title: S129,liver,HFD,EsrrgChIP-seq, rep3

city: Houston

first: Dongyin

last: Guan

address: 1 Baylor Plaza, ABBR

institute: Baylor College of Medicine

data_processing:
ChIPseq reads were aligned to mm10 using bowtie2 alignment software. Only one uniquely aligning read per genomic position was kept for further analysis. Peak calling was performed with HOMER (Heinz et al., 2010).The tag directories were established, and normalized read counts were measured using Homer.
Assembly: mm10
Supplementary files format and content: Processed data file format is .bed file contained all samples merged resize into 200bp peaks
Supplementary files format and content: UCSC genome browser tracks (bigwig files) for each samples.

extract_protocol:
ChIP experiments were performed as described (Guan et al. 2018) with minor changes. Mouse livers were harvested and cross-linked in 1% formaldehyde for 20min, followed by quenching with 1/20 volume of 2.5M glycine solution for 5 min, and two washes with 1×PBS. Nuclear extracts were prepared by Dounce homogenization in ChIP buffer (50mM Tris-HCl pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% NaDOC). Chromatin fragmentation was performed by sonication in lysis buffer (50mM Tris-HCL ph 8.0, 0.1%SDS, 10mM EDTA), using the Bioruptor (Diagenode). Proteins were immune-precipitated in ChIP buffer, and crosslinking was reversed overnight at 65°C in SDS buffer (50mM Tris-HCL, 10mM EDTA, 1% SDS, pH 8), and DNA isolated using phenol/chloroform/isoamyl alcohol.
DNA was amplified according to ChIP Sequencing Sample Preparation Guide provided by Illumina, using adaptor oligo and primers from Illumina, enzymes from New England Biolabs and PCR Purification Kit and MinElute Kit from Qiagen.

value: Liver

value: Esrrg

value: wild type

value: High-fat Diet

value: male


title: B6, Liver,NC,EsrrgKO

status: Public on Aug 25 2025

type: SRA

state: TX

country: USA

description: Library name: EsrrgChIPseq_B6_Esrrg_KO

library_source: genomic

data_processing:
ChIPseq reads were aligned to mm10 using bowtie2 alignment software. Only one uniquely aligning read per genomic position was kept for further analysis. Peak calling was performed with HOMER with B6 NC EsrrgKO as input(Heinz et al., 2010).The tag directories were established, and normalized read counts were measured using Homer.
Assembly: mm10
Supplementary files format and content: Processed data file format is .bed file contained all samples merged resize into 200bp peaks
Supplementary files format and content: UCSC genome browser tracks (bigwig files) for each samples.

molecule: genomic DNA

organism: Mus musculus

source_name: Liver

tag: tissue

tag: antibody

tag: strain

tag: genotype

value: Esrrg KO

tag: diet

value: Normal Chow

tag: Sex


title: S129,liver,HFD,EsrrgChIP-seq, rep2


title: Thalamus_Pcoat+MB-_Replicate1

city: Singapore

first: Benoit

last: Malleret

address: 5 Science Drive 2, Level 5, MD4-05-03C

department: Microbiology and Immunology

country: Singapore

institute: National University of Singapore, Yong Loo Lin School of Medicine

library_source: transcriptomic

data_processing:
The raw reads were aligned to the Refseq Macaca mulatta genome assembly (NCBI accession number: GCF_000772875.2_Mmul_8.0.1) using STAR aligner.
The reads were also quantified against transcript sequences obtained from the same Refseq assembly using Salmon in mapping-free (alignment-free) mode to quantify transcript abundance.
Transcript abundances were then summarized into gene abundances using the tximport R/Bioconductor package.
Normalized gene abundances were expressed in transcript per million (TPM) after trimmed mean of M values (TMM) normalization.
Assembly: GCF_000772875.2_Mmul_8.0.1
Supplementary files format and content: tab-delimited text file includes count, abundance, length, and counts from abundance for each sample

molecule: total RNA

organism: Macaca mulatta

source_name: Thalamus

extract_protocol:
Brain, heart, kidney and liver tissues were collected immediately post-mortem in multiple repeats of 1 mm2 squares and preserved in RNAlater (Sigma-Aldrich). Total RNA was isolated by homogenizing in TRIzol (Thermo Fisher Scientific) and performing double extraction with guanidinium thiocyanate-phenol-chloroform followed by a Qiagen RNeasy Micro clean-up procedure. 500 ng of total RNA was subjected to Ribo-Zero (Illumina) ribosomal RNA depletion, and used for construction of sequencing libraries.
Lexogen SENSE Total RNA-seq libraries were prepared according to the manufacturer’s protocol.

value: Thalamus

tag: age

tag: disease state

value: Pcoat+MB-

tag: treatment


title: S129,liver,HFD,EsrrgChIP-seq, rep1


title: Thalamus_Pcoat-MB-_Replicate3

value: Pcoat-MB-


title: S129,liver,NC,EsrrgChIP-seq, rep3


title: subject 1, non-inf, 14W

city: Tokyo

first: Mayu

last: Suzuki

address: 2-1-1 Nihonbashi-Honcho, Chuo-ku

department: Japan Medical Office

country: Japan

institute: Takeda Pharmaceutical Co., Ltd.

description:
Library name: IonXpress_013
IonXpress_013

data_processing:
Torrent Suite 5.12.1
Assembly: hg19
Supplementary files format and content: spreadsheet file with the .xls extension includes raw counts for each sample

organism: Homo sapiens

source_name: colonic mucosa

extract_protocol:
RNA samples were prepared using RNeasy Mini Kit (Qiagen).
AmpliSeq libraries were prepared using Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Themo Fisher Scientific) with 10 ng of RNA.

value: colonic mucosa


title: subject 1, inf, 54W

description:
Library name: IonXpress_017
IonXpress_017


title: B6, Liver,HFD,EsrrgChIP-seq, rep3


title: B6, Liver,HFