title: brain cortex, PHF6 IP

description:
Phf6_vs_IgG_peaks.bed
Phf6_vs_IgG_summits.bed

data_processing:
Raw reads were aligned to the mouse genome assembly version mm10 withbowtie2(version 2.3.4.1) using the“--very-sensitive-local” mode.
Duplicate reads were removed usingsamtools(version 1.9).
ChIP-seq peaks were identified usingMACS(version 1.4) with a permissive p-value threshold of 0.001, using “--nomodel” option. Fragment size was specified using “--shiftsize” argument, with the fragment length obtained by cross-correlation analysis usingphantompeakqualtools.
Bigwig files were created from BAM files using bamCoverage (version 3.5.1, --binSize 10).
Assembly: mm10
Supplementary files format and content: bed

extract_protocol:
developing cortex of mouse embryos at embryonic day 17 to 18 was extracted for ChIP-seq
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

value: homogenous mix

tag: chip antibody

value: Novus Biological, NB100-68262


title: Yumm3.3_ActA_12H_R4

status: Public on Feb 07 2024

type: SRA

city: Lausanne

first: Giovanna

last: Ambrosini

address: Station 19

country: Switzerland

institute: EPFL

library_source: transcriptomic

data_processing:
Sequencing data were demultiplexed using the BRB-seq tools suite from Deplancke Lab (https://github.com/DeplanckeLab/BRB-seqTools)
Raw reads were aligned to the mouse (GRCm39) genome using STAR (2.7.10b)
Gene counts were generated using FeatureCounts and differential expression analysis was performed with the DESeq2 (v.1.36.0) package from Bioconductor (v3.15)
Assembly: mm39
Supplementary files format and content: raw_counts.txt

molecule: total RNA

organism: Mus musculus

source_name: skin

growth_protocol: YUMM3.3 mouse melanoma cells were maintained in DMEM/F12 (Gibco) medium supplemented with 10% fetal bovine serum, 50 µg/mL gentamicin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 1% GlutaMAX (Gibco).

extract_protocol:
Total RNA was extracted using Quant-iT™ RiboGreen™ RNA Assay Kit from Invitrogen R11490
Transcriptomes were prepared using MERCURIUS BRB-seq kit library

treatment_protocol: YUMM3.3 cells were seeded in 6 well plates in quadruplicates at density of 0.5x106 and treated with 20 ng/ml IFN-γ and 40 ng/ml Activin-A individually or together for 4 or 12 hrs.

tag: tissue

value: skin

tag: cell line

tag: cell type

value: mouse melanoma cell line

tag: treatment

value: ActA

tag: time


title: cortical progenitors, siCtl

city: Ottawa

first: Arezu

last: Jahani-Asl

state: Ontario

address: 451 Smyth Road

department: Department of Cellular and Molecular Medicine

country: Canada

institute: University of Ottawa

data_processing:
RNA-seq raw reads were mapped to mm10 genome usingHISAT2, followed by duplicate read removal usingsamtools.
Gene-level read counts were obtained byHTSeq, using gene annotations from GENCODE (release M9).
Bigwig files were created from BAM files using bamCoverage (version 3.5.1, --binSize 10).
Assembly: mm10
Supplementary files format and content: TAB delimited file with counts

source_name: brain cortex

extract_protocol:
Cortical progenitors were established from the cortex and were maintained in culture for 5 days to expand the homogeneous population prior to submitting samples for mRNA-Seq.
RNA-Seq libraries were constructedfrom total RNA with the TruSeq RNA v2 kit (Illumina).

value: brain cortex

value: progenitor cells

value: cortical progenitors

tag: genotype

value: WT

value: siCtl

tag: Sex

value: M


title: brain cortex, IgG CTL

library_source: genomic

molecule: genomic DNA

tag: age

value: Cell Signaling, #3900S


title: PNIBD55_C, colonic tissue

city: Chapel Hill

first: Terrence

last: Furey

state: NC

address: 7320 Medical Biomolecular Research Building, CB# 7032, 111 Mason Farm Road

department: Center for Gastrointestinal Biology and Disease

country: USA

institute: University of North Carolina at Chapel Hill

data_processing:
Transcript expression was quantified using Salmon with default parameters.
Supplementary files format and content: Processed files are comma-separated, rows representing raw counts for genes (as gene symbols or as Ensembl ID's if no gene symbol available)

organism: Homo sapiens

source_name: colon

extract_protocol:
Macroscopically uninflamed mucosal samples from the ascending colon and terminal ileum were obtained at the time of initial iagnosis, before therapy was started. These samples were preserved as fresh frozen paraffin-embedded (FFPE) tissue. RNA was isolated from FFPE tissue using the Quick-RNA FFPE MiniPrep (Zymo Research, Irvine, CA). Total RNA was then purified using the MagMAX kit in the KingFisher system (ThermoFisher, Carlsbad, CA).
RNA-seq libraries were prepared using TruSeq Stranded Total RNA with Ribo-Zero (Illumina, San Diego, CA). Paired-end (50bp) sequencing was processed on the NovaSeq 6000 platform using default parameters (Illumina, San Diego, CA).

value: colon

value: F

tag: status

value: nonIBD

tag: diagnosis age

tag: location montreal

tag: batch

tag: b montreal

tag: perianal disease

tag: rectosigmoid involvement

tag: family history

tag: surgery

tag: remission


title: Yumm3.3_IFNy_12H_R2

tag: tissue

tag: cell line

value: IFN-gamma


title: RIAILs_Batch7_QX490

first: Gaby

last: El Alam

state: Vaud

address: Station 15

department: SV LISP

institute: École polytechnique fédérale de Lausanne

data_processing:
FastQC (default parameters) was used to verify the quality of the reads. No low-quality reads were present and no trimming was needed.
Alignment was performed against worm genome (WBcel235 sm-toplevel) following the STAR (version 2.73a) manual guidelines
Counts for each sample features (outputed by STAR) where read into R
The STAR gene-counts for each alignment were analyzed for differentially expressed genes using the R package DESeq2
Assembly: WBcel235 sm-toplevel
Supplementary files format and content: tab-delimited text files include raw mapped read counts per feature (generated by STAR - per strand and combined strands)

organism: Caenorhabditis elegans

growth_protocol: Worms of each strain were cultured on plates seeded with E. coli OP50, then eggs were obtained by alkaline hypochlorite treatment of gravid adults. A synchronized L1 population was obtained by incubating the egg suspension in M9 butter overnight at room temperature. Approximately 2000 L1 worms were transferred onto plates seeded with E. coli HT115. L4 worms were harvested after 50 h by three times of washing with M9 buffer. Tubes containing worm pellets were immediately submerged in liquid nitrogen for snap freezing and stored at -80ºC until use.

extract_protocol:
On the day of the extraction, 1 mL of TriPure Isolation Reagent was added to each tube. The samples were then frozen and thawed quickly eight times with liquid nitrogen and 37ºC water bath to rupture cell membranes. RNA was then extracted by using a column-based kit from Macherey-Nagel.
BGISeq-500 library construction Protocol

treatment_protocol: Synchronized worms from each strain were few with E. coli HT115

tag: strain

tag: treatment

value: Cntrl


title: Yumm3.3_ActA_IFNy_12H_R3

tag: cell type

tag: time


title: Yumm3.3_ActA_12H_R3


title: PNIBD49_C, colonic tissue

tag: Sex


title: PNIBD53_C, colonic tissue

tag: status


title: PB104_I, ileal tissue

source_name: ileum

value: ileum

value: CD

tag: diagnosis age


title: RIAILs_Batch7_QX571


title: RIAILs_Batch6_QX520


title: Yumm3.3_ActA_12H_R1

value: skin


title: RIAILs_Batch6_QX516


title: Yumm3.3_ActA_12H_R2

value: mouse melanoma cell line


title: PNIBD54_C, colonic tissue

tag: location montreal


title: PNIBD47_C, colonic tissue

tag: batch


title: PNIBD45_C, colonic tissue

tag: b montreal


title: RIAILs_Batch6_QX518


title: RIAILs_Batch7_QX495


title: PB101_I, ileal tissue

tag: perianal disease


title: PNIBD44_C, colonic tissue

tag: rectosigmoid involvement


title: RIAILs_Batch6_QX517


title: Yumm3.3_NT_12H_R3

value: NT


title: Yumm3.3_NT_12H_R2


title: PNIBD43_C, colonic tissue

tag: family history


title: RIAILs_Batch6_QX514


title: Yumm3.3_NT_12H_R1


title: RIAILs_Batch6_QX512


title: RIAILs_Batch7_CB4856


title: Yumm3.3_ActA_IFNy_4H_R4


title: RIAILs_Batch6_QX511


title: PNIBD42_C, colonic tissue

tag: surgery


title: PNIBD41_C, colonic tissue

tag: remission


title: Yumm3.3_ActA_IFNy_4H_R3

tag: tissue


title: Yumm3.3_NT_12H_R4

tag: cell line


title: PNIBD40_C, colonic tissue


title: Yumm3.3_ActA_IFNy_4H_R2


title: RIAILs_Batch6_QX509

tag: strain


title: PNIBD39_C, colonic tissue


title: Yumm3.3_ActA_IFNy_4H_R1

tag: treatment


title: PNIBD37_C, colonic tissue


title: RIAILs_Batch6_QX506


title: Yumm3.3_IFNy_4H_R4

tag: cell type


title: Yumm3.3_IFNy_4H_R3

tag: time


title: RIAILs_Batch6_QX505


title: RIAILs_Batch6_QX504


title: PNIBD36_C, colonic tissue

tag: Sex


title: PNIBD38_C, colonic tissue

tag: status


title: PNIBD35_C, colonic tissue

tag: diagnosis age


title: Yumm3.3_IFNy_4H_R2


title: PNIBD34_C, colonic tissue

tag: location montreal


title: Yumm3.3_ActA_4H_R4

value: ActA


title: Yumm3.3_IFNy_4H_R1


title: RIAILS_Batch6_N2


title: RIAILS_Batch6_CB4856


title: PNIBD33_C, colonic tissue

tag: batch


title: RIAILs_Batch4_QX564


title: Yumm3.3_ActA_4H_R3


title: RIAILs_Batch6_QX510


title: PNIBD30_C, colonic tissue

tag: b montreal


title: Yumm3.3_ActA_4H_R2


title: Yumm3.3_ActA_4H_R1


title: RIAILs_Batch4_QX563


title: PNIBD28_C, colonic tissue

tag: perianal disease


title: Yumm3.3_NT_4H_R4


title: RIAILs_Batch4_QX546


title: PNIBD26_C, colonic tissue

tag: rectosigmoid involvement


title: RIAILs_Batch4_QX544


title: Yumm3.3_NT_4H_R3


title: Yumm3.3_NT_4H_R2


title: RIAILs_Batch4_QX543


title: RIAILs_Batch6_QX500


title: PNIBD25_C, colonic tissue

tag: family history


title: PNIBD23_C, colonic tissue

tag: surgery


title: shFBXO38#2_3

city: shanghai

first: Yijia

last: Wu

address: 800 Dongchuan RD. Minhang District, Shanghai, China

country: China

institute: shanghai jiaotong university

data_processing:
Sequence reads were aligned to the reference Mus musculus, GRCh38 using STAR (2.7.0d).
With the cufflinks program and corresponding sequence reads data we estimated the expression level for each gene for Mus musculus.
hg38
Supplementary files format and content: tab-delimited text files include count values for each Sample

source_name: tissue

extract_protocol:
RNA samples were prepared using TRIzol reagent (Ambion,15596-026) following the manufacturer's protocol.
The library was qualified using the Agilent 2100 bioanalyzer and quantified by Qubit and qPCR. The produced libraries were sequenced on the illumina Novaseg 6000 platform.

value: tissue

tag: genotype

value: FBXO43 knockdown


title: PNIBD27_C, colonic tissue

tag: tissue

tag: remission


title: shFBXO38#2_2

tag: cell line

value: FBXO42 knockdown


title: RIAILs_Batch4_QX541

tag: strain

tag: treatment


title: PNIBD31_C, colonic tissue


title: PNIBD24_C, colonic tissue


title: PNIBD22_C, colonic tissue


title: PNIBD21_C, colonic tissue


title: RIAILs_Batch4_QX562


title: Yumm3.3_NT_4H_R1

tag: cell type

tag: time


title: shFBXO38#1_3

value: FBXO40 knockdown


title: PNIBD20_C, colonic tissue

tag: Sex

tag: status


title: RIAILs_Batch4_CB4856


title: RIAILs_Batch4_QX538


title: shFBXO38#1_1

value: FBXO38 knockdown


title: shFBXO38#1_2

value: FBXO39 knockdown


title: RIAILs_Batch3_QX560


title: PNIBD19_C, colonic tissue

value: colon

tag: diagnosis age

tag: location montreal


title: RIAILs_Batch4_QX542


title: PNIBD17_C, colonic tissue

value: F

tag: batch

tag: b montreal


title: shcontrol_2

value: WT


title: shcontrol_3


title: RIAILs_Batch4_QX540


title: RIAILs_Batch3_QX557


title: shFBXO38#2_1

value: FBXO41 knockdown


title: shcontrol_1


title: RIAILs_Batch3_QX556

value: Cntrl


title: PNIBD15_C, colonic tissue

value: nonIBD

tag: perianal disease

tag: rectosigmoid involvement


title: MEL Cells, 2000 nM Folic Acid, AICAR, 72hr, 3

city: Boston

first: Naama

last: Kanarek

state: Massachusetts

address: 61 Binney St Rm 1126, Enders Building

department: Pathology

institute: Boston Children's Hospital

data_processing:
quality control was performed via fastQC
Reads were mapped to the mouse or human transcriptome using STAR aligner
Transcript quantification was performed using RSEM
Transcript abundance was imported into R using tximport
differential gene expression analysis was performed using DeSeq2
Assembly: hg38 and mm10
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

source_name: MEL

growth_protocol: K562 and MEL were maintained in RPMI + 10% fetal bovine serum (FBS) with antibiotics in humidified incubator at 37C and 5% CO2. Two dyas prior to experiment start, K562 and MEL cells were switched to folate free RPMI + 10% dialyzed FBS + 2,000 nM folic acid with antibiotics.

extract_protocol:
Rnazol RT (Sigma) was used to harvest RNA from cell samples.
Azenta Life Sciences prepared libraries

treatment_protocol: At t=0, cells were plated at 100,000 cells / mL in folate free RPMI + 10% dialyzed FBS + 2,000 nM or 100 nM folic acid. Treatment was performed with or without SHIN1 (1.25 uM) or AICAR (500 nM).

value: MEL

value: Erythroid Leukemia Cells

value: 2000 nM folic acid + AICAR (500 nM)


title: RIAILs_Batch4_QX539


title: PNIBD14_C, colonic tissue

tag: family history

tag: surgery


title: RIAILs_Batch3_QX555


title: PNIBD13_C, colonic tissue

tag: tissue


title: RIAILs_Batch3_QX552


title: MEL Cells, 2000 nM Folic Acid, AICAR, 72hr, 1

tag: genotype


title: PNIBD12_C, colonic tissue

tag: remission


title: PNIBD18_C, colonic tissue

value: M


title: MEL Cells, 2000 nM Folic Acid, SHIN1, 72hr, 3

tag: cell line

value: 2000 nM folic acid + SHIN1 (1.25uM)


title: MEL Cells, 2000 nM Folic Acid, SHIN1, 72hr, 2

tag: treatment


title: RIAILs_Batch3_QX550

tag: strain


title: PNIBD11_C, colonic tissue


title: RIAILs_Batch3_QX548


title: PNIBD09_C, colonic tissue


title: PNIBD16_C, colonic tissue


title: RIAILs_Batch3_QX559


title: PNIBD08_C, colonic tissue


title: MEL Cells, 2000 nM Folic Acid, SHIN1, 72hr, 1

tag: cell type


title: MEL Cells, 100 nM Folic Acid, Vehicle, 72hr, 3

value: 100 nM folic acid + Vehicle


title: RIAILs_Batch3_QX554


title: MEL Cells, 100 nM Folic Acid, Vehicle, 72hr, 2


title: PNIBD07_C, colonic tissue

tag: Sex


title: RIAILs_Batch3_N2


title: RIAILs_Batch3_QX547


title: RIAILs_Batch3_CB4856


title: PNIBD06_C, colonic tissue

tag: status


title: RIAILs_Batch2_QX594


title: PNIBD05_C, colonic tissue

tag: diagnosis age


title: PNIBD03_C, colonic tissue

tag: location montreal


title: MEL Cells, 100 nM Folic Acid, Vehicle, 72hr, 1


title: MEL Cells, 2000 nM Folic Acid, Vehicle, 72hr, 3

value: 2000 nM folic acid + Vehicle


title: MEL Cells, 2000 nM Folic Acid, AICAR, 72hr, 2


title: RIAILs_Batch2_QX589


title: PNIBD02_C, colonic tissue

tag: batch


title: RIAILs_Batch2_QX588


title: MEL Cells, 2000 nM Folic Acid, Vehicle, 72hr, 2


title: PB319_C, colonic tissue

value: CD

tag: b montreal


title: PB318_C, colonic tissue

tag: perianal disease


title: RIAILs_Batch2_QX531


title: KP tumor-2

city: Shanghai

first: Meng

last: Qiao

address: No.507 Zhengmin Road, Yangpu District

institute: Shanghai Pulmonary Hospital

data_processing:
Raw data (raw reads) of fastq format were processed through in-house perl scripts of Novogene Co., Ltd (https://cn.novogene.com/). In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the edgeR R package (3.22.5). The P values were adjusted using the Benjamini & Hochberg method. Corrected P-value of 0.05 and absolute foldchange of 2 were set as the threshold for significantly differential expression.
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.20.0). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include FPKM counts for each Sample

source_name: subcutaneous tumor

growth_protocol: A total of 2 × 106 KP or KL cells were suspended in 200 μL PBS and then injected subcutaneously into 6 week old male C57BL/6 mice in the right flank.

extract_protocol:
The mouse tumor tissue was harvested for total RNA extraction with TRIzol (Invitrogen). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations.
The RNA libraries for RNA-seq were generated with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs Inc)

treatment_protocol: The tumor volumes were measured by caliper and calculated with a formula of π/6 × long diameter × short diameter × short diameter. Mice were sacrificed when tumor volume reached 700-800mm3. ce were sacrificed when tumor volume reached 700-800mm3. Tumors from KP or KL cell-bearing mice were excised for further analysis.

value: subcutaneous tumor

value: tumor cell

value: KRAS-mutated and TP53 knockdown

value: when tumor volume reached 700-800mm3


title: KP tumor-3


title: RIAILs_Batch2_QX530


title: KP tumor-1


title: PB317_C, colonic tissue

value: colon

tag: rectosigmoid involvement


title: MEL Cells, 2000 nM Folic Acid, Vehicle, 72hr, 1

value: WT


title: PB314_C, colonic tissue

tag: family history


title: RIAILs_Batch2_CB4856


title: PB308_C, colonic tissue

value: F

tag: surgery


title: PB311_C, colonic tissue

tag: tissue


title: RIAILs_Batch2_QX528

value: Cntrl


title: DIFF_miR-NC_H3

city: Brussels

first: Océane

last: Perdaens

address: avenue Mounier 53

country: Belgium

institute: UCLouvain

data_processing:
Raw reads of fastq format were processed through fastp software. Clean reads were obtained by removing reads containing adapter or ploy-N and low quality reads from raw data. Q20, Q30 and GC content of the clean data were calculated. Sample DIFF_NC_H2 was excluded as it appeared to be an outlier on principal component analysis, masking the effect of every other sample.
Paired-end clean reads were aligned to the improved rat reference genome (mRatBN7.2) using HISAT2 v2.0.5. The mapped reads of each sample were assembled by StringTie (v1.3.3b).
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Differential expression analysis of two conditions/groups was performed using the DESeq2 R package (1.20.0).
Gene Ontology enrichment analysis of differentially expressed genes was implemented with the cluster Profiler R package, with correction of gene length bias.
The p-values were adjusted by the Benjamini and Hochberg’s approach for controlling the false discovery rate. Differentially expressed genes (DEG) and enriched GO terms were significant if the adjusted p-value was ≤ 0.05
Assembly: mRatBN7.2
Supplementary files format and content: xlsx files containing one sheet with the gene information, the gene count and gene fpkm of each sample separately
Supplementary files format and content: xlsx files containing several sheets with the differentially expressed genes (DEG) or enriched Gene Ontology (GO) terms of each microRNA vs each control (on grouped replicates) (all data included, both significant and not significant)

organism: Rattus norvegicus

growth_protocol: CG-4 cells were passaged every 3-4 days in poly-L-lysine coated flasks in a proliferation medium (DMEM with 100 UI/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin,  supplemented with 30% B104 conditioned medium, 50 µg/mL human apo-transferrin, 9.8 ng/mL biotin, 40 ng/mL sodium selenite, 5 µg/mL human insulin and 10 mM Hepes)

extract_protocol:
For RNA extraction, TriPure Isolation Reagent (Roche) was added to the washed wells and the plates were stored for minimum 30 minutes at -80°C to help with the cell lysis. RNA was hereafter extracted following the manufacturer’s protocol except for the precipitation step, samples were stored for 2h in isopropanol at -20°C to help with RNA precipitation before being centrifuged.
For library preparation, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first cDNA strand was synthesized using random hexamer primers, the second using either dUTP for directional library or dTTP for non-directional library, followed by end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion (for the directional library only), amplification, and purification. The libraries, quantified with Qubit and real-time PCR and verified by bioanalyzer for size distribution, were pooled and sequenced on Illumina NovaSeq 6000.

treatment_protocol: CG-4 cells were plated at a cell density of 250,000 cells per well in a pre-coated 6-well plate in a proliferation medium on D-1. On D0, the cells were transfected with the microRNA mimic (Qiagen, 40nM) using Lipofectamine 2000 (3 µl) for 6h.The medium was then replaced by the differentiation medium and the cells were cultured for an additional 48h before being washed (1x with DPBS) and collected for RNA extraction. (Differentiation medium: DMEM with penicillin-streptomycin, 1% GlutaMAX supplement, and supplemented with 5 µg/mL bovine insulin, 50 µg/mL human holo-transferrin, 2% B-27 supplement, 0.5% fetal bovine serum, 0.05 µg/mL recombinant rat ciliary neurotrophic factor, 1% OL-supplement and 10 mM Hepes)

value: oligodendrocyte progenitor cells

tag: genotype

value: differentiation medium + miRNA negative control (miRCURY LNA microRNA mimic, Qiagen)


title: KL tumor-1

value: KRAS-mutated and LKB1 knockdown


title: PB306_C, colonic tissue

tag: remission


title: KL tumor-2

tag: cell line


title: DIFF_miR-NC_H1

tag: treatment


title: YTHDF2 RIP rep3

city: Hefei

first: Ding

middle: feng

last: Li

address: Huang shan road 443

institute: USTC

description: YTHDF2 RIP RNA

data_processing:
Illumina Casava1.7 software used for basecalling.
Bowtie2 v2.2.3 was used for building the genome index, and Clean Data was then aligned to the reference genome using HISAT2 v2.1.0
Reads Count for each gene in each sample was counted by HTSeq v0.6.0
Assembly: mm10
Supplementary files format and content: tab-delimited txt files include normalized gene expression for each group

source_name: mPFC

growth_protocol: Standard protocol for mPFC for RNA-seq

extract_protocol:
Total RNA was extracted by Trizol.Total amount of 2 ¦Ìg RNA per sample was used as input material for the RNA
sample preparations. Sequencing libraries were generated using NEBNext? Ultra? RNA
Library  Prep  Kit  for  Illumina following the manufacturer¡¯s
recommendations  and  index  codes  were  added  to  attribute  sequences  to  each  sample"
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: strain

value: mPFC


title: YTHDF2 RIP rep2

description: YTHDF2 RIP RNA


title: DIFF_miR-214-3p_G3

tag: cell type

value: differentiation medium + miR-214-3p mimic (miRCURY LNA microRNA mimic, Qiagen)


title: PB227_C, colonic tissue

tag: Sex


title: PB305_C, colonic tissue

value: M

tag: status


title: RIAILs_Batch2_QX536


title: DIFF_miR-214-3p_G2


title: YTHDF2 RIP rep1

description: YTHDF2 RIP RNA


title: DIFF_miR-145-5p_F3

value: differentiation medium + miR-145-5p mimic (miRCURY LNA microRNA mimic, Qiagen)


title: DIFF_miR-214-3p_G1


title: YTHDF1+2 OE rep2


title: PB223_C, colonic tissue

tag: diagnosis age


title: YTHDF1+2 OE rep1


title: DIFF_miR-145-5p_F2


title: KL tumor-3


title: PB222_C, colonic tissue

tag: location montreal


title: DIFF_miR-145-5p_F1


title: 22M insoluble rep3


title: PB303_C, colonic tissue

tag: batch


title: PB216_C, colonic tissue

tag: b montreal


title: DIFF_miR-124-5p_E3

value: differentiation medium + miR-124-5p mimic (miRCURY LNA microRNA mimic, Qiagen)


title: PB218_C, colonic tissue

value: CD

tag: perianal disease


title: 22M insoluble rep2


title: PB210_C, colonic tissue

tag: rectosigmoid involvement


title: 22M insoluble rep1


title: PB209_C, colonic tissue

tag: family history


title: Optic nerve, crush, 3 months, mouse 5

city: Saint Louis

first: David

last: Gutmann

state: MO

address: Box 8111, 660 S Euclid Ave

department: Neurology

institute: Washington University in St. Louis School of Medicine

data_processing:
Partek Flow v 10.0
Sequence reads quantified to assembly mm10, annotation model Ensembl release 102 using Partek Flow (default parameters)
Median normalization conducted using Partek Flow to generate normalized gene count matrix
Assembly: mm10
Supplementary files format and content: tab-delimited text file containing gene count matrix (features in row, samples in columns)

source_name: optic nerve

extract_protocol:
RNA was isolated from the optic nerves of transcardially perfused mice using the NucleoSpin® RNA Plus kit (Takara-740984.205) following manufacturer's instructions. 10ng of total RNA was used for sequencing library construction.
RNA libraries were constructed using using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer's protocol.

value: optic nerve

tag: time

value: 3 months

value: crush


title: YTHDF1+2 OE rep3

tag: tissue


title: Optic nerve, crush, 3 months, mouse 6

tag: genotype


title: PB207_C, colonic tissue

tag: surgery

tag: remission


title: Optic nerve, crush, 3 months, mouse 4

tag: treatment


title: DIFF_miR-34c-5p_D3

tag: cell line

value: differentiation medium + miR-34c-5p mimic (miRCURY LNA microRNA mimic, Qiagen)


title: PB206_C, colonic tissue

value: colon


title: DIFF_miR-34c-5p_D2

tag: cell type

value: WT


title: PB205_C, colonic tissue

tag: Sex


title: DIFF_miR-34c-5p_D1


title: PB204_C, colonic tissue

tag: status


title: DIFF_miR-33-3p_C3

value: differentiation medium + miR-33-3p mimic (miRCURY LNA microRNA mimic, Qiagen)


title: Optic nerve, sham, 3 months, mouse 2

value: sham


title: Optic nerve, sham, 3 months, mouse 3


title: PB150_C, colonic tissue

value: F

tag: diagnosis age


title: DIFF_miR-33-3p_C2

value: oligodendrocyte progenitor cells


title: DIFF_miR-33-3p_C1


title: Optic nerve, sham, 3 months, mouse 1


title: DIFF_miR-124-5p_E2


title: PB201_C, colonic tissue

tag: location montreal


title: PB149_C, colonic tissue

tag: batch


title: DIFF_miR-124-5p_E1


title: DIFF_Vehicle_Control_B2

value: differentiation medium + vehicle (Lipofectamine 2000)


title: PB148_C, colonic tissue

tag: b montreal


title: E15.5 brain, female, KO3

city: Setagaya

first: Shiori

last: Kinoshita

state: Tokyo

department: Department of Maternal-Fetal Biology

country: Japan

institute: National Research Institute for Child Health and Development

data_processing:
All 150 bp paired-end reads were trimmed to a length of 75 bp using FASTX-toolkit (v0.0.14) to 103 ensure the same analytical conditions as the 75 bp paired-end reads.
Reads with adapter sequences were trimmed by cutadapt (v.2.6).
Reads were aligned to the mouse genome (mm10) using HISAT2 (v.2.1.0).
Transcripts were assembled and quantified using Stringtie (v.2.0.6).
Gene counts matrix were generated by the Python script “prepDE.py” to extract read count information from StringTie output.
Assembly: mm10
Supplementary files format and content: Gene counts matrix from Stringtie in comma-separated file format.

source_name: brain

extract_protocol:
Total RNA was extracted from E15.5 brain in WT and Nsd2 KO mice using AllPrep DNA/RNA Mini Kit (QIAGEN).
Ribosomal RNA was depleted using NEBNext rRNA Depletion Kit or Kit v2 (Human/Mouse/Rat) (New England Biolabs) and cDNA libraries were generated using NEBNext Ultra Ⅱ Directional RNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturer’s protocol.

value: brain

value: Nsd2 knockout

tag: developmental stage

value: female


title: E15.5 brain, female, KO1


title: E15.5 brain, female, KO2


title: PB146_C, colonic tissue

value: M

tag: perianal disease

tag: rectosigmoid involvement


title: DIFF_Vehicle_Control_B1


title: DIFF_Vehicle_Control_B3


title: PROL_Vehicle_Control_A3

value: proliferation medium + vehicle (Lipofectamine 2000)


title: PB142_C, colonic tissue

value: CD

tag: family history


title: PB145_C, colonic tissue

tag: tissue


title: E15.5 brain, female, WT2

tag: genotype


title: PROL_Vehicle_Control_A1


title: PROL_Vehicle_Control_A2


title: E15.5 brain, female, WT1


title: 21_CC_24_V_Rep3

city: Jena

first: Sascha

last: Schäuble

address: Beutenbergstraße 11a

country: Germany

institute: Leibniz Institute for Natural Product Research and Infection Biology

description:
Dual-RNAseq
CcCa_Calbicans_counts.xls
CcCa_Calbicans_counts_mrn.xls
CcC2_Hsapiens_counts.xls
CcC2_Hsapiens_counts_mrn.xls

data_processing:
quality control using multiQC (v1.7)
quality trimming of raw data using trimmomatic (v0.36)
mapping using HiSat2 (v2.1.0)
read counts using featureCounts (v1.28.0)
Assembly: Homo sapiens: Ensembl_GRCh38; C_albicans_SC5314_version_A22
Supplementary files format and content: *counts.xls

organism:
Candida albicans
Homo sapiens

source_name: IECs (C2BBe1)

growth_protocol: The intestinal epithelial Caco-2 brush border expressing 1 cell line (C2BBe1; ATCC, CRL2102)  was cultivated and differentiated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum , 10µg/ml Holotransferrin , and 1% non-essential amino acids at 37°C with 5% CO2 for 12 d in collagen-coated 6-well plates (5×10^5 cells/well). Candida albicans overnight cultures were cultured for 16 h in YPD broth (1% yeast extract, 2% peptone, 2% ᴅ-glucose) at 30°C with shaking at 180 rpm.

extract_protocol:
At each respective time point, the medium was removed, unadhered Candida cells were washed away, and 650 µl of RLT buffer (RNeasy Minikit; Qiagen) was added to each well. The plates were frozen with liquid nitrogen and thawed at room temperature (RT). Each well was scraped and the suspensions were centrifuged for 8 min (20,000×g) to pellet the Candida cells. The supernatant was removed and the host RNA was isolated using the RNeasy Minikit (Qiagen) according to the manufacturer’s instructions.
After poly(A) enrichment, mRNA was fragmented and random-primed cDNA synthesis was performed followed by adaptor ligation and adaptor-specific PCR amplification. Single sequence reads of 50 bp were produced.

treatment_protocol: Differentiated intestinal epithelial cells (IECs) were infected with 2×10^6 Candida cells/well and incubated at 37°C and 5% CO2 in DMEM medium for 0.75, 3, 6, 12, or 24 h. In parallel, C2BBe1 and C. albicans cells were cultured individually in 6-well plates in DMEM medium. Prior to infection, individual C. albicans and C2BBe1 samples were harvested (0h).

tag: strain

value: IECs (C2BBe1)

value: C.alb. + C2BBe1 co-incubation (infection)

tag: time

tag: oxygen condition

value: normoxic


title: LPS-induced ALI

city: Guangzhou

first: chen

middle: wei

last: fen

address: 4F, Building 1, Standard Property Unit 3, International Biotech Island

institute: Forevergen

description: RNA-seq

data_processing:
Illumina CASAVA 1.8 software used for basecalling.
Low quality which have more than 20% low base quality bases or adapter sequence contamination reads were discarded. Then high quality reads were aligned to the reference genome using BWA mem alignment module. CircRNA identify and characterize was used the CIRI2 software based on the following process. First, reads aligning contiguously to the genome without any trimming were discarded. The remaining reads were used for circRNA candidate detection. Next, terminal sequences (anchors) were extracted and re-aligned independently to find unique anchor positions. Anchors that aligned in the reverse orientation (head-to-tail) indicated circRNA splicing (back-splicing).
The following criteria were used to filter the resulting splicing events: 1) GT/AG signal flanking the splice sites; 2) unambiguous breakpoint detection; 3) no more than two mismatches in the extension procedure; 4) the breakpoint reside a maximum of two nucleotides inside an anchor; 5) less than two independent reads support the head-to-tail splice junction.
Counts of reads mapping across an identified back-splice are normalized by number of reads mapping (spliced tags reads per million mapping, TPM), which allows expression comparisons between two circRNAs. Differential expression analysis of two samples was performed using the previous statistics model. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. CircRNA with an adjusted P-value <0.05 were assigned as differentially expressed.
Assembly: rn6

source_name: lung

growth_protocol: A total of 12 SD rats (~ 300g) purchased from Guangdong Medical Laboratory Animal Center were randomly assigned to 2 groups (sham and LPS group), receiving sham (PBS) treatment or LPS (2 mg/kg, Sigma, MO, USA) induction through intratracheal spray. Rats were then returned to the cages and were free to food and clean water. After 24h, rats were anesthetized and killed. Lung tissues were ressected for subsequently experiments. All the animal experiments were approved by Ethics Committee of Sun Yat-sen University.

extract_protocol:
Total RNA from the cells was isolated using the Trizol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions, followed by a DNase I digestion for 30 minutes to remove genomic DNA
Total RNA was treated with the RiboMinus kit (Life Technologies) to remove the ribosomal RNAs; RNAs were incubation with 16 units RNase R (Sigma–Aldrich, St. Louis, MO, USA) for 15 min at 37℃ to deplete linear and enrich circRNAs; The remaining RNAs were fragmented and reverse-transcribed using random primers and reverse transcriptase; Second strand cDNA synthesis was completed using DNA polymerase I and RNase H. Sixth, the Illumina series KAPA Library Preparation Kit (Kapa Biosystems) was used to perform end repair, dA-tailing, and adapter ligation; The dUTP-marked second strand was digested at 37 °C for 30 min using Uracil Specific Excision Reagent (USER) enzyme (New England Biolabs); The cDNA was enriched by PCR amplification to create the final cDNA libraries.

value: lung

tag: treatment

value: LPS-induced ALI


title: PB139_C, colonic tissue

value: colon

tag: surgery

tag: remission


title: 21_CC_24_II_Rep2

tag: cell line


title: PB140_C, colonic tissue

tag: Sex

tag: status

tag: diagnosis age


title: PB136_C, colonic tissue

tag: location montreal

tag: batch

tag: b montreal


title: Sham Lung

description: RNA-seq

value: Sham


title: PB135_C, colonic tissue

tag: perianal disease

tag: rectosigmoid involvement


title: 21_CC_24_IV_Rep1


title: PB132_C, colonic tissue

tag: tissue

tag: family history


title: 21_CC_12_V_Rep3

tag: strain


title: 21_CC_12_II_Rep2

tag: time


title: 21_CC_12_IV_Rep1

tag: oxygen condition


title: PB131_C, colonic tissue


title: HT_3

city: Nijmegen

first: Ivo

last: Rieu

address: Heyendaalseweg 135

department: Radboud Institute for Biological and Environmental Sciences

country: Netherlands

institute: Radboud University

data_processing:
Reads were trimmed for adapter sequences by Novogene
Reads were  filtered against low quality reads and reads with adapters by Novogene, based on the following criteria: (1)Remove reads with adaptor contamination, (2)Remove reads when uncertain nucleotides constitute more than 10 percent of either read (N > 10%), (3)Remove reads when low quality nucleotides (Base Quality less than 5) constitute more than 50 percent of the read.
Reads were mapped against the reference genome by Novogene using the HISAT2 algorithm
Read count was normalised by Novogene using the using the DESeq2 R package
Assembly: TAIR10
Supplementary files format and content: Excel file (.xlsx), contains FPKM for each sample
Supplementary files format and content: Excel file (.xlsx), contains DESeq2-normalised readcounts for each sample

molecule: polyA RNA

organism: Arabidopsis thaliana

source_name: root tip

growth_protocol: Seeds were sown on solid agar medium containing 0.5X Murashige and Skoog salts with Gamborg B5 (Duchefa, Haarlem, The Netherlands, 1 % (w/v) sucrose, 0.05 % (w/v) MES monohydrate and 1 % (w/v) plant agar (Duchefa), pH5.7. Seeds on plates were stratified for 2 days at 4 °C in the dark and then transferred to a climate chamber to grow vertically with long-day period (16 h light / 8 h dark, fluorescent tubes) at 22 °C.

extract_protocol:
Total RNA was isolated using RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
RNA libraries were prepared from mRNA using standard protocols (no amplification, the first strand of cDNA was synthesized using random hexamer primers and the second strand of cDNA was synthesized with dUTP to generate a directional library)

treatment_protocol: For heat treatment, plates with 5-day-old seedlings were transferred to 30 °C. For control treatment, plates remained at 22 °C.

value: root tip

tag: genotype

tag: treatment

value: 48 hours at 30 degrees Celcius


title: HT_4


title: PB130_C, colonic tissue

value: F

tag: surgery

tag: remission


title: PB129_C, colonic tissue

tag: Sex


title: HT_2


title: 21_CC_6_V_Rep3

tag: cell line

value: 6 h


title: CT_4

value: 48 hours at 22 degrees Celcius


title: CT_2


title: PB125_C, colonic tissue

tag: status

tag: diagnosis age


title: 21_CC_6_II_Rep2


title: E15.5 brain, female, WT3

value: brain

value: WT

tag: developmental stage


title: PB122_C, colonic tissue

tag: location montreal

tag: batch

tag: b montreal


title: HT_1

tag: tissue


title: 21_CC_3_V_Rep3

value: 3 h


title: 21_CC_45_II_Rep2

value: 45 min


title: 21_CC_6_IV_Rep1


title: CT_1


title: PB120_C, colonic tissue

value: CD

tag: perianal disease

tag: rectosigmoid involvement

tag: family history


title: 21_CC_45_IV_Rep1

tag: strain

value: IECs (C2BBe1)

tag: time

tag: oxygen condition


title: PB126_C, colonic tissue

value: M

tag: surgery


title: 21_Ca_C2_24_V_Rep3

tag: treatment

value: individual C.alb. and C2BBe1 samples combined for dual-RNAseq

value: normoxic


title: PB124_C, colonic tissue

tag: Sex

tag: remission


title: 21_CC_3_II_Rep2

tag: cell line

value: C.alb. + C2BBe1 co-incubation (infection)


title: CT_3

tag: genotype


title: PB123_C, colonic tissue

value: colon

tag: status

tag: diagnosis age


title: PB115_C, colonic tissue

tag: location montreal

tag: batch


title: 21_CC_3_IV_Rep1


title: Human transdiferentiated astrocytes, CMV-Null, Control 3

city: Madison

first: Marcelo

last: Vargas

state: WI

address: 600 Highland Avenue

institute: UW-Madison

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using Trimmomatic v.0.36
Trimmed sequence reads were mapped to GRCh38 using the STAR aligner v.2.5.2b
Read count extraction was performed using Subread Package v.1.5.2 and differential gene expression analysis was performed using DESeq2
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include count values for each sample

source_name: Human iPSC-derived astrocytes

growth_protocol: An induced pluripotent stem cell (iPSC) line from healthy control subjects  (iPSC ID# FA0000011) was obtained from a comercial vendor. iPSCs were differentiated into induced neural progenitor cells (NPCs) using an embryoid body formation protocol as we previously described (PMID: 33508151). Astrocyte differentiation was achieved by culturing induced NPCs for 3 weeks in astrocyte differentiation media (STEMdif Astrocyte Differentiation Media, Stemcell), followed by 3 more weeks in astrocyte maturation media (STEMdif Astrocyte Maturation Media, Stemcell). Differentiation of NPCs into astrocytes was confirmed by assessing GFAP, S100B, and ALDH1L1 gene expression. Following differentiation, iPSC-derived astrocytes (iAs) were maintained in complete astrocyte medium from ScienCell.

extract_protocol:
RNA was harvested using TriReagent. 0.5 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using Illumina Stranded mRNA Prep.

treatment_protocol: Confluent iAs cultures were transduced with adenovirus vectors expressing wild-type mouse FABP7 or a Null orf under a CMV promoter. Cultures were transduced at a multiplicity of infection of 50.

tag: cell type

value: Human iPSC-derived astrocytes

tag: disease state

value: Non-diseased

value: Adenovirus CMV-Null transduction

value: 72hs-post tranduction


title: PB116_C, colonic tissue

tag: tissue

tag: b montreal

tag: perianal disease

tag: rectosigmoid involvement


title: 21_CC_45_V_Rep3

tag: strain

tag: time

tag: oxygen condition


title: Human transdiferentiated astrocytes, CMV-Fabp7-2

tag: treatment

value: Adenovirus CMV-Fabp7 transduction


title: Human transdiferentiated astrocytes, CMV-Fabp7-3


title: 21_Ca_C2_24_II_Rep2


title: PB121_C, colonic tissue

tag: Sex

tag: family history

tag: surgery

tag: remission


title: Human transdiferentiated astrocytes, CMV-Fabp7-1


title: Human transdiferentiated astrocytes, CMV-Null, Control 4


title: M2 THP-1_m6A [IP_MIL-4+IL-13_2]

city: Camperdown

first: RENHUA

last: SONG

state: NSW

address: Charles Perkins Centre - D17, Level 4 West. The University of Sydney Johns Hopkins Drive

country: Australia

institute: Centenary Institute

data_processing:
Raw reads were trimmed to remove adaptor sequences and low-quality reads using Trimmomatic with the default setting. Read quality was assessed using FastQC. Clean reads were aligned to the human reference genome hg38 (ENSEMBL version 86) using the STAR aligner.
To minimise the rate of false positives, only uniquely mapped reads were selected using samtools.
Peaks enriched in immunoprecipitated over corresponding input samples were called as expected peaks using MACS2. Peaks enriched in input samples were called as observed (background) peaks using MACS2.
Peaks identified in both biological replicates were merged using the mergePeaks command in the HOMER software and overlapping peaks were mapped to the RefSeq gene annotation using intersectBed from BEDTools.
Enriched m6A motifs were identified using de novo motif search with the HOMER software (version 4.9.1). Motifs with the most significant P-values were visualised using WebLogo. DESeq2 (108) was used to identify m6A peak levels that were significantly different (1.5-fold) between two samples with a Benjamini-Hochberg correction (p <0.05).
bigWig files were generated using the deepTools bamCoverage by --normalizeUsing BPM and binsize=1. narrowPeak files were generated using MACS v2 with the parameters of -q 0.05.
Assembly: hg38
Supplementary files format and content: bigWig, narrowPeak
Library strategy: m6A-IP-Seq

source_name: THP! M2

growth_protocol: 5 µg of DNAse treated total RNA were subjected to fragmentation with ZnCl2 incubation at 70°C for 3min. Following precipitation, fragmented RNA size distribution was assessed using RNA 6000 Nano Bioanalyzer kit (Agilent).

extract_protocol:
Following extensive washing, RNA was eluted from the beads using RLT buffer and RNeasy mini kit (Qiagen). RNA was quantified using RNA 6000 Pico Bioanalyzer kit (Agilent).
library preparation was performed using the SMARTER Stranded Total RNA Seq kit v2-Pico Input Mammalian kit (Takara Bio) following the manufacturer’s instructions. Libraries were then sequenced using HiSeq2500 (Illumina) and a minimum of 20 million paired-end reads were obtained per sample. This experiment was performed in two replicates for each sample.

treatment_protocol: Approximately 500ng of sample were stored as input control. Fragmented RNA was subjected to two rounds of m6A immunoprecipitation for 2 hours each using an anti-m6A antibody (Merck) previously conjugated to protein-A magnetic beads (Thermo Fisher Scientific) and of protein-G magnetic beads (Thermo Fisher Scientific) by incubation at 4°C for at least 6 hours.

tag: cell line

value: THP-1 M2-like cells (polarised)

tag: antibody

value: an anti-m6A antibody (Merck)


title: 21_Ca_C2_12_V_Rep3


title: Human transdiferentiated astrocytes, CMV-Null, Control 2


title: Human transdiferentiated astrocytes, CMV-Null, Control 1


title: 21_Ca_C2_12_II_Rep2


title: M2 THP-1_m6A [IP_MIL-4+IL-13_1]


title: PB111_C, colonic tissue

tag: status

tag: diagnosis age

tag: location montreal

tag: batch


title: 21_Ca_C2_24_IV_Rep1


title: 21_Ca_C2_12_IV_Rep1


title: M2 THP-1_m6A [Input_MIL-4+IL-13_1]

tag: cell type


title: M2 THP-1_m6A [Input_MIL-4+IL-13_2]


title: PB114_C, colonic tissue

tag: tissue

tag: b montreal

tag: perianal disease


title: 21_Ca_C2_6_II_Rep2

tag: strain

tag: time

value: 6 h


title: PB108_C, colonic tissue

value: F

tag: rectosigmoid involvement


title: M1 THP-1_m6A [IP_MLPS+IFN-γ_1]

source_name: THP! M1

value: THP-1 M1-like cells (polarised)


title: PB107_C, colonic tissue

tag: Sex

value: CD


title: M1 THP-1_m6A [Input_MLPS+IFN-γ_2]


title: PB113_C, colonic tissue

tag: family history


title: 21_Ca_C2_6_IV_Rep1

value: IECs (C2BBe1)

tag: treatment

tag: oxygen condition


title: M1 THP-1_m6A [Input_MLPS+IFN-γ_1]

tag: cell line


title: PB112_C, colonic tissue

value: M

tag: surgery

tag: remission


title: 21_Ca_C2_3_II_Rep2

value: individual C.alb. and C2BBe1 samples combined for dual-RNAseq

value: 3 h


title: PB104_C, colonic tissue

tag: status

tag: diagnosis age


title: PB110_C, colonic tissue

tag: location montreal

tag: batch


title: 21_Ca_C2_6_V_Rep3

value: normoxic


title: M1 THP-1_m6A [IP_MLPS+IFN-γ_2]

tag: cell type

tag: antibody


title: 17525N

city: Bethesda

first: Joshua

last: Stone

state: Maryland

address: 37 Convent Drive Room 3050

institute: NIH

data_processing:
The sequencing quality of each sample was assessed using FastQC.
Read- and alignment-level quality were assessed using FastQ Screen, Preseq, Picard tools, RSeQC and QualiMap.
Reads were trimmed for adapter sequences and low-quality bases using Cutadapt prior to alignment against the human reference genome, hg38, using STAR in two-pass mode with GENCODE v19 annotation
Gene expression levels were quantified using TPM.
Assembly: hg38

source_name: esophageal tissue

extract_protocol:
Total RNA was extracted from frozen lung tissues using TRIzol (Invitrogen) according to the manufacturer’s protocol. Only RNA samples with RNA sufficient RNA were included for analysis.
Libraries suitable for paired-ended sequencing were generated using Illumina’s TruSeq Stranded Total RNA Library Prep Kit.

tag: tissue

value: Non-tumor


title: Mϕ THP-1_m6A [IP_Mϕ_1]

source_name: THP1 Mϕ

value: THP-1-derived macrophage


title: 21_Ca_C2_3_IV_Rep1

tag: strain


title: 17304N


title: 21_Ca_C2_3_V_Rep3

tag: time


title: 21_Ca_C2_45_II_Rep2

value: 45 min


title: 17300N


title: Mϕ THP-1_m6A [Input_Mϕ_2]


title: PB106_C, colonic tissue

value: colon

tag: b montreal

tag: perianal disease

tag: rectosigmoid involvement


title: 16592BF

value: Barretts esophagus


title: 21_Ca_C2_45_IV_Rep1

tag: treatment


title: PB105_C, colonic tissue

tag: Sex

tag: family history


title: Mϕ THP-1_m6A [IP_Mϕ_2]

tag: cell line


title: Mo THP-1_m6A [IP_Mo_1]

source_name: THP-1 Mo

value: Monocytic cell line


title: 17823N


title: Hela cells, shControl

first: Nestor

last: Zhang

address: Dongchuan Road

institute: Shanghai Jiao Tong University

data_processing:
Sequence reads were aligned to the reference human genome, GRCh38 using STAR (2.7.0d).
With the cufflinks program and corresponding sequence reads data we estimated the expression level for each gene for Homo sapiens.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: cell line

extract_protocol:
RNA was isolated with EZ-press RNA Purification Kit (B0004D, EZBioscience) and submitted to the Personalbio Laboratory for stranded library preparation.
Sequencing libraries were generated by using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) and the quality was assessed on the Agilent Bioanalyzer 2100 system. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.

value: cell line

value: Hela

value: human cervical cancer cell line

tag: genotype

value: WT


title: 21_Ca_C2_0_II_Rep2

value: 0 h

tag: oxygen condition


title: 15752T

source_name: esophageal tumor

value: Tumor


title: 21_Ca_C2_0_IV_Rep1

value: IECs (C2BBe1)


title: PB103_C, colonic tissue

source_name: colon

value: M

tag: status

value: CD

tag: diagnosis age

tag: surgery

tag: remission


title: Mϕ THP-1_m6A [Input_Mϕ_1]

tag: cell type

tag: antibody


title: 17206T

tag: tissue


title: Hela cells, shFBXO38 3

value: FBXO38 knockdown


title: Mo THP-1_m6A [IP_Mo_2]

value: an anti-m6A antibody (Merck)


title: 21_Ca_C2_0_V_Rep3

tag: strain

value: individual C.alb. and C2BBe1 samples combined for dual-RNAseq

tag: time

value: normoxic


title: HCT116 cells, shControl

value: human colorectal cancer cell line


title: M2_ THP-1_Riboseq [MIL-4+IL-13_3]

data_processing:
Truseq3-PE adapter and poor-quality sequences were assessed using FastQC were trimmed by Trimmomatic using the default settings.
Trimmed reads were then aligned to the hg38 (ENSEMBL version 86) reference genome using STAR.
FeatureCounts was subsequently employed to convert aligned short reads into read counts for each sample.
The data were then analysed using R and DESeq2.
Differentially enriched mRNAs undergoing translation in the polysomes were identified using Wald statistical test, with fold-change > 1.5 and p <0.05 after Benjamini-Hochberg correction.
Expressed genes were identified as those with RPKM greater than 1 for at least one group of samples. Differentially expressed genes between two groups were identified using Wald statistics, with fold-change > 1.5 and p <0.05 after Benjamini-Hochberg correction.
Assembly: hg38
Supplementary files format and content: bigWig, txt (count table)
Library strategy: Ribosome profiling

source_name: THP-1 M2

growth_protocol: cell lysates in CHAPS buffer containing RNAse inhibitors were separated by size exclusion chromatography on a Thermo Dionex BioRs UHPLC and Agilent SEC-5 7.8 x 300 mm HPLC column with 2000Å pores and 5 mm particles.

extract_protocol:
Polysome fractions were harvested in TRIzol LS reagent (10296028, Invitrogen) and RNA was extracted following the manufacturer’s protocol.
RNA-Seq was then performed on RNA from the polysome-bound fraction.

value: THP-1 M2-like cells (polarised)


title: tps1 pistil day +1, Rep 2

city: West Lafayette

first: Ryan

last: Patrick

state: Indiana

address: 625 Agriculture Mall Dr

institute: Purdue University

description:
tps1_dp1_02
pistil_tps1_counts.txt

data_processing:
Adapters were trimmed using Cutadapt and reads were aligned to the Petunia axillaris v.1.6.2 genome using TopHat2. Counts were assigned to genome features using htseq-count.
Assembly: Petunia axillaris v1.6.2
Supplementary files format and content: a tab-delimited file with normalized gene counts for P. axillaris genes

organism: Petunia x hybrida

source_name: pistil

extract_protocol:
Total RNA was extracted from wild type and PhTPS1 pistils collected at 14:00hr on day -1 and +1 postanthesis using the Spectrum Plant Total RNA Kit (Sigma-Aldrich).
PolyA library prep and sequencing was performed by the Purdue Genomics Core Facility at Purdue University.

tag: cultivar

value: Mitchell

value: pistil

value: day +1


title: M2_ THP-1_Riboseq [MIL-4+IL-13_2]


title: tps1 pistil day +1, Rep 1

description:
tps1_dp1_01
pistil_tps1_counts.txt


title: K562 - IgG

city: Stanford

first: Thomas

last: Koehnke

state: CA

address: 265 Campus Drive

institute: Stanford University

description: CUT&RUN sequencing IgG Control of K562 cells

data_processing:
Library strategy: CUT&RUN
ATAC-Seq: Read trimming and quality control was performed using TrimGalore (https://doi.org/10.5281/zenodo.5127899) and reads were aligned to the GRCh38 reference genome using bowtie2 (Langmead and Salzberg, 2012) and the --very-sensitive option. Aligned reads were converted to bam format, sorted, deduplicated, mitochondrial reads removed and indexed using SAMTools (Li et al., 2009) and Picard (http://broadinstitute.github.io/picard). ATAC-Seq reads at TSSs were quantified and differential testing was performed using csaw and edgeR for R (Chen et al., 2016; Lun and Smyth, 2016).
RNA-Seq: Reads were trimmed and quality control was performed using TrimGalore (https://doi.org/10.5281/zenodo.5127899). The cDNA index was built using Kallisto Bustools (Melsted et al., 2021) using gene annotations downloaded from Ensembl (release 104) and the GRCh38 reference genome. Pseudo-alignment was performed using Kallisto (Bray et al., 2016).
CUT&RUN: After quality control and read trimming using TrimGalore (https://doi.org/10.5281/zenodo.5127899), reads were aligned to the GRCh38 reference genome using bowtie2 (Langmead and Salzberg, 2012) using the following settings: --dovetail --very-sensitive --local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 2000. Alignment was converted to bam format, sorted and indexed using SAMTools (Li et al., 2009) and Picard (http://broadinstitute.github.io/picard). BigWig files were generated using deeptools2 (Ramírez et al., 2016).
Genome_build: GRCh38
Supplementary_files_format_and_content: ATAC-Seq abundance for TSS regions was generated with csaw (version 1.24.3) for R (version 4.0.2) and represent normalized abundance. Differential testing was performed with edgeR (version 3.32.1) for R (version 4.0.2).
Supplementary_files_format_and_content: Kallisto abundance estimates were generated using Kalisto (version 0.44.0) and represent abundance estimates per transcript
Supplementary_files_format_and_content: BigWig files were generated using deeptools2 (version 3.3.1). Scores represent normalized abundance.

