA_WW_210804_22-24h


title: H9, H3K27me3 Rep1


title: H9, H3K27me3 Rep2


title: Micro-Capture-C DMSO_8


title: FTW-mXENs-Co rep2

value: Extra-embryonic

value: Mouse XENs


title: RNA_WW_210722_22-24h


title: RNA_WW_210722_20-22h


title: FTW-mXENs 2# rep2


title: FTW-mXENs 2# rep1


title: H9, H3K27ac Rep3

value: H3K27ac(Abcam, ab4729)


title: Micro-Capture-C DMSO_9


title: H9, H3K4me1 Rep2


title: FTW-mXENs-Co rep1


title: Micro-Capture-C DMSO_12


title: FTW-mTSCs 2# rep2

value: Mouse TSCs


title: RNA_WW_210622_22-24h

value: wastewater

value: Concentration


title: H9, H3K27ac Rep2


title: RNA_WW_210819_20-22h


title: Micro-Capture-C DMSO_7


title: Micro-Capture-C DMSO_6


title: RNA_WW_210622_20-22h


title: H9, H3K27ac Rep1


title: FTW-mTSCs 2# rep1


title: Micro-Capture-C DMSO_5


title: FTW-mXENs 1# rep1


title: TDF, MSL1 KO ATAC Rep2

data_processing:
The ATAC-seq reads were processed for mapping by trimming for adapters using the Trimmomatic tool, followed by aligning to the hg38 genome through local Bowtie2(version) with these parameters for pair-end mapping: --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700 (Alteration in -X to 2000was done to allow the mapping of reads for H9 cells).
Bam files obtained were sorted, merged and uniqued and subjected to bigwig generation via deepTools (version 3.5.1) bamCoverage tool with --binSize 20 –normalize Using BPM or CPM --scaleFactor=1.0 --smoothLength 60 --extendReads 150 --centerReads options. The signal was normalized through bigwigCompare with option –operation subtract. For H9, signal for H9 Scr was subtracted from MSL3 KD and for TDF, signal for icas9 was subtracted from MSL1 KO.
Assembly: hg38
Supplementary files format and content: bigWig

growth_protocol: H9 hPSC line were grown on geltrex coated plates (ThermoFisher Scientific, A1413302) in mTeSR™ Plus medium (StemCell Technologies, #100-0276) supplemented with 100 U/ml penicillin-streptomycin (Gibco, 15140122) and passaged every 3-4 days with ReLeSR™ (StemCell Technologies, 100-0484) according to manufacturer’s protocols.   TDF MSL-LOF and WT lines were grown in MEM (Gibco, 11095080) supplemented with 10% FBS (Sigma, F7524), 1X Glutamax (Gibco, 35050061), 1X non-essential amino acid solution (Sigma, M7145) and 100 U/ml penicillin-streptomycin.

extract_protocol:
ATAC-seq is performed as described in (2) with the modifications. The freshly harvested 50,000 cells were washed in PBS and resuspended in a resuspension buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2). Cells were resuspended and incubated on ice for 3 min in 50ul of cold lysis buffer (0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin in resuspension buffer). Nuclei were washed in 1 ml of wash buffer (990ul resuspension buffer, 0.1% Tween-20) by inverting three times. Nuclei were pelleted by centrifuging at 500 xg for 10 min at 4C.  The nuclei were resuspended in 47.5 ml of Nextera Tagmentation buffer (Nextera DNA Sample Preparation Kit) and incubated with 2.5 μl of the Tn5 transposase (Nextera kit, Illumina) at 37°C for 30 min. The resulting DNA fragments were purified using a miniElute column (Qiagen) and amplified by NEBNext High-Fidelity PCR Master Mix in a total volume of 50 μl. The thermocycling protocol for this reaction was 72°C for 5 min, 98°C for 30 s and five cycles of 98°C for 10 s, 63°C for 30 s and 72°C for 1 min.
Using the universal adapter primer and a unique barcoded adapter primer (same as CUT&Tag primers). To avoid over-amplification, after the initial five cycles, the number of remaining cycles required was estimated for each sample using qPCR by adding SYBRGreen and using five μl of the previous PCR as a template. The number of additional cycles was determined to be the number it took for the qPCR to reach one-third maximal fluorescence. The original PCR was then resumed, and each sample cycled as necessary. After amplification, the samples were purified using AMPure XP beads. The libraries were sequenced as a Minimum of 50 million 150 bp paired-end reads in Novoseq (Novogene PLC).

value: TDF

value: Transformed Dermal Fibroblasts

value: MSL1 KO


title: RNA_WW_210610_22-24h

value: Trizol/Zymo DirectZol


title: Micro-Capture-C DMSO_4

value: Epithelial Tumor Colon

value: DMSO


title: TDF, MSL1 KO ATAC Rep1


title: RNA_WW_210610_20-22h


title: Micro-Capture-C DMSO_3

value: human colon cancer cells


title: RNA_WW_210604_22-24h


title: FTW-mXENs 1# rep2

value: Separately cultured cells


title: Micro-Capture-C DMSO_2


title: FTW-mTSCs 1# rep1


title: TDF, icas9 ATAC Rep1


title: RNA_WW_210604_20-22h


title: Micro-Capture-C DMSO_1


title: FTW-mES cells rep2

value: mouse ES cells


title: RNA_WW_210527_22-24h


title: H9, MSL3 KD ATAC Rep3

value: Human embryonic stem cells

value: MSL3 KD


title: FTW-mES cells rep1

value: Extra-embryonic


title: Cut&Tag IgG_dTAG

data_processing:
Basecalls were performed using bcl2fastq v2.17 for NextSeq550 output.
Cut&Tag data analysis was performed as described in the Methods section of the paper.
Paired-end reads were processed for adapter removal and filtering of duplicates and were mapped to hg38 human genome using Bowtie2.
Assembly: hg38
Supplementary files format and content: Normalised bigwig files
Library strategy: Cut&Tag

extract_protocol:
Cells were mildly fixed with 0.3% paraformaldehyde for 2 minutes.
Antibody incubations, washes, tagmentation reactions, DNA extraction, indexing of libraries were done according to the manufacturer’s instructions (Active Motif, 53160).

value: dTAG


title: H9, MSL3 KD ATAC Rep2


title: H9, MSL3 KD ATAC Rep1


title: AKFP-RNA-2

data_processing:
RNA-seq reads were aligned to the mouse genome available in Ensembl (release 95) with HISAT2 (v2.1.0)
StringTie (v1.3.3b) for transcript assembly
The quantitative gene expression (FPKM) was obtained with Ballgown
Genome_build: mm10
Supplementary_files_format_and_content: csv

extract_protocol:
For mice, intestinal crypts were isolated and digested with Tryple, Lgr5-high or Lgr5-low cells were sorted by flow cytometry. For organoids, organoids were digested with Tryple and alive cells were sorted by flow cytometry. Total RNA was extracted with RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions.
The library were prepared for sequecing using NEBNext Ultra RNA library Prep Kit for Illumina.

treatment_protocol: Mettl3fl/fl mice and Mettl3fl/fl; Lgr5-EGFP-IRES-CreERT2; Villin-CreERT2 mice were injected with 20mg/ml tamoxifen for four days, and mice were euthanized at indicated time. Organoids derived from Mettl3fl/fl; Lgr5-EGFP-IRES-CreERT2; Villin-CreERT2 mice were infected with AKF(Ascl2, Klf9, Fosb)-OE retrovirus and AKFP(Ascl2, Klf9, Fosb, Purb)-OE retrovirus. Organoids were selected out under puromycin treatment.Organoids were treated with 4-OHT to induce Mettl3 deletion and AKF or AKFP overexpression.

value: RNA-Seq

value: Treated with 4-OHT for 2 days

value: Intestinal organoid cells


title: Cut&Tag IgG_DMSO


title: RNA_WW_210527_20-22h

value: wastewater

value: Concentration


title: Cut&Tag dTAG_5


title: FTW-mTSCs 1# rep2

value: Mouse TSCs


title: H9, Scr ATAC Rep3

value: Scramble KD


title: MLL-AF9,replicate1,scRNA-seq

city: Guangzhou

first: Suqi

last: Deng

state: Guangdong

address: Huangpu Avenue, Tianhe District, Guangzhou, China

institute: Jinan University

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: m39
Supplementary files format and content: Tab-separated values files and matrix files

source_name: bone marrow

growth_protocol: MLL-AF9/GFP cells were thawed in PBS and tail-vein injected in CD45.1 recipient mice.

extract_protocol:
Leukaemia cells (GFP+) were sorted by flow cytometry(BD FACSArial III) from MLL-AF9-driven AML mice.
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics).

value: bone marrow

value: acute myeloid leukemia cells

value: Lin- bone marrow cells from healthy CD45.2 C57BL/6 mice were transduced with MLL-AF9-GFP retroviruses vector, followed by tansplanting into CD45.1 mice through tail-vain injection. After 2nd transplantation, bone marrow cells from moribund MLL-AF9-driven AML CD45.1 mice were collected and frozen.


title: TDF, icas9 ATAC Rep2

value: TDF

value: Transformed Dermal Fibroblasts


title: H9, Scr ATAC Rep2


title: Cut&Tag dTAG_4

value: Epithelial Tumor Colon

value: human colon cancer cells


title: AKFP-RNA-1


title: RNA_WW_210507_22-24h

value: Trizol/Zymo DirectZol


title: AKF-RNA-2


title: Cut&Tag dTAG_3


title: H9, Scr ATAC Rep1

value: Human embryonic stem cells


title: Cut&Tag dTAG_2

value: dTAG


title: AKF-RNA-1

value: RNA-Seq


title: RNA_WW_210427_22-24h


title: Cut&Tag dTAG_1


title: RNA_WW_210507_20-22h


title: FTW-hXENs-dif rep2

source_name: Naïve ES converted

value: Separately cultured cells

value: Extra-embryonic

value: differentiated human XENs


title: FTW-hXENs-sep rep1

value: human XENs


title: FTW-hXENs-sep rep2


title: RNA_WW_210427_20-22h

value: wastewater

value: Concentration


title: KO-m3-flag-RNA-2

value: Treated with 4-OHT for 2 days

value: Intestinal organoid cells


title: KO-m3-flag-RNA-1


title: RNA_WW_210427_16-18h


title: Cut&Tag DMSO_5

value: DMSO


title: Cut&Tag DMSO_4


title: WT-m3-flag-RNA-2

value: Wild type

value: Treated with EtOH for 2 days


title: FTW-hXENs-dif rep1


title: WT-m3-flag-RNA-1


title: RNA_WW_210519_22-24h


title: RNA_WW_210427_12-14h


title: RNA_WW_210422_22-24h


title: RNA_WW_210519_20-22h


title: FTW-hiPS cells-sep rep2

source_name: Fibroblast

value: Embryonic

value: human iPS cells


title: Cut&Tag DMSO_1

value: Epithelial Tumor Colon

value: human colon cancer cells


title: Cut&Tag DMSO_3


title: FTW-hiPS cells-sep rep1


title: Cut&Tag DMSO_2


title: KO-6d-RNA-1

value: Treated with Tamoxifen for 4 days

value: Intestinal stem cells from tissue


title: RNA_WW_210422_20-22h

value: Trizol/Zymo DirectZol


title: KO-6d-RNA-2

value: RNA-Seq


title: RNA_WW_210415_22-24h


title: FTW-hXENs-Co rep2

value: Co-cultured cells

value: Extra-embryonic


title: FTW-hXENs-Co rep1

value: human XENs


title: RNA_WW_210415_20-22h

value: wastewater

value: Concentration


title: KO-4d-Lgr5-low-RNA-2


title: Capture C dTAG+I-BET-3

description:
ANGPT2.normalised.tsv
DCBLD2.normalised.tsv
DDIT4.normalised.tsv
EREG.normalised.tsv
ERRIF1.normalised.tsv
HMGA2.normalised.tsv
H3S3T1.normalised.tsv
IRS1.normalised.tsv
ITPRID2.normalised.tsv
KITLG.normalised.tsv
KRR1.normalised.tsv
KRT19.normalised.tsv
KRT80.normalised.tsv
MTAP.normalised.tsv
MYC.normalised.tsv
PCDH7.normalised.tsv
PHLDA1.normalised.tsv
SPRY4.normalised.tsv
TRIB1.normalised.tsv
VTI1A.normalised.tsv

data_processing:
Basecalls were performed using bcl2fastq v2.17 for NextSeq550 output.
Capcture-C data analysis was performed using CapCruncher pipeline (https://github.com/sims-lab/CapCruncher; Downes et al, 2022)
Assembly: hg38
Supplementary files format and content: tsv files containing number of normalized interactions per restricition fragment
Library strategy: Capture-C

extract_protocol:
Cells were fixed with 2% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin was digested by DpnII and religated. Decrosslinking and DNA extraction was performed. Samples were sonicated using a S220 focused-ultrasonicator (Covaris).
Capture-C libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Libraries were amplified using 6 cycles on the thermocycler. Capture enrichment was done with IDT biotinylated DNA oligonucleotides and washes were performed using Roche KAPA HyperCapture Reagent Kit Post amplification libraries were purified using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity NGS Analysis Kit.

value: dTAG+I-BET


title: FTW-hiPS cells-Co rep1


title: KO-4d-Lgr5-high-RNA-2


title: RNA_WW_210408_20-22h


title: Capture C dTAG+I-BET-2


title: Capture C dTAG_3

value: dTAG


title: Capture C dTAG_2


title: Capture C dTAG+I-BET-1


title: KO-4d-Lgr5-low-RNA-1


title: KO-4d-Lgr5-high-RNA-1


title: RNA_WW_210408_22-24h


title: KO-3d-Lgr5-low-RNA


title: RNA_WW_210325_KW2_22-24


title: RNA_WW_210325_KW2_morning


title: KO-3d-Lgr5-high-RNA


title: Capture C dTAG_1


title: WT-Lgr5-low-RNA-2

value: Wild type

value: Untreated


title: RNA_WW_210304_KW6


title: WT-Lgr5-low-RNA-1


title: RNA_WW_210304_KW3


title: Capture C DMSO_3

value: DMSO


title: FTW-hiPS cells-Co rep2

value: Embryonic

value: human iPS cells


title: Whole-body Cells collected at L3 stage (P6.p 2-cell) 1

city: Durham

first: David

middle: R

last: Sherwood

state: North Carolina

address: Science Drive

department: Biology

country: USA

institute: Duke University

data_processing:
FASTQC was used to check the quality of the raw sequence data
Trim Galore was used to remove any low-quality bases and adapter sequences
Trimmed reads mapped using STAR alignment. Parameters: outFilterMultimaoNmax 1, outSAMstrandField intronMOtif, alignSJoverhangMin 500
HTSeq used to quantify gene expression data
Downstream Differential Expression analysis performed in R version 4.0.2 using DESeq2 v.1.30.1
Assembly: WBcel235
Supplementary files format and content: .count files

organism: Caenorhabditis elegans

source_name: whole-body

growth_protocol: Synchronized population of worms plated on 100mM NEP plates seeded with NA22 bacteria and grown at 20C. Worms collected at the P6.p 2-cell stage (mid L3 larval stage)

extract_protocol:
RNA extracted using phenol-chloroform. 8-10ng of RNA was extracted
Libraries were prepared using Ovation RNA-Seq Sytem V2 Kit (Part No. 7102) according to the manufacturer’s suggested practices

treatment_protocol:
SDS-DTT solution used to break down cuticle, followed by 15mg/mL Pronase E treatment and mechanical disruption to release cells. Cells settled in ice-cold L-15/FBS solution and passed through a 5 μm filter.
Then cells were resuspended in ice-cold PBS + 2% FBS + 10 mM EDTA.

value: whole-body

value: Whole-body Cells

value: WT


title: Anchor Cells collected at L3 stage (P6.p 2-cell) 2

source_name: Uterine

value: Uterine

value: Anchor Cells


title: Whole-body Cells collected at L3 stage (P6.p 2-cell) 3


title: Anchor Cells collected at L3 stage (P6.p 2-cell) 3


title: Whole-body Cells collected at L3 stage (P6.p 2-cell) 2


title: Capture C DMSO_2

value: Epithelial Tumor Colon

value: human colon cancer cells


title: WT-Lgr5-high-RNA-2

value: RNA-Seq

value: Intestinal stem cells from tissue


title: Capture C DMSO_1


title: WT-Lgr5-high-RNA-1


title: RNA_WW_210304_KW1

value: wastewater

value: Concentration

value: Trizol/Zymo DirectZol


title: No template DSP-1001660005232-A-A01

status: Public on Apr 12 2023

city: La Jolla

first: Judith

last: Varner

state: CA

address: 3855 Health Sciences Dr.

department: Moores Cancer Center

institute: University of California, San Diego

description: No template

data_processing:
Counts from barcodes corresponding to RNA probes were normalized with internal positive and negative controls and then normalized to ROI area.
Data were pre-processed in nanoString DSP Software v 2.2.0.123 with Q3 normalization. ROIs were classified according to predominant cell type (epithelium, granulocyte, macrophage) in one of two conditions (COVID-19 positive and normal). The R BioConductor packages edgeR (Robinson et al., 2010) and limma (Ritchie et al., 2015) were used to implement the limma-voom (Law et al., 2014) method for differential expression analysis within and across cell types and condition. Trimmed mean of M-values (TMM) (Robinson and Oshlack, 2010) normalization was applied. The experimental design was modeled upon cell type and condition (~0 + cellType_condition). The voom method was employed to model the mean-variance relationship, after which lmFit was used to fit per-gene linear models and empirical Bayes moderation was applied with the eBayes function. Significance was defined by using an adjusted p-value cut-off of <0.05 after multiple testing correction (Benjamini and Hochberg, 1995) using a moderated t-statistic in limma.
Genome_build: GCA_000001405.15
Supplementary_files_format_and_content: Nanostring GeoMx Q3 normalized data file as csv file,  Included relative expression levels of genes

organism: blank sample

source_name: No Template Control

extract_protocol:
nanoString GeoMx digital spatial profiling was applied to 3 COVID-19  and 1 normal lung tissues to acquire spatially resolved, quantitative measurements of gene expression in human lung macrophages, granulocytes and epithelium in FFPE tissue sections. Three cases of post-mortem COVID lung tissues and one case of surgically resected normal lung tissue were sectioned, deparaffinized, rehydrated and treated with low pH retrieval solution. nanoString GeoMX Digital Spatial ImmuneOncology and COVID-19 RNA probes were hybridized to sections overnight. Sections were then stained with a cocktail of immunofluorescent antibodies comprised of Alexa fluor 532-labeled anti-CD68 (Clone KP1, 1:100, Novus Biologicals, USA), Alexa fluor 647-labeled Myeloperoxidase (Clone 2C7, 1:500, Novus Biologicals, USA), Alexa fluor 594 labelled anti-PanCytokeratin (C AE-1, AE-3) and Alexa fluor 488 SYTO13 Nuclear stain.  Digital scanning of slides was performed on the GeoMX Digital Profiler (nanoString Technologies, Inc.). Regions of Interest (ROI) and cell type with a spatial resolution of approximately 10 mm were selected, exposed to UV light for RNA probe cleavage and oligonucleotide collection.
Samples were sequenced on a NovaSeq6000.

tag: geomx oligo set

value: Cancer Transcriptome Atlas, (v1.0) GeoMx COVID-19

tag: roi number

value: no template control

tag: ffpe

value: no template


title: CUT&RUN - WT72_d5_X - IgG

city: Leuven

first: Bernard

middle: Klaas

last: van der Veer

state: Vlaams-Brabant

address: Herestraat 49 - box 804

department: Department of Development and Regeneration

country: Belgium

institute: KU Leuven

description:
CUTeRUN_WT72_d5_X_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed

data_processing:
CUT&RUN reads were first trimmed on quality (PHRED > 20) and length (> 20 bp) using TrimGalore! in paired-end mode. Trimmed reads were aligned to GENCODE mm10 GRCm38.p6 using bowtie with specific settings “--local --very-sensitive-local --no-unal --no-mixed --no-discordant -I 10 -X 700”, specifically for CUT&RUN (33), and deduplicated with Picard MarkDuplicates (v2.23.0) using default settings. The duplication rate was < 1%. SEACR was used to call peaks, using stringent mode and normalized against IgG R (v4.0.5) (44). Diffbind (v3.0.15) was used to define differentially enriched regions using default settings. Genes were associated with differentially enriched H3K27me3 regions using the rGREAT (v1.22.0) package. For visualization, bam files of merged replicates were made into bigwig files using BamCoverage (v3.4.3) with settings “--binSize 20 --smoothLength 60 --minMappingQuality 3 --normalizeUsing BPM”. Profile plots and heatmaps were made uinsg DeepTools (v3.4.3).
Assembly: mm10
Supplementary files format and content: .bed file with called peaks
Supplementary files format and content: .bigwig file for pooled reads
Supplementary files format and content: .bigwig file for sample
Supplementary files format and content: .bed file for loss-in-KO
Supplementary files format and content: .bed file for loss-gain-in-KO
Supplementary files format and content: .bed file for gain-in-KO
Supplementary files format and content: .bed file for gain-in-KO d5+X
Library strategy: CUT&RUN

source_name: mESC B6129S6F1 Tet1<tm1Koh>

growth_protocol: Anterior neural progenitor differentiation was performed as previously described (Cruz-Molina et al., 2017; Gouti et al., 2014; Luo et al., 2020). Feeder depleted serum cultured ESCs were plated on gelatin coated plates at 10,000 cells/cm2 (90,000 cells per well in a 6-well plate) in advanced N2B27 defined media compromising a 1:1 mix of 1:1 DMEM/F12 (Invitrogen, 12634-010, Invitrogen) and Neurobasal medium (Invitrogen, 10888-022) supplemented with 0.5x B27 without vitamin A (Invitrogen, 12587-010), 0.5x N2 (Invitrogen, 17502-048), 2 mM L-glutamine (Invitrogen, 25030-024), 40 mg/ml BSA fraction V (Invitrogen, 15260037), 0.1 mM 2-mercaptoethanol (Invitrogen, 31350-010) and 100:100 units:µg/ml penicillin:streptomycin (Sigma-Aldrich, P4333; or Gibco, 15140122). Cells were supplemented with 10 ng/ml bFGF (Cat#100-18C, Peprotech) from day 0 until day 3. At day 3 the medium was changed to medium without bFGF. In some experiments, 5 µM of the Wnt inhibitor XAV939 (Sigma-Aldrich, X3004), 2.5 µM of the TGF-β receptor inhibitor SB431542 (Sigma-Aldrich, S4317), 0.1 µM BMP inhibitor LDN193189 (Sigma-Aldrich, SML0559), 0.3 µM or 3 μM GSK3 β inhibitor CHIR99021 (Axon Medchem BV,1408), 5 or 25 ng/ml of Activin A (Peprotechm 120-14E), or equivalent amount of DMSO (vehicle control) were added from day 2 until day 5. The medium was changed every day during differentiation, except on day 1. At day 4, 1.5x amount of medium was added. The experimental end-point is day 5.

extract_protocol: CUT&RUN was performed using the CUTANATM KIT (Epicypher, 14-1048) according to the manufacturer’s instructions with minor modifications (33,34). Briefly, 5 x 105 cells were collected as a single cell suspension and washed three times with 100 μl/sample wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine and 1 Roche complete tablet/50ml). Cells were then bound to activated Concanavalin A beads and incubated for 10 minutes at RT, followed by incubation with 0.5 μg anti-H3K27me3 (Invitrogen, MA5-11198) antibody or IgG (Epicypher, 18-1401) diluted in antibody buffer on a nutator overnight at 4°C. After 3 washes of magnet-bound beads with digitonin buffer, pAG/MNase was added and incubated for 10 minutes at RT, washed and resuspended in digestion buffer for a 30-minute incubation on ice. The reaction was stopped by addition of STOP buffer and incubated for 10 minutes at 37 °C. The supernatant containing enriched DNA was then extracted using the supplied DNA Clean-up Columns. Libraries were prepared using the NEBNext® Ultra II DNA library Prep Kit for Illumina (New England Biolabs®, NEB E7645L) according to Epicypher’s instructions, and amplified by PCR using NEBNext Dual Index Primers (New England Biolabs, E6440S) with the following settings: 98°C 45 sec; 14 x 98°C 15 sec, 60°C 10 sec; 72°C 1 min. Library concentration was quantified using Qubit HS dsDNA Quant kit (ThermoFisher, Q32851).Quality and size distribution were further assessed on an Agilent 2100 Bioanalyzer using a High Sensitivity DNA Analysis Kit (Agilent, 5067-4626). Libraries were diluted to 4nM and pooled together for PE50 sequencing on NextSeq500 or NextSeq2000 for minimally 5 million reads per sample.

value: mESC B6129S6F1 Tet1<tm1Koh>

value: mESC

value: d5 + XAV

value: IgG


title: CUT&RUN - WT72_d5 - IgG

description:
CUTeRUN_d5_WT72_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed

value: WT


title: CUT&RUN - WT72_d5_X - H3K27me3

description:
CUTeRUN_WT72_d5_X_H3K27me3.stringent.bed
d5_X_WT_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - WT72_d3 - IgG

description:
CUTeRUN_d3_WT72_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - WT70_d5_X - IgG

description:
CUTeRUN_WT70_d5_X_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - WT72_d5 - H3K27me3

description:
CUTeRUN_d5_WT72_H3K27me3.stringent.bed
d5_WT_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - WT72_d3 - H3K27me3

description:
CUTeRUN_d3_WT72_H3K27me3.stringent.bed
d3_WT_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - WT70_d5_X - H3K27me3

description:
CUTeRUN_WT70_d5_X_H3K27me3.stringent.bed
d5_X_WT_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - WT70_d5 - H3K27me3

description:
CUTeRUN_d5_WT70_H3K27me3.stringent.bed
d5_WT_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - WT70_d3 - H3K27me3

description:
CUTeRUN_d3_WT70_H3K27me3.stringent.bed
d3_WT_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: RNA_WW_210304_KW2

data_processing:
For metagenomics annotation, kaiju (Menzel et al., 2016) was used.
The xHYB data was quantified by aligning raw fastq reads to the reference sequences on which the xHYB panel was built.
Trinity (Haas et al., 2013) and SPAdes (Prjibelski et al., 2020) were used for transcript assembly. Transcript assemblies were then used for detection of novel virus contigs.
Supplementary files format and content: tab-separated values
Supplementary files format and content: fasta files

extract_protocol:
Raw influx was filtered by 2 µm and 0.2 µm. Filtrate was either concentrated using Centricon units (60 ml), or used for Ceres nanotrap based extraction according to the manufacturer's protocol (10 ml) with addition of the ER2 reagent.
RNA was extracted using Trizol/Trizol LS (Thermo Fisher) and the Zymo DirectZol kit (cat# R2050), or with the Zymo Biomics RNA Magbead kit (cat# R2137) using either the RNA isolation protocol from this kit's manual or the RNA/DNA combination isolation protocol from the manual of the Zymo Biomics MagBead DNA/RNA kit (cat# R2135).
DNA was extracted using the Zymo RNA Magbead kit (cat# R2137) with the RNA/DNA combination protocol from the manual of the MagBead DNA/RNA kit (cat# R2135), or with the Zymo Biomics DNA Miniprep Kit (cat# D4300)
RNA sequencing libraries were constructed using the SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara). DNA sequencing libraries were prepared by first applying the ResolveDNA Whole Genome Amplification kit (BioSkryb) and subsequently the Nextera XT DNA Library Preparation Kit (Illumina).
A subset of RNA sequencing libraries was subjected to enrichment using the xHYB adventitious agent panel (Qiagen) where indicated in the sample description.

tag: geographic location


title: CUT&RUN - WT70_d5 - IgG

description:
CUTeRUN_d5_WT70_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed

tag: tissue


title: CUT&RUN - WT70_d3 - IgG

description:
CUTeRUN_d3_WT70_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - KO15_d5_X - IgG

description:
CUTeRUN_KO15_d5_X_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed

value: Tet1 KO


title: CUT&RUN - KO15_d5 - IgG

description:
CUTeRUN_d5_KO15_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - KO15_d3 - IgG

description:
CUTeRUN_d3_KO15_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - KO15_d5 - H3K27me3

description:
CUTeRUN_d5_KO15_H3K27me3.stringent.bed
d5_KO_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - KO1_d5_X - IgG

description:
CUTeRUN_KO1_d5_X_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - KO1_d5 - H3K27me3

description:
CUTeRUN_d5_KO1_H3K27me3.stringent.bed
d5_KO_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - KO1_d3 - H3K27me3

description:
CUTeRUN_d3_KO1_H3K27me3.stringent.bed
d3_KO_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - KO1_d5 - IgG

description:
CUTeRUN_d5_KO1_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - KO1_d5_X - H3K27me3

description:
CUTeRUN_KO1_d5_X_H3K27me3.stringent.bed
d5_X_KO_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - KO1_d3 - IgG

description:
CUTeRUN_d3_KO1_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - CM69_d5_X - H3K27me3

description:
CUTeRUN_CM69_d5_X_H3K27me3.stringent.bed
d5_X_CM_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed

source_name: mESC B6129S6F1 Tet1<tm1Koh>; CRISPR HDR

value: mESC B6129S6F1 Tet1<tm1Koh>; CRISPR HDR

value: Tet1 MUT


title: CUT&RUN - CM69_d5 - IgG

description:
CUTeRUN_d5_CM69_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - CM69_d3 - IgG

description:
CUTeRUN_d3_CM69_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - CM69_d5 - H3K27me3

description:
CUTeRUN_d5_CM69_H3K27me3.stringent.bed
d5_CM_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - CM69_d3 - H3K27me3

description:
CUTeRUN_d3_CM69_H3K27me3.stringent.bed
d3_CM_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - CM59_d5_X - IgG

description:
CUTeRUN_CM59_d5_X_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - CM59_d5_X - H3K27me3

description:
CUTeRUN_CM59_d5_X_H3K27me3.stringent.bed
d5_X_CM_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - CM59_d5 - IgG

description:
CUTeRUN_d5_CM59_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - CM59_d5 - H3K27me3

description:
CUTeRUN_d5_CM59_H3K27me3.stringent.bed
d5_CM_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: ATACseq -WT7 - day 5 + XAV

description:
WT_d5_X.pooled.fc.signal.bigwig
antNPC.d5_X.DOR.KO_d5_XvsWT_d5_X.open.order.asc.bed
antNPC.d5_X.DOR.KO_d5_XvsWT_d5_X.close.order.asc.bed

data_processing:
ATACseq reads were analyzed using ENCODE ATACseq pipeline (http://doi.org/10.5281/zenodo.156534) developed by Anshul Kundaje’s laboratory, which performs quality and adapter trimming, alignment, deduplication, peak calling and quality control in a fully automated manner. The resulting peak files and alignment files were used by DiffBind (v3.0.15) to find differentially accessible regions (DARs) using settings specific for ATACseq; “summits = 150” in peak defining and “background = true” in read normalization. Transcription factor motif enrichment in DARs was determined using HOMER (v2) package with default settings. Profile plots and heatmaps were made using DeepTools (v3.4.3) from bigwig files generated from merged replicate reads with the ATACseq pipeline.
Assembly: mm10
Supplementary files format and content: narrowPeak file with peaks per sample
Supplementary files format and content: bigwig file per sample
Supplementary files format and content: bigwig file for pooled reads
Supplementary files format and content: bed file with differentially accesbile reagions (open)
Supplementary files format and content: bed file with differentially accesbile reagions (close)