source_name: ATCC

growth_protocol: K562 cells were cultured in IMDM supplemented with 10% FBS

extract_protocol:
100,000 cells were washed twice in CUT&RUN wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM Spermidine, supplemented with Roche EDTA-free cOmplete Protease Inhibitor). Next, activated Concanavalin A-coated magnetic beads (BioMagPlus Concanavalin A, Bang Laboratories, IN, USA) were added to the cell suspension and cells were allowed to bind to the beads for 10 min at room temperature under gentle agitation. After washing, the cells/bead mixture was resuspended in antibody buffer (consisting of wash buffer supplemented with 0.1% digitonin, 2 mM EDTA and 0.5 % BSA) and H2AK119Ub antibody (Clone D27C4) or IgG control (Clone DA1E, both Cell Signaling Technologies, MA, USA) at a 1:50 dilution and incubated with gentle agitation at 4°C overnight. After washing twice to remove unbound antibody, Protein A/G-MNase fusion protein (Epicypher, NC, USA) was added to the cell/bead mixture and allowed to bind for 1h at 4°C. Cells were washed twice to remove unbound antibody, resuspended in wash buffer supplemented with 0.1% digitonin, allowed to cool to 0° C in a chilled ice block before adding CaCl2 to a final concentration of 2 mM to initiate cleavage. After 30 min, the reaction was quenched with EDTA and EGTA and cleaved fragments were allowed to be released into the supernatant by incubating the cells at 37°C for 30 min.
Fragments were cleaned up using Monarch PCR & DNA Cleanup Kit (NEB, MA, USA), sequencing libraries were prepared using the Ovation Ultralow V2 DNA-Seq Library Preparation Kit (Tecan, Männedorf, Switzerland) according to the manufacturer's protocol.

tag: cut&run antibody

value: IgG Control (Cell Signaling Technology, Clone DA1E)


title: 21_Ca_C2_45_V_Rep3

tag: treatment

value: 45 min


title: Hela cells, shFBXO38 2

value: cell line

value: Hela

value: human cervical cancer cell line


title: 13922BO

value: Barretts esophagus


title: 16210T

value: Tumor


title: HCT116 cells, shFBXO38 2


title: Mo THP-1_m6A [Input_Mo_2]

value: Monocytic cell line


title: K562 - ASXL1:WT - H2AK119Ub - Rep3

description: CUT&RUN sequencing H2AK119Ub of K562 cells (corrected genotype ASXL1:WT)

value: H2AK119Ub (Cell Signaling Technology, Clone D27C4)


title: PB102_C, colonic tissue

first: Terrence

last: Furey

address: 7320 Medical Biomolecular Research Building, CB# 7032, 111 Mason Farm Road

institute: University of North Carolina at Chapel Hill

data_processing:
Transcript expression was quantified using Salmon with default parameters.
Supplementary files format and content: Processed files are comma-separated, rows representing raw counts for genes (as gene symbols or as Ensembl ID's if no gene symbol available)

extract_protocol:
Macroscopically uninflamed mucosal samples from the ascending colon and terminal ileum were obtained at the time of initial iagnosis, before therapy was started. These samples were preserved as fresh frozen paraffin-embedded (FFPE) tissue. RNA was isolated from FFPE tissue using the Quick-RNA FFPE MiniPrep (Zymo Research, Irvine, CA). Total RNA was then purified using the MagMAX kit in the KingFisher system (ThermoFisher, Carlsbad, CA).
RNA-seq libraries were prepared using TruSeq Stranded Total RNA with Ribo-Zero (Illumina, San Diego, CA). Paired-end (50bp) sequencing was processed on the NovaSeq 6000 platform using default parameters (Illumina, San Diego, CA).

value: colon

tag: Sex

value: F

value: CD

tag: location montreal

tag: batch

tag: b montreal

tag: perianal disease

tag: rectosigmoid involvement


title: Mo THP-1_m6A [Input_Mo_1]

tag: cell line


title: 15180N

value: Non-tumor


title: WT pistil day +1, Rep 2

description:
wt_dp1_02
pistil_tps1_counts.txt

tag: genotype

value: WT


title: K562 - ASXL1:WT - H2AK119Ub - Rep1


title: K562 - ASXL1:WT - H2AK119Ub - Rep2


title: 14303N


title: 14303T


title: HCT116 cells, shFBXO38 1

value: human colorectal cancer cell line

value: FBXO38 knockdown


title: M1_THP-1_Riboseq [MLPS+IFN-γ_1]

source_name: THP-1 M1

value: THP-1 M1-like cells (polarised)


title: M2_ THP-1_Riboseq [MIL-4+IL-13_1]

value: THP-1 M2-like cells (polarised)


title: WT pistil day +1, Rep 3

description:
wt_dp1_03
pistil_tps1_counts.txt

value: Mitchell

value: pistil

value: day +1


title: 13812T


title: 13732BO


title: K562 - ASXL1:Y591X - H2AK119Ub - Rep2

description: CUT&RUN sequencing H2AK119Ub of K562 cells (parental genotype ASXL1:Y591X)


title: Mϕ_THP-1_Riboseq [Mϕ_3]

value: THP-1-derived macrophage


title: tps1 pistil day -1, Rep 2

description:
tps1_dm1_02
pistil_tps1_counts.txt

value: day -1


title: Mϕ_THP-1_Riboseq [Mϕ_2]


title: 13850N


title: K562 - ASXL1:Y591X - H2AK119Ub - Rep1


title: tps1 pistil day -1, Rep 3

description:
tps1_dm1_03
pistil_tps1_counts.txt


title: tps1 pistil day -1, Rep 1

description:
tps1_dm1_01
pistil_tps1_counts.txt


title: M1_THP-1_Riboseq [MLPS+IFN-γ_3]


title: Mϕ_THP-1_Riboseq [Mϕ_1]


title: tps1 pistil day +1, Rep 3

description:
tps1_dp1_03
pistil_tps1_counts.txt


title: M1_THP-1_Riboseq [MLPS+IFN-γ_2]


title: K562 - ASXL1:Y591X - H2AK119Ub - Rep3


title: WT pistil day +1, Rep 1

description:
wt_dp1_01
pistil_tps1_counts.txt


title: 13367T

value: Tumor


title: CB-derived HSPCs - ASXL1 Trunc - RNASeq - Rep6

description: mRNA sequencing of edited cord-blood derived HSPCs

data_processing:
ATAC-Seq: Read trimming and quality control was performed using TrimGalore (https://doi.org/10.5281/zenodo.5127899) and reads were aligned to the GRCh38 reference genome using bowtie2 (Langmead and Salzberg, 2012) and the --very-sensitive option. Aligned reads were converted to bam format, sorted, deduplicated, mitochondrial reads removed and indexed using SAMTools (Li et al., 2009) and Picard (http://broadinstitute.github.io/picard). ATAC-Seq reads at TSSs were quantified and differential testing was performed using csaw and edgeR for R (Chen et al., 2016; Lun and Smyth, 2016).
RNA-Seq: Reads were trimmed and quality control was performed using TrimGalore (https://doi.org/10.5281/zenodo.5127899). The cDNA index was built using Kallisto Bustools (Melsted et al., 2021) using gene annotations downloaded from Ensembl (release 104) and the GRCh38 reference genome. Pseudo-alignment was performed using Kallisto (Bray et al., 2016).
CUT&RUN: After quality control and read trimming using TrimGalore (https://doi.org/10.5281/zenodo.5127899), reads were aligned to the GRCh38 reference genome using bowtie2 (Langmead and Salzberg, 2012) using the following settings: --dovetail --very-sensitive --local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 2000. Alignment was converted to bam format, sorted and indexed using SAMTools (Li et al., 2009) and Picard (http://broadinstitute.github.io/picard). BigWig files were generated using deeptools2 (Ramírez et al., 2016).
Genome_build: GRCh38
Supplementary_files_format_and_content: ATAC-Seq abundance for TSS regions was generated with csaw (version 1.24.3) for R (version 4.0.2) and represent normalized abundance. Differential testing was performed with edgeR (version 3.32.1) for R (version 4.0.2).
Supplementary_files_format_and_content: Kallisto abundance estimates were generated using Kalisto (version 0.44.0) and represent abundance estimates per transcript
Supplementary_files_format_and_content: BigWig files were generated using deeptools2 (version 3.3.1). Scores represent normalized abundance.

source_name: Human umbilical cord blood

growth_protocol: CD34+ HSPCs were cultured in low-density conditions (≤200,000 cells/mL), low oxygen conditions (5% O2), in StemSpan SFEM II (Stemcell Technologies, Vancouver, Canada) base media supplemented with 100ng/mL of TPO, SCF, FLT3L, IL-6 (Peprotech) and the small molecule UM-171 (35nM)

extract_protocol:
RNA was isolated from 100,000 edited CB-derived HSPCs using RNeasy Kits
mRNA was purified using oligo(dT) coupled magnetic beads and first strand cDNA synthesis was performed using random hexamers followed by second strand cDNA synthesis, end repair, A-tailing, adapter ligation, size selection and amplification

value: cord-blood derived HSPCs

value: ASXL1 Truncation


title: WT pistil day-1, Rep 2

description:
wt_dm1_02
pistil_tps1_counts.txt


title: WT pistil day-1, Rep 1

description:
wt_dm1_01
pistil_tps1_counts.txt


title: 13622BO

value: Barretts esophagus


title: CB-derived HSPCs - ASXL1 Trunc - RNASeq - Rep7


title: CB-derived HSPCs - ASXL1 Trunc - RNASeq - Rep4


title: 13041T


title: WT pistil day-1, Rep 3

description:
wt_dm1_03
pistil_tps1_counts.txt


title: CB-derived HSPCs - ASXL1 Trunc - RNASeq - Rep3


title: M2 THP-1_5hmC [5hmC_MIL-4+IL-13_3]

data_processing:
Rraw reads were trimmed to remove adaptor sequences and low-quality reads using Trimmomatic with the default setting. Read quality was assessed using FastQC. Clean reads were aligned to the human reference genome hg38 (ENSEMBL version 86) using the STAR aligner.
To minimise the rate of false positives, only uniquely mapped reads were selected using samtools.
Peaks enriched in immunoprecipitated over corresponding input samples were called as expected peaks using MACS2. Peaks enriched in input samples were called as observed (background) peaks using MACS2.
Peaks identified in both biological replicates were merged using the mergePeaks command in the HOMER software and overlapping peaks were mapped to the RefSeq gene annotation using intersectBed from BEDTools.
Enriched 5hmC motifs were identified using de novo motif search with the HOMER software (version 4.9.1). Motifs with the most significant P-values were visualised using WebLogo. DESeq2 (108) was used to identify 5hmC peak levels that were significantly different (1.5-fold) between two samples with a Benjamini-Hochberg correction (p <0.05).
bigWig files were generated using the deepTools bamCoverage by --normalizeUsing BPM and binsize=1. narrowPeak files were generated using MACS v2 with the parameters of -q 0.05.
Assembly: hg38
Supplementary files format and content: bigWig, narrowPeak
Library strategy: 5hmC-IP-Seq

growth_protocol: 1 mg of DNAse treated total RNA was chemically fragmented by incubating RNA in fragmentation buffer (10 mM Tris-HCl pH7, 100 mM ZnCl2) at 94°C for 40 seconds, the reaction was stopped by the addition of 50 mM EDTA. Fragmented RNA was ethanol precipitated and resuspended in nuclease-free water.