extract_protocol:
Although viability of the cells is high (> 90-95%), we treated them in suspension nonetheless with 0.1 mg/ml DNase I (Worthington, LS002005) for 30 minutes at 37 ºC directly in the medium and added digestion buffer (0.5 mM CaCl2, 2.5 mM MgCl2). Cells were washed with PBS and incubated for 5 minutes with TrypLE which was subsequently quenched with medium.
We used the omni-ATAC protocol to prepare ATACseq libraries as described before (Buenrostro et al., 2013; Corces et al., 2017).  After two washes with ice-cold PBS, 50,000 cells were pelleted in a pre-cooled 1.5 mltube by centrifugation for 5 minutes at 600 rcf at 4 ºC in a microcentrifuge. Supernatant was removed and 50 µl of ATAC-lysis buffer was added (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, 0.01% digitonin) and incubated on ice for 3 minutes, followed by addition of 1 ml of ATAC-wash buffer (ATAC-lysis buffer, without NP40 and digitonin) and the tube was inverted 3 times. The nuclei were pelleted for 10 minutes at 600 rcf at 4 ºC in a microcentrifuge. Supernatant was removed, and nuclei were resuspended in 50 µl transposition reaction buffer (5 µl H2O, 15 µl PBS, 0.5 µl 10% Tween-20, 0.5 µl 1% digitonin, 25 µl TD buffer, 2.5 µl Tagment DNA Enzyme 1) (Tagment DNA Enzyme and Buffer Small Kit, Illumina, 20034197) and incubated for 30 minutes at 37 ºC at 1000 rpm in a thermomixer. The transposed DNA was cleaned up immediately following transposition ZymoDNAClean& Concentrator-5 kit (Zymo D4014) following manufacturer instructions. The transposed DNA was amplified in one pre-amplification step (5 cyles) and a final amplification (5 to 7 cycles) in the same 50 µl reaction using 1x NEBNext High-fidelty PCR master mix (Bioké/NEB, M0541S) and 125 nM of Ad1_noMX primer and Ad2.index primer with the following program: 72 ºC 5 min, 98 ºC 30 sec; 5 to 7x 98 ºC 10 sec, 63 ºC 30 sec, 72 ºC 1 min; 72 ºC 1 min. For each library, the optimal amplification was optimized using qPCR; 1/5th of the pre-amplified libraries was added to amplification master mix containing SYBR Green and run on a Applied Biosystems StepOnePlus Real-Time PCR system  for an additional 20 cycles. Relative log-fluorescence vs amplification cycle was plotted out to manually determine the appropriate amplification cycles. The number of cycles was selected to reach 1/3 of the final relative fluorescence unit value. Following amplification, the libraries were purified using ZymoDNAClean& Concentrator-5 kit (Zymo D4014) followed by 0.55x-1.75x dual size slection using AMPure XP beads. The quality of the libraries was assessed using an Agilent Bioanalyzer 2100 with the High Sensitivity DNA analysis kit (Agilent, 5067-4626) and concentration was determined using Qubit™ dsDNA HS Assay kit (Invitrogen, Q32854). The libraries were diluted to 4 nM and pooled together for SE50 sequencing on an Illumina Hiseq4000, aiming for minimally 25 million mapped and unique reads.

value: day 5 + XAV


title: CUT&RUN - CM59_d3 - H3K27me3

description:
CUTeRUN_d3_CM59_H3K27me3.stringent.bed
d3_CM_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - KO15_d5_X - H3K27me3

description:
CUTeRUN_KO15_d5_X_H3K27me3.stringent.bed
d5_X_KO_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: CUT&RUN - CM59_d3 - IgG

description:
CUTeRUN_d3_CM59_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: ATACseq -KO1 - day 5 + XAV

description:
KO_d5_X.pooled.fc.signal.bigwig
antNPC.d5_X.DOR.KO_d5_XvsWT_d5_X.open.order.asc.bed
antNPC.d5_X.DOR.KO_d5_XvsWT_d5_X.close.order.asc.bed


title: ATACseq -WT34 - day 5 + XAV


title: CUT&RUN - KO15_d3 - H3K27me3

description:
CUTeRUN_d3_KO15_H3K27me3.stringent.bed
d3_KO_H3K27me3.merged.bw
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: ATACseq -KO15 - day 5 + XAV


title: CUT&RUN - CM69_d5_X - IgG

description:
CUTeRUN_CM69_d5_X_H3K27me3.stringent.bed
H3K27me3.loss-loss-in-KO.bed
H3K27me3.loss-gain-in-KO.bed
H3K27me3.gain-gain-in-KO.bed
H3K27me3.gain-in-KO_d5_X.bed


title: LNA Malat1 n1

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

data_processing:
The resulting raw reads were assessed for quality, adapter content and duplication rates with FastQC
Trimmomatic version 0.39 was employed to trim reads after a quality drop below a mean of Q20 in a window of 10 nucleotides. Only reads of at least 15 nucleotides were cleared for subsequent analyses.
Trimmed and filtered reads were aligned versus the Ensembl mouse genome version mm10 (ensemble release 101) using STAR 2.7.7a with the parameters “--outFilterMismatchNoverLmax 0.1 --alignIntronMax 200000”.
The number of reads aligning to genes was counted with featureCounts 1.6.5 from the Subread package. Only reads mapping at least partially inside exons were admitted and aggregated per gene. Reads overlapping multiple genes or aligning to multiple regions were excluded.
Differentially expressed genes were identified using DESeq2 version 1.30.0. Only genes with a minimum fold change of +- 1.5 (log2 +-0.59), a maximum Benjamini-Hochberg corrected p-value of 0.05, and a minimum combined mean of 5 reads were deemed to be significantly differentially expressed.
Assembly: mm10
Supplementary files format and content: library size normalized counts

source_name: Mouse HL1 cell line

growth_protocol: The murine atrial cardiomyocyte  cell line HL-1 (Sigma-Aldrich, SCC065) was cultured in Claycomb medium (Sigma-Aldrich, 51800C), supplemented with 10 % fetal bovine serum (FBS) (Invitrogen), 100 μg/ml penicillin/streptomycin (Roche), 0.1 mM norepinephrine (Sigma-Aldrich) and 2 mM GlutaMAX (Thermo Fisher). Cells were cultured in cell culture dishes coated with 0.02 % gelatin (Millipore) and 0.1 % fibronectin from human plasma (Sigma-Aldrich) at 37°C and 5 % CO2.

extract_protocol:
RNA isolation from mouse HL1 cells was done using Qiagen RNeasy mini kits (Qiagen, 74104) including an on-column DNase digestion step according to the manufacturer’s instructions.
1µg of total RNA was used as input for SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Takara Bio).

treatment_protocol: For transfection experiments, cells were seed 24 hours before transfection and were transfected with a final concentration of 50nM of LNA Gapmers or 50nM of siRNA using RNAimax. Cells were collected 48 hours after transfection.

value: cardiomyocyte like cell line


title: LNA control n3


title: LNA Malat1 n3


title: LNA Malat1 n2


title: LNA control n1


title: WT-Avi-6d_2

city: Tianjin

first: Jingen

last: Li

address: Xiqi Dao 32, Tianjin Airport Economic Area

institute: Tianjin institute of industrial biotechnology,Chinses Academy of Sciences

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence with NGSQCToolkit (v2.3).
Clear reads were mapped to Myceliophthora thermophila ATCC 42464 genome using Tophat (version 2.0.8b) with parameters -i 10 -g 1 --segment-length 24 --b2-very-sensitive --no-convert-bam --library-type fr-unstranded.
The counts of reads uniquely mapped to only one gene for each gene were calculated by HTseq-count (v0.5.4p2).
Assembly: Myceliophthora thermophila ATCC 42464 (https://genome.jgi.doe.gov/Spoth2/Spoth2.home.html).
Supplementary files format and content: RNA-seq-Readcounts.xlsx: Excel file includes reads count for each sample.

organism: Thermothelomyces thermophilus ATCC 42464

source_name: Myceliophthora thermophila ATCC 42464

growth_protocol: M. thermophila strains, including WT, ΔlaeA, Δgrf-1, Δgrf-2, Δgrf-3, and Δcre-1, were inoculated into 1×Vogel’s salt with 2% glucose and cultivated at 45 °C for 24h. When grown on 2% Avicel, the cultures of strains WT and ΔlaeA were collected after the cultivation of 2 d, 4 d, and 6 d.

extract_protocol:
Mycelia were harvested by vacuum filtration with Whatman filter paper, frozen immediately in liquid nitrogen. Total RNA from frozen samples was isolated using TRIzol reagent and an additional clean-up was performed for DNA degradation using the RNeasy mini kit (Qiagen).
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: Mycelium

tag: condition

value: Avicel, 6d


title: ΔlaeA-Avi-6d_1

value: <delta>laeA


title: ΔlaeA-Avi-6d_2


title: WT-Avi-4d_2

value: Avicel, 4d


title: WT-Avi-4d_1


title: Anchor Cells collected at L3 stage (P6.p 2-cell) 1

value: Uterine

value: Anchor Cells


title: WT-Avi-6d_1


title: ΔlaeA-Avi-4d_2


title: ΔlaeA-Avi-2d_2

value: Avicel, 2d


title: WT-Avi-2d_2


title: Δcre-1-Glu_2

value: Glucose


title: Δcre-1-Glu_1


title: ΔlaeA-Avi-2d_1


title: Δgrf-3-Glu_2


title: LNA control n2


title: Δgrf-2-Glu_2


title: Δgrf-3-Glu_1


title: Δgrf-2-Glu_1


title: Δgrf-1-Glu_2


title: Δgrf-1-Glu_1


title: ΔlaeA-Glu_2


title: WT-Glu_2


title: WT-Glu_1


title: ΔlaeA-Avi-4d_1


title: RNA sequencing of K562 clone 5, CRISPRoff [K562-SC1-5-OFF-LRRC8C-Rep1]

city: Seattle

first: Xiaoyi

state: WA

address: 3720 15th Ave NE

department: Department of Genome Sciences

institute: University of Washington

description: Stranded mRNA library

data_processing:
Sequencing reads were demultiplexed using the bcl2fastq software (Illumina). For calculating TPM of genes, sequencing reads from the unstranded RNA libraries were aligned to the GRCh38 reference genome (Gencode V43) using Salmon (v1.9.0). For the CRISPRoff experiment, reads were aligned to the GRCh38 reference genome and counted against all Ensembl genes using STAR (2.7.6a). Raw counts were analyzed with DESeq2.
Assembly: GRCh38
Supplementary files format and content: K562_WT_TPM.txt contains expression levels of genes in TPM (transcript per million)
Supplementary files format and content: CRISPRoff_RNAseq_count.txt cotains expression levels of genes in raw counts

molecule: polyA RNA

growth_protocol: K562 cells (CCL-243) were purchased from ATCC and maintained in RPMI 1640 medium (Gibco) supplied with 10% FBS (Hyclone) and penicillin/streptomycin (Gibco, 100 U/ mL).

extract_protocol:
Total RNAs were purified from cells using TRIzol LS Reagent (Invitrogen) following manufacturer’s instructions, treated with DNase (QIAGEN or Invitrogen) and cleaned up using RNeasy Mini kit (QIAGEN).
For K562 wild-type cells, 1 ug total RNA (RNA integrity >= 9.7) were used for unstranded mRNA library preparation using the TruSeq RNA Library Prep Kit v2. For the CRISPRoff experiment, 1 ug total RNA (RNA integrity >= 9.9) were used for stranded mRNA library preparation using the Illumina TruSeq® Stranded mRNA Library Prep kit. The RNA libraries were indexed using the Illumina TruSeq RNA UD Indexes.

treatment_protocol: For the CRISPRoff experiment, on Day 0, 2 x 106 clone 5 cells were transfected with CRISPRoff-v2.1 (3,000 ng), pU6-Sp-dual-gRNA (1,000 ng) and pmax-GFP (500 ng, Lonza) using the SF Cell Line 4D-Nucleofector X kit L (Lonza). On Day 2, cells were sorted based on high GFP expression (top 20%) on a flow cytometer and expanded. On Day 11, cells were collected for RNA extraction.

value: lymphoblast

value: Clone 5

value: CRISPRoff v2, LRRC8C


title: RNA sequencing of K562 clone 5, CRISPRoff [K562-SC1-5-OFF-METTL2A-Rep2]

description: Stranded mRNA library

value: CRISPRoff v2, METTL2A


title: ΔlaeA-Glu_1


title: RNA sequencing of K562 clone 5, CRISPRoff [K562-SC1-5-OFF-LRRC8C-Rep2]

description: Stranded mRNA library


title: RNA sequencing of K562 clone 5, CRISPRoff [K562-SC1-5-OFF-Ctrl-Rep1]

description: Stranded mRNA library

value: CRISPRoff v2, Non-targeting gRNA


title: RNA sequencing of K562 clone 5, CRISPRoff [K562-SC1-5-OFF-Ctrl-Rep2]

description: Stranded mRNA library


title: RNA sequencing of K562 clone 5, CRISPRoff [K562-SC1-5-OFF-USP7-Rep2]

description: Stranded mRNA library

value: CRISPRoff v2, USP7


title: WT-Avi-2d_1

value: WT


title: Prime editing library of endogenous genes after CRISPRa [K562-EGFR-Rep2-XLP167]

description:
Amplicon sequencing of engogenous genes
CRISPRa_CXCR4_IL2RB_EGFR_CDKL5.csv

data_processing:
T7-assisted reporter mapping: Sequencing reads were first demultiplexed using the bcl2fastq software. Under the current library design and sequencing scheme, Read1 started from genomic sequence and extended into the integrated synHEK3 construct, while Read2 contained reporter barcode information. Therefore, 1) for each sequencing read pair, the 16-bp reporter barcode was extracted from Read 2 and attached to the read name of Read 1. 2) Read1 was trimmed using cutadapt (v4.1) with following parameters: --cores=4 --discard-untrimmed -e 0.2 -m 10 -O 8 -a CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG. The adapter corresponded to the 3’ITR of piggyBac transposon. Since parameter “--discard-untrimmed” was used, only reads spanning insertion junctions were kept and trimmed. 3) Trimmed sequences were aligned to the GRCh38 reference genome using bwa mem (v0.7.17). The “-Y” option was used to enable soft clipping for supplementary alignments. 4) Reads uniquely (without XA:Z tag) and contiguously mapped near putative piggyBac landing pads (TTAA motifs) were kept using samtools (v1.9) and a custom script. 5) Aligned reads were converted to BED format using the sam2bed function in bedops (v2.4.35). Reads aligned to standard chromosomes were kept. 6) Insertion points were calculated for all reads based on strand of alignment. And reads were sorted by the insertion coordinates. 7) The first 8-bp of reads were used as UMIs. A custom script was used to collapse reads at a per-location, per-barcode, per UMI basis. A barcode-location-UMI count table was generated. 8) synHEK3 barcodes <3 Levenshtein Distance at each location were collapsed and barcodes with > 3 UMIs were kept. 9) The count table was converted to a GenomicRanges object in R. Coordinates of the last 4 base pairs of aligned reads were designated as genomic locations of the inserted synHEK3 reporters. 10) “Landing pads” of the mapped synHEK3 reporters were retrieved using the getSeq() function in the BSgenome package. Reporters that didn’t have a TTAA sequence (which could be due to PCR error, mapping error, or the use of non-canonical landing pads) were removed. 11) SynHEK3 barcodes mapped to more than one location were removed.
SynHEK3 amplicon sequencing library: Sequencing reads from the Illumina NextSeq platforms were first demultiplexed using the bcl2fastq software. The 16-bp reporter barcodes were extracted from read and attached to its read name. Sequences around the CRISPR cut site were extracted for all reads. A barcode-editing outcome table was generated. For prime editing experiments, a custom script was used to align sequences to a reference sequence and count mutation frequency for every barcode. For Cas9 mutagenesis analysis, all sequences were aggregated and editing outcomes (alleles) with the highest number of counts were selected. These most frequent alleles were then aligned to reference sequences using needleall with the following parameters: -gapopen 20 -gapextend 0.5 -endopen 20. A custom script was used to annotate the mutational events.
Amplicon sequencing of endogenous genes: Sequencing reads were demultiplexed using the bcl2fastq software (Illumina). A custom script was used to determine frequencies of the introduced substitutions.
Assembly: GRCh38
Supplementary files format and content: T7-500-1_S1_ITR_corrected.txt contains processed, barcode collapsed synHEK3 integration raw counts
Supplementary files format and content: T7-500-1_processed.csv contains fully processed, unique synHEK3 reporters and relevent information
Supplementary files format and content: K562-HEK3-insCTT-DMSO_count.txt contains processed prime editing outcomes of all synHEK3 barcodes
Supplementary files format and content: K562-HEK3-Cas9-Day1-XL281_S68_barcode_edit.txt contains processed Cas9 indel frequency of all synHEK3 barcodes on Day 1
Supplementary files format and content: K562-HEK3-Cas9-Day2-XL281_S70_barcode_edit.txt contains processed Cas9 indel frequency of all synHEK3 barcodes on Day 2
Supplementary files format and content: K562-HEK3-Cas9-Day4-XL281_S72_barcode_edit.txt contains processed Cas9 indel frequency of all synHEK3 barcodes on Day 4
Supplementary files format and content: sg1_top_alleles_count.txt contains Cas9 editing allele count for all synHEK3 barcodes on Day 4
Supplementary files format and content: sg1_top_alleles_info.txt contains annotations of the most frequent alleles for Cas9 editing on Day 4
Supplementary files format and content: CRISPRoff_LRRC8C.csv contains processed prime editing frequencies of synHEK3 reporters in the CRISPRoff experiment, LRRC8C vs. non-targeting control
Supplementary files format and content: CRISPRoff_METTL2A.csv contains processed prime editing frequencies of synHEK3 reporters in the CRISPRoff experiment, METTL2A vs. non-targeting control
Supplementary files format and content: CRISPRoff_USP7.csv contains processed prime editing frequencies of synHEK3 reporters in the CRISPRoff experiment, USP7 vs. non-targeting control
Supplementary files format and content: CRISPRa_CXCR4_IL2RB_EGFR_CDKL5.csv contains prime editing frequencies at endogenous loci before and after CRISPR activation
Supplementary files format and content: shrna_freq_table.csv contains prime editing frequencies of synHEK3 reporters measured in cells transduced with individual shRNAs (GFP positive and GFP negative)