extract_protocol:
RNA was then incubated at 4C overnight with or without 12.5 μg of anti-5hmC antibody (Diagenode) in freshly prepared 1X Immunoprecipitation (IP) buffer (50 mM Tris-HCl pH 7.4, 750 mM NaCl and 0.5% Igepal CA-630, RNasin 400 U/ml and RVC 2 mM) supplemented with protease inhibitor (complete EDTA free, Roche). 60 μL of equilibrated Dynabeads Protein G (Invitrogen) were added to each sample and incubated for 2.5 hours at 4C. After three washes with 1X IP buffer, samples were eluted by addition of 1 mL of TriPure Reagent (Roche) as per manufacturer’s instructions.
6 pM of DNA library spiked with 1% PhiX viral DNA was clustered on cBot (Illumina) and then sequenced on a HiScanSQ module (Illumina).

treatment_protocol: Fragmentation efficiency was checked by running a Nano RNA Bioanalyzer chip (Agilent) on the Agilent 2100 Bioanalyzer. RNA was then incubated at 4C overnight with or without 12.5 μg of anti-5hmC antibody (Diagenode) in freshly prepared 1X Immunoprecipitation (IP) buffer (50 mM Tris-HCl pH 7.4, 750 mM NaCl and 0.5% Igepal CA-630, RNasin 400 U/ml and RVC 2 mM) supplemented with protease inhibitor (complete EDTA free, Roche). 60 μL of equilibrated Dynabeads Protein G (Invitrogen) were added to each sample and incubated for 2.5 hours at 4C. After three washes with 1X IP buffer, samples were eluted by addition of 1 mL of TriPure Reagent (Roche) as per manufacturer’s instructions.

value: anti-5hmC antibody (Diagenode)


title: Mo THP-1_Riboseq [Mo_3]

value: Monocytic cell line


title: 13460N

value: Non-tumor


title: CB-derived HSPCs - ASXL1 Trunc - RNASeq - Rep5


title: 13367N


title: CB-derived HSPCs - ASXL1 Trunc - RNASeq - Rep2


title: CB-derived HSPCs - ASXL1 Trunc - RNASeq - Rep1


title: YERS053, Resistant, Time 60 minutes, Replicate A

city: Frederick

first: Christopher

middle: Patrick

last: Stefan

state: MD

address: 1425 Porter Street

institute: USAMRIID

data_processing:
FASTQ files were imported into CLC Genomics workbench and analyzed using the RNA-Seq Analysis workflow. Reads were mapped using mapping setting: mismatch cost (2), insertion cost (2), deletion cost (3), length fraction (0.8), and similarity fraction (0.8). Paired reads were counted as two when calculating transcripts per million (TPM).
Assembly: GCF_000008445.1, GCF_000222975.1
Supplementary files format and content: Excel file containing raw TPM counts from CLC genomics workbench

organism: Yersinia pestis

source_name: culture

growth_protocol: Glycerol stocks were plated on sheep’s blood agar plates, grown overnight at 37°C, and selected for recovery the following day in tryptic soy broth (TSB). Cells were recovered in TSB for approximately 3 hours then diluted to a McFarland standard of 0.5 using the DensiChek Plus

extract_protocol:
2 volumes of RNAprotect Bacteria Reagent was added to each tube and incubated at room temperature for 5 minutes at indicated time points. For all reactions tubes were centrifuged at 5000 x g for 10 minutes. The supernatant was decanted, and pellets were extracted or stored at -80°C until processing. Pellets were resuspended in 100µl 1xTE containing 15 mg/ml lysozyme and 10 µl Proteinase K. Suspended pellets were incubated at room temperature for 10 minutes. Then, 700 µl of buffer RLT was added to suspended pellets along with approximately 100 µl of 0.5 mM glass beads and cells were incubated at 95°C for 5 minutes, bead beaten for 10 minutes, then centrifuged at max speed for 30 seconds. 760 µl of supernatant was added to new 2 ml Eppendorf tubes containing 590 µl of 80% ethanol. Samples were then extracted utilizing the RNeasy kit protocol and an on-column DNase treatment according to manufacturer’s protocols
Extracted RNA was quantified using the Qubit 2.0 Fluorometer and Qubit RNA HS assay. RNA was spiked with the ERCC control, an external RNA control, and input into the QIAseq FastSelect – 5s/16s/23s kit to remove bacterial ribosomal RNA prior to sequencing. Sequencing libraries were prepared using the QIAseq Stranded total RNA library kit according to manufacturer’s instructions. Libraries were quantified using the High Sensitivity D1000 kit on the 4200 Tapestation System. Four barcoded libraries were combined and diluted to a 4 nM library pool, denatured with NaOH, and further diluted to a final library concentration of 12 pM prior to sequencing. Pooled libraries were sequenced using the MiSeq Instrument and MiSeq reagent kit version 3–150 cycles.

treatment_protocol: One milliliter of diluted cells were treated with indicated concentrations of ciprofloxacin and incubated at 37°C for 60 minutes (or indicated time) in 5 ml Eppendorf tubes (Thermo Fisher Scientific) with constant agitation.

value: 60 minutes

tag: resistance

value: Ciprofloxacin resistant

value: 0.25 ng/ul Ciprofloxacin


title: 12997T


title: Mo THP-1_Riboseq [Mo_1]

tag: cell type


title: Mo THP-1_Riboseq [Mo_2]


title: 13460T


title: YERS053, Resistant, Time 60 minutes, Replicate C


title: 13008T


title: M2 THP-1_5hmC [INP_MIL-4+IL-13_3]

tag: antibody


title: YERS053, Resistant, Time 60 minutes, Replicate B


title: 13008N

tag: tissue


title: CB-derived HSPCs - AAVS1 - RNASeq - Rep5

value: AAVS1 control


title: YERS053, Resistant, Time 40 minutes, Replicate C

value: 40 minutes

tag: strain


title: M1 THP-1_5hmC [INP_MLPS+IFN-γ_3]

value: THP-1 M1-like cells (polarised)


title: M2 THP-1_5hmC [5hmC_MIL-4+IL-13_2]

value: THP-1 M2-like cells (polarised)


title: YERS053, Resistant, Time 20 mintues, Replicate C

tag: time

value: 20 minutes


title: M2 THP-1_5hmC [INP_MIL-4+IL-13_2]


title: CB-derived HSPCs - AAVS1 - RNASeq - Rep7


title: 12767BO


title: YERS053, Resistant, Time 40 minutes, Replicate B


title: M2 THP-1_5hmC [INP_MIL-4+IL-13_1]


title: CB-derived HSPCs - AAVS1 - RNASeq - Rep3


title: 12943T


title: 12943N


title: M2 THP-1_5hmC [5hmC_MIL-4+IL-13_1]


title: YERS053, Resistant, Time 40 minutes, Replicate A


title: 12672T


title: CB-derived HSPCs - AAVS1 - RNASeq - Rep4


title: YERS053, Resistant, Time 0 minutes, Replicate C

value: 0 minutes

value: 0 ng/ul Ciprofloxacin


title: CB-derived HSPCs - AAVS1 - RNASeq - Rep1


title: M1 THP-1_5hmC [5hmC_MLPS+IFN-γ_3]


title: YERS053, Resistant, Time 0 minutes, Replicate B


title: M1 THP-1_5hmC [INP_MLPS+IFN-γ_2]


title: YERS053, Resistant, Time 20 mintues, Replicate B


title: YERS053, Resistant, Time 20 mintues, Replicate A


title: 12997N


title: 12631N


title: M1 THP-1_5hmC [5hmC_MLPS+IFN-γ_2]


title: CB-derived HSPCs - AAVS1 - RNASeq - Rep2


title: CB-derived HSPCs - AAVS1 - RNASeq - Rep6


title: 12672N


title: 12841N


title: 11802N


title: CB-derived HSPCs - ASXL1 Trunc - H2AK119Ub - Rep3

description: CUT&RUN sequencing H2AK119Ub of edited cord-blood derived HSPCs

value: H2AK119Ub (Cell Signaling Technology, Clone D27C4)


title: M1 THP-1_5hmC [5hmC_MLPS+IFN-γ_1]


title: YERS053, Resistant, Time 0 minutes, Replicate A


title: 12631T

value: Tumor


title: CB-derived HSPCs - ASXL1 Trunc - H2AK119Ub - Rep2

value: cord-blood derived HSPCs

value: ASXL1 Truncation


title: Mϕ THP-1_5hmC [5hmC_Mϕ_2]

value: THP-1-derived macrophage

value: anti-5hmC antibody (Diagenode)


title: 11802BO

value: Barretts esophagus


title: Mϕ THP-1_5hmC [INP_Mϕ_3]


title: YERS052, Suceptable, Time 60 minutes, Replicate C

value: 60 minutes

value: Ciprofloxacin suceptable


title: 12306N

value: Non-tumor


title: 11738N


title: Mϕ THP-1_5hmC [5hmC_Mϕ_3]


title: CB-derived HSPCs - ASXL1 Trunc - ATAC-seq - Rep2.2

description: Bulk ATAC-seq of edited cord-blood derived HSPCs. Replicate 2

extract_protocol:
Nuclei were isolated from 5,000-50,000 cells resuspended in 50 µL of ATAC-seq resuspension buffer (RSB: 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water) with 0.1% NP40, 0.01% digitonin, and 0.1% Tween-20 and incubated on ice for three minutes. After lysis, 1 mL of ATAC-seq RSB with 0.1% Tween-20 was added and tubes were inverted six times to mix. Isolated nuclei were then spun down.
Transposition reactions were incubated at 37°C for 30 minutes in a thermomixer with shaking at 1000 rpm. Reactions were cleaned up using Qiagen MinElute Reaction Cleanup kits and amplified with a target concentration of 20 μl at 4 nM


title: M1 THP-1_5hmC [INP_MLPS+IFN-γ_1]


title: Mϕ THP-1_5hmC [INP_Mϕ_2]


title: YERS052, Suceptable, Time 60 minutes, Replicate A

value: 0.25 ng/ul Ciprofloxacin


title: Mϕ THP-1_5hmC [5hmC_Mϕ_1]


title: CB-derived HSPCs - AAVS1 - H2AK119Ub - Rep3

value: AAVS1 control


title: Mo THP-1_5hmC [INP_Mo_3]