extract_protocol:
gDNA of K562 cells was purified using the DNeasy Blood & Tissue Kit. Alternatively, cells were lysed in a lysis buffer [100 mM Tris-HCl pH8.0, 0.05% SDS and 0.04 mg/mL proteinase K(Thermo Scientific)], and incubated at 50 oC 60 min and 80 oC 30 min.
T7-assisted reporter mapping: gDNA of K562 cells was purified using the DNeasy Blood & Tissue Kit (QIAGEN) and in vitro transcribed with the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs). Briefly, each reaction (20 uL) contained 0.3~1 ug gDNA, NTPs (10 mM each) and 2 ul T7 RNA Polymerase Mix. The reaction mixture was incubated at 37 oC for 16 hours. Then, gDNA was digested with 2.5 uL DNase (Qiagen) in a 100 uL reaction at room temperature for 30 min. RNA was extracted with TRIzol LS Reagent (Invitrogen), and aqueous phase was precipitated with 1 volume of isopropanol and 5 ug Glycogen (Invitrogen) at -80 oC for 1 hour. RNA pellet was collected by centrifugation at 21,000 x g at 4 oC for 1 hour. The pellet was washed with 80% ice-cold ethanol and resuspended in 11.5 uL nuclease-free water. For reverse transcription, RNA was incubated with 0.5 uL 100 uM RT primer p6 (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNN-3’) and 1 uL 10 mM dNTP at 65 oC for 5 min and cooled on ice. Then, 4 uL 5X RT buffer, 1 uL 100 mM DTT, 1 uL RNaseOUT (40 U/uL) and 1 uL SuperScript IV RT (200 U/uL, Invitrogen) were added and the reaction mixture was incubated at 23 oC for 10 min, 50  oC for 15 min and 80 oC for 10 min. cDNA library was amplified with KAPA2G Robust HotStart polymerase, using primers p7 (5’- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAAGGAAGCCCTGCTTCCTCCAGAGGG-3’, 0.5 uM) and p8 (5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’, 0.5 uM). PCR reaction was performed as follows: 95 oC 3 min; 95 oC 15 s, 65 oC 15 s, 72 oC 30 s, 16~18 cycles; and 72 oC 1 min. The PCR product was subjected to double-sided size selection (0.5X, 0.9X) and cleaned up with AMPure XP beads. The resulting product ranged from 200 to 1000 bp. To prepare Illumina sequencing libraries, 5-10 ng PCR product was re-amplified with the Nextera P5 and TruSeq P7 library index primers for 5 cycles. The final PCR product underwent another round of double-sided size selection (0.5X, 0.9X) and cleaned up with AMPure XP beads. The library was sequenced on an Illumina MiSeq in paired-end mode (Read 1: 254 bp; Read2: 55bp).  Amplicon sequencing of synHEK3 reporters: 100~250 ng gDNA or cell lysates were amplified using KAPA2G Robust HotStart ReadyMix with primers p9 (5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTACCCCGACCACATGAAGCAGC-3’, 0.5 uM) and p10 (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNGACCATGTCATCGCGCTTCTCGT-3’, 0.5 uM). PCR reaction was performed as follows: 95 oC 3 min; 95 oC 15 s, 68-N oC 15 s, 72 oC 30 s, for 9 cycles (N was cycle number); 95 oC 15 s, 65 oC 15 s, 72 oC 30 s, for 11 cycles; and 72 oC 1 min. To ensure enough coverage and accurate measurement of editing efficiencies, for the K562 synHEK3 pool, we pooled products from at least 16 PCR reactions. The PCR product was purified with AMPure XP beads. 5 ng PCR product was re-amplified with the Nextera P5 and TruSeq P7 library index primers for 5 cycles.   Other amplicon sequencing: Cell lysates were directly used for PCR with the KAPA2G Robust HotStart ReadyMix with primers (0.5 uM each) designed for the endogenous targets. PCR reaction was performed as follows: 95 oC 3 min; 95 oC 15 s, 66-N oC 15 s, 72 oC 40 s, for 8 cycles (N was cycle number); 95 oC 15 s, 60 oC 15 s, 72 oC 40 s, for 11 cycles; and 72 oC 1 min. The PCR product was purified with AMPure XP beads. 5 ng PCR product was re-amplified with the Nextera P5 and TruSeq P7 library index primers for 5 cycles.

treatment_protocol: T7-assisted reporter mapping: We randomly integrated a complex library of the synHEK3 reporters into a monoclonal K562 cell line constitutively expressing the PE2 prime editor. We further bottlenecked the pool to ~500 clones, each containing a unique combination of randomly inserted reporters, and performed T7-assisted reporter mapping on this population.  SynHEK3 amplicon sequencing for prime editing-mediated CTT insertion and Cas9 editing: the K562 synHEK3 pool was transfected with the pU6-Sp-pegRNA-HEK3-insCTT plasmid or Cas9 RNP using the SF Cell Line 4D-Nucleofector X kit L (Lonza).  CRISPRoff experiments: On Day 0, 2 x 106 clone 5 cells were transfected with CRISPRoff-v2.1 (3,000 ng), pU6-Sp-dual-gRNA (1,000 ng) and pmax-GFP (500 ng, Lonza) using the SF Cell Line 4D-Nucleofector X kit L (Lonza). On Day 2, cells were sorted based on high GFP expression (top 20%) on a flow cytometer and expanded. On Day 11, 4 x 105 cells were transfected with the pU6-Sp-pegRNA-HEK3-insCTT plasmid (1,000 ng) using the SF Cell Line 96-well Nucleofector Kit (Lonza). Program code was FF-120.  CRISPRa experiments: On Day 0, 4 x 105 K562 dCas9-VP64 cells were transfected with PB-CMV-MCP-XTEN80-p65-Rta-3xNLS-P2A-T2A-mPlum (600 ng) and paired pU6-Sp-gRNA-2XMS2 (200 ng each) plasmids targeting the same promoter. On Day2, 4 x 105 cells from the previous transfection were transfected with pCMV-PEmax (Addgene: 174820)11 or  PB-PEmax-P2A-mCherry-EF1a-Puro (800 ng) and the pU6-Sp-pegRNA plasmids (400 ng). All transfections were performed using the SF Cell Line 96-well Nucleofector Kit (Lonza) with program FF-120.  For prime editing in shRNA treated cells: we transduced 2 monoclonal line (Clone 3 and Clone 5) individually with lentiviral shRNAs against candidate genes. After 2 days of doxycycline treatment to induce shRNA and GFP expression. For each shRNA, we pooled the 2 clones and transfected the cells with pegRNAs to induce 6-bp insertions. Cells were sorted into GFP positive and negative populations. Amplicon sequencing were performed on the sorted cells.