value: Monocytic cell line


title: CB-derived HSPCs - AAVS1 - H2AK119Ub - Rep2


title: YERS052, Suceptable, Time 40 minutes, Replicate C

value: 40 minutes


title: CB-derived HSPCs - AAVS1 - H2AK119Ub - Rep1


title: CB-derived HSPCs - AAVS1 - ATAC-seq - Rep2.1

description: Bulk ATAC-seq of edited cord-blood derived HSPCs. Replicate 1


title: YERS052, Suceptable, Time 40 minutes, Replicate B


title: Mϕ THP-1_5hmC [INP_Mϕ_1]


title: CB-derived HSPCs - ASXL1 Trunc - H2AK119Ub - Rep1


title: YERS052, Suceptable, Time 60 minutes, Replicate B


title: CB-derived HSPCs - ASXL1 Trunc - ATAC-seq - Rep1.2


title: 12291T


title: YERS052, Suceptable, Time 40 minutes, Replicate A


title: 11455T


title: 11267N


title: CB-derived HSPCs - ASXL1 Trunc - ATAC-seq - Rep1.1


title: CB-derived HSPCs - ASXL1 Trunc - ATAC-seq - Rep2.1


title: YERS052, Suceptable, Time 20 mintues, Replicate C

value: 20 minutes


title: YERS052, Suceptable, Time 20 mintues, Replicate B


title: Mo THP-1_5hmC [5hmC_Mo_3]


title: Mo THP-1_5hmC [INP_Mo_2]


title: YERS052, Suceptable, Time 20 mintues, Replicate A


title: 11362N


title: 11267T


title: NIH-3T3 cells, methanol fixed cells, 800 µM 4sU, rep B

status: Public on Feb 06 2024

city: Würzburg

first: Kevin

last: Berg

address: Versbacher Str. 7

institute: University of Würzburg

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using cutadapt, reads with length < 18 were removed
Trimmed sequence reads were mapped to rRNA (U13369.1) and Mycoplasma database using bowtie2 (v. 2.3.0). Mapped reads were filtered out
Trimmed and filtered sequence reads were mapped to the mouse genome (ensembl 102) using STAR v. 2.7.10b (parameters: --outFilterMismatchNmax 20 --outFilterScoreMinOverLread 0.4 --outFilterMatchNminOverLread 0.4 --alignEndsType Extend5pOfReads12 --outSAMattributes nM MD NH –outSAMunmapped Within)
Subsequent analyses were conducted in R.
Assembly: mus musculus (ensembl 90)
Supplementary files format and content: Tab-separated values files
Library strategy: SLAM-seq

growth_protocol: NIH-3T3 cells were grown in DMEM supplemented with 100 IU/mL Penicillin and, 100 g/mL Streptomycin and supplemented with 10% NCS at 37°C, 5% CO2

extract_protocol:
Cells subject to 4sU-labeling were harvested using TRI reagent (Sigma) and RNA isolation was conducted using the Zymo DirectZol RNA-microprep kit (R2062) as described by the manufacturer and re-suspended in 1X PBS buffer
SLAM-seq conversion was performed in tubes or in methanol fixed cells. It was conducted using IAA to mediate U>C conversions at 50oC/30 minutes, conversion in “Tube” samples was done at 37°C/1h. The reaction was quenched using excess DTT. RNA was then purified using RNeasy Mini elute kit (Qiagen)3

treatment_protocol: Cells were treated with 200 or 800 µM 4sU for 2 hours.

value: fibroblast cells

value: WT

value: Methanol fixation, 800 uM 4sU

value: 2 hours

tag: protocol

value: SLAM-seq


title: Mo THP-1_5hmC [INP_Mo_1]


title: Mo THP-1_5hmC [5hmC_Mo_2]


title: YERS052, Suceptable, Time 0 minutes, Replicate C

value: 0 minutes

value: 0 ng/ul Ciprofloxacin


title: YERS052, Suceptable, Time 0 minutes, Replicate B


title: 10153N


title: NIH-3T3 cells, methanol fixed cells, no4sU, rep B

value: Methanol fixation, no4sU


title: CB-derived HSPCs - AAVS1 - ATAC-seq - Rep1.2

value: cord-blood derived HSPCs

tag: cut&run antibody


title: NIH-3T3 cells, in Tube, 200 µM 4sU, rep B

tag: treatment

value: 200 uM 4sU


title: NIH-3T3 cells, methanol fixed cells, no4sU, rep A


title: CB-derived HSPCs - AAVS1 - ATAC-seq - Rep2.2


title: Mo THP-1_5hmC [5hmC_Mo_1]

value: anti-5hmC antibody (Diagenode)


title: NIH-3T3 cells, in Tube, 800 uM 4sU, rep A

value: 800 uM 4sU


title: CB-derived HSPCs - AAVS1 - ATAC-seq - Rep1.1


title: NIH-3T3 cells, in Tube, no4sU, rep B


title: NIH-3T3 cells, in Tube, 200 µM 4sU, rep A


title: YERS052, Suceptable, Time 0 minutes, Replicate A

value: Ciprofloxacin suceptable


title: NIH-3T3 cells, in Tube, no4sU, rep A


title: NIH-3T3 cells, methanol fixed cells, 200 µM 4sU, rep B

value: Methanol fixation, 200 uM 4sU


title: NIH-3T3 cells, methanol fixed cells, 800 µM 4sU, rep A


title: TPL_WT_30m_input

city: Cambridge

first: Larry

last: Melidis

state: Not US or Canada

address: Robinson Way

department: CRUK-CI

country: United Kingdom

institute: University of Cambridge

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

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

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

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

value: Human embryonic kidney

tag: chip antibody


title: NIH-3T3 cells, methanol fixed cells, 200 µM 4sU, rep A


title: NIH-3T3 cells, in Tube, 800 uM 4sU, rep B


title: TPL_RNAPIIS5P_WT_30m_B3

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


title: TPL_RNAPIIS5P_WT_1h_B2


title: TPL_RNAPIIS5P_WT_2h_B1


title: TPL_RNAPIIS5P_WT_1h_B1


title: TPL_WT_0h_input


title: TPL_RNAPIIS5P_WT_0h_B1


title: TPL_WT_2h_input


title: TPL_BG4_WT_30m_B3


title: TPL_WT_1h_input


title: TPL_RNAPIIS5P_WT_30m_B2


title: TPL_BG4_WT_1h_B1


title: TPL_BG4_WT_2h_B1


title: TPL_BG4_WT_2h_B3


title: TPL_RNAPIIS5P_WT_2h_B3


title: TPL_BG4_WT_1h_B3


title: 11362T

value: Tumor


title: TPL_BG4_WT_0h_B3


title: TPL_RNAPIIS5P_WT_2h_B2


title: TPL_RNAPIIS5P_WT_0h_B3


title: TPL_RNAPIIS5P_WT_30m_B1


title: TPL_RNAPIIS5P_WT_0h_B2


title: TPL_RNAPIIS5P_WT_1h_B3


title: TPL_BG4_WT_0h_B1


title: TPL_BG4_WT_30m_B2


title: TPL_BG4_WT_1h_B2


title: TPL_BG4_WT_0h_B2


title: TPL_BG4_WT_2h_B2


title: SG4_SWP_Bio1_Tech2


title: SG4_WT_Bio2_Tech1


title: SG4_SWP_Bio3_Tech1


title: SG4_WT_Bio2_Tech2


title: SG4_SWP_Bio1_Tech1


title: SG4mut_R105A_WT_Bio1


title: SG4_WT_Bio1_Tech1


title: SG4_WT_Bio3_Tech1


title: SG4mut_R105A_SWP_Bio2


title: SG4mut_R105A_SWP_Bio1


title: SG4mut_R105A_SWP_Bio3


title: TPL_BG4_WT_30m_B1


title: SG4mut_R105A_MUT_Bio3


title: SG4_MUT_Bio3_Tech1


title: SG4mut_R105A_WT_Bio2


title: SG4_MUT_Bio1_Tech2


title: RNaseH_WT_Rloop

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

treatment_protocol: As R-loop CUT&Tag controls, RNase A and RNase H treatments were performed prior to primary antibody incubation. 10mg of RNase A (Takara) or 100 U RNase H (NEB, M0297S) in 50mL Dig-wash buffer were added to the bead-bound cells and incubated at 37°C for 40 min.

value: Anti-DNA-RNA Hybrid, clone S9.6, Cat. No. MABE1095


title: SG4_MUT_Bio1_Tech1


title: RNaseA_WT_Rloop


title: SG4_MUT_Bio2_Tech1


title: SG4mut_R105A_MUT_Bio1


title: RNaseA_SWP_Rloop


title: RNaseA_MUT_Rloop


title: RNaseH_SWP_Rloop


title: SG4_SWP_Bio2_Tech1


title: SG4_WT_Bio1_Tech2


title: RNAPIIS5P_SWP_Bio3


title: RNAPIIS5P_SWP_Bio2


title: SG4mut_R105A_MUT_Bio2


title: RNAPIIS5P_SWP_Bio1


title: RNAPIIS2P_SWP_Bio2

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


title: RNAPIIS2P_SWP_Bio3


title: Rloop_WT_Bio1_Tech2


title: Rloop_WT_Bio1_Tech1


title: Rloop_SWP_Bio3_Tech2


title: RNAPIIS2P_SWP_Bio1


title: Rloop_WT_Bio3_Tech2


title: Rloop_SWP_Bio3_Tech1


title: Rloop_WT_Bio2_Tech2


title: Rloop_WT_Bio3_Tech1


title: Rloop_WT_Bio2_Tech1


title: Rloop_SWP_Bio2_Tech2


title: RNaseH_MUT_Rloop


title: Rloop_SWP_Bio2_Tech1


title: MYCFlip_StrandSwapClonitoTech2

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


title: Rloop_SWP_Bio1_Tech2


title: Rloop_MUT_Bio3_Tech2


title: Rloop_MUT_Bio1_Tech1


title: Rloop_MUT_Bio2_Tech2


title: Rloop_SWP_Bio1_Tech1


title: FV_RNAPIIS2P_WT_30m_B3


title: MYCFlip_StrandSwapClonitoTech1

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


title: Rloop_MUT_Bio1_Tech2


title: FV_RNAPIIS2P_WT_0h_B2


title: FV_RNAPIIS2P_WT_0h_B3


title: FV_RNAPIIS2P_WT_2h_B1


title: FV_RNAPIIS2P_WT_30m_B2


title: FV_RNAPIIS2P_WT_2h_B3


title: FV_RNAPIIS2P_WT_1h_B1


title: FV_BG4_WT_2h_B3


title: FV_BG4_WT_30m_B1


title: FV_RNAPIIS2P_WT_30m_B1


title: FV_BG4_WT_2h_B1


title: FV_RNAPIIS2P_WT_0h_B1


title: FV_BG4_WT_30m_B2


title: FV_RNAPIIS2P_WT_2h_B2


title: FV_BG4_WT_1h_B3


title: FV_BG4_WT_1h_B1


title: FV_BG4_WT_0h_B1


title: FV_BG4_WT_30m_B3


title: FV_BG4_WT_1h_B2


title: FV_BG4_WT_2h_B2


title: FV_RNAPIIS2P_WT_1h_B3


title: Rloop_MUT_Bio2_Tech1


title: FV_BG4_WT_0h_B3


title: FV30m_input


title: Rloop_MUT_Bio3_Tech1


title: FV_BG4_WT_0h_B2


title: FV0h_input


title: FV2h_input


title: FV1h_input