value: Wild type

value: CRISPRa, EGFR


title: Prime editing library of endogenous genes after CRISPRa [K562-IL2RB-Rep2-XLP166]

value: CRISPRa, IL2RB


title: Prime editing library of endogenous genes after CRISPRa [K562-EGFR-Rep1-XLP167]


title: Prime editing library of endogenous genes after CRISPRa [K562-CDKL5-Rep2-XLP169]

value: CRISPRa, CDKL5


title: Prime editing library of endogenous genes after CRISPRa [K562-EGFR-Rep1-XLP169]


title: Prime editing library of endogenous genes after CRISPRa [K562-CDKL5-Rep1-XLP169]


title: Prime editing library of endogenous genes after CRISPRa [K562-EGFR-Rep2-XLP169]


title: Prime editing library of endogenous genes after CRISPRa [K562-CXCR4-Rep1-XLP166]

value: CRISPRa, CXCR4


title: Prime editing library of endogenous genes after CRISPRa [K562-CXCR4-Rep2-XLP166]


title: Prime editing library of endogenous genes after CRISPRa [K562-CDKL5-Rep1-XLP167]


title: Prime editing library of endogenous genes after CRISPRa [K562-CDKL5-Rep2-XLP167]


title: Prime editing library of endogenous genes after CRISPRa [K562-IL2RB-Rep1-XLP166]


title: Prime editing library of endogenous genes after CRISPRa [K562-IL2RB-Rep2-XLP165]


title: Prime editing library of endogenous genes after CRISPRa [K562-IL2RB-Rep1-XLP165]


title: Prime editing library of endogenous genes after CRISPRa [K562-CXCR4-Rep2-XLP165]


title: RNA sequencing of K562 clone 5, CRISPRoff [K562-SC1-5-OFF-METTL2A-Rep1]

description: Stranded mRNA library


title: Cas9 editing library in synHEK3 reporters (Day 4) [K562-HEK3-Cas9-Day4-XL281_S72]

description:
synHEK3 amplicon sequencing library
K562-HEK3-Cas9-Day4-XL281_S72_barcode_edit.txt
sg1_top_alleles_count.txt
sg1_top_alleles_info.txt


title: Prime editing library of endogenous genes after CRISPRa [K562-CXCR4-Rep1-XLP165]


title: Cas9 editing library in synHEK3 reporters (Day 1) [K562-HEK3-Cas9-Day1-XL281_S68]

description:
synHEK3 amplicon sequencing library
K562-HEK3-Cas9-Day1-XL281_S68_barcode_edit.txt


title: Prime editing library of CTT insertions in synHEK3 reporters [K562-HEK3-insCTT]

description:
synHEK3 amplicon sequencing library
K562-HEK3-insCTT-DMSO_count.txt


title: Cas9 editing library in synHEK3 reporters (Day 2) [K562-HEK3-Cas9-Day2-XL281_S70]

description:
synHEK3 amplicon sequencing library
K562-HEK3-Cas9-Day2-XL281_S70_barcode_edit.txt


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-12H]

description: sci-RNA-seq3 library

data_processing:
For the sci-RNA-seq3 transcriptome libraries, sequencing reads from the Illumina NextSeq platforms were first demultiplexed based on PCR index using the bcl2fastq software. Reads were filtered based on RT and ligation index, by allowing <2 hamming distance from reference. Filtered reads were trimmed with TrimGalore (v0.6.6) with parameters: -a AAAAAAAA --three_prime_clip_R1 1. Reads were then aligned to the GRCh38 reference genome using STAR (v2.7.6a). PCR duplicates were collapsed using the RT index, ligation index, UMI sequence and end coordinate of reads. Reads were further demultiplexed based on the combination of the RT, ligation and PCR index and split into files for individual cells. To generate gene expression count matrices, reads were assigned to the exonic and intronic region of closest genes with HTseq (v.2.0.2). Reads with ambiguous assignments were discarded. Cells were further filtered based on total UMI (> 100) and mitochondrial reads percentage (<10%). Cells with the number of features detected between 10% and 90% percentile of all cells were kept and considered high-quality cells. The single cell analysis was performed using the Seurat (v4.0.0) package in R.
For capture libraries of the synHEK3 and shRNA constructs, sequencing reads from the Illumina NextSeq platforms were first demultiplexed based on PCR index using the bcl2fastq software. A custom script was used to further demultiplex and filter the synHEK3 and shRNA libraries based on the combination of RT, ligation and PCR index (cell ID), by allowing <2 hamming distance from reference. For the synHEK3 library, reporter barcodes (<2 hamming distance to the reference barcode set), prime editing outcomes and read UMIs were extracted. For the shRNA library, shRNA sequences (<2 hamming distance to the reference barcode set) and read UMIs were extracted. UMIs <3 hamming distance were collapsed.   A series of pre-processing steps were applied to the data. 1) We matched cell IDs between the sci-RNA-seq3 transcriptome libraries and the capture libraries, and only kept high-quality cells (number of features between 10% and 90% percentile). 2) For the shRNA library, cells with <3 or >200 UMIs or >20 shRNA were removed. 3) For the synHEK3 library, cells with <4 UMIs were first removed. We counted UMIs for synHEK3 reporters belonging to the two clones and calculated a clone UMI/total UMI ratio. If the ratio was >80% for a specific clone, the cell is assigned to the corresponding clone. Cells with extremely high UMIs (cutoff between 100 and 250 UMIs, depending on library complexity and sequencing depth of the plate) were also removed for downstream analysis. 4) For a cell-synHEK3 barcode combination, if multiple insertion sequences were observed, sequences <3 hamming distance were collapsed. Cell-synHEK3 barcode pairs having conflicting editing outcomes were discarded. 5) A cell-synHEK3 barcode-editing outcome-shRNA matrix was generated for a common set of cells identified by the shRNA and synHEK3 libraries.
Assembly: GRCh38
Supplementary files format and content: cell_synHEK3_shRNA.csv.gz contains a cell-synHEK3-shRNA capture matrix

extract_protocol: We performed a modified version of sci-RNA-seq3 (Martin et al., 2022). In brief, K562 cells were lysed with 5 mL of a hypotonic, PBS-based lysis buffer containing DEPC. The resulting nuclei were fixed with 4 volume of ice-cold methanol. After rehydrating and washing the nuclei carefully in a sucrose/PBS/triton buffer (SPBST), the nuclei were treated with T7 polymerase in an NTP mix buffer at 37oC for 30 min. Then nuclei were washed, sonicated briefly and counted. The nuclei were distributed to a 96-well plate for reverse transcription, with a cocktail of three indexed primers: oligo-dT, HEK3 and CS1 -- specific for the cellular transcriptome, and RNAs produced from the synHEK3 reporter and the shRNA constructs, respectively. After reverse transcription, nuclei were pooled, washed in SPBST and redistributed to 4 fresh plates for ligation of the second index primer with T4 DNA ligase. Nuclei were then again pooled, washed, and redistributed to 4 final plates at two different concentrations (1300 cell per well for 2 plates and 2500 cell per well for 2 plates) for second strand synthesis and extraction. Nuclear lysates were split in half, with one half undergoing normal sci-RNA-seq3 transcriptome library preparation (tagmentation and PCR to add the third index) and the other half subjected to indexed enrichment PCR for both the synHEK3 and shRNA constructs. Products were pooled by PCR plate, size-selected and sequenced on an Illumina Nextseq 2000 or Nextseq 550.

treatment_protocol: Two monoclonal K562 PE2-Puro lines (clone 3 and clone 5) containing multiple randomly integrated chromatin sensors for prime editing (synHEK3) were transduced with Lenti-rtTA-P2A-Blast viruses and selected in 10 ug/mL Blasticidin for 7 days. Monoclonal lines were derived and further transduced with a DNA damage response gene-focused lentiviral shRNA library containing 304 hairpins targeting 76 genes. Cells were transdcued at either low MOI (<1) or high MOI (~10) and selected in 800 ug/mL Geneticin for 7 days. For preparing sci-RNA-seq3 libraries, at the RT step, the low MOI cells are placed in the first 6 columns of the 96-well plate, while the high MOI cells were placed in the 7-12 columns of the 96-well palte. Low MOI and High MOI transduced cells can be distiguished by RT indices.

value: lentiviral transduction of a DDR-focused shRNA library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-12F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-12C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-12B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-12A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-12E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-11H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-11G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-11C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-11E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-11F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-12G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-10G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-10D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-10H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-10E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-11A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-11D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-12D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-11B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-10C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-10A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-10B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-09G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-09H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-09E]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-09D]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-09C]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-08F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-09B]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-08H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-09A]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-10F]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-08G]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-07H]

description: sci-RNA-seq3 library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-07G]

description: sci-RNA-seq3 library


title: RNA sequencing of K562 clone 5, CRISPRoff [K562-SC1-5-OFF-USP7-Rep1]

description: Stranded mRNA library


title: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate2500-3_TX_P1-08D]

description: sci-RNA-seq3 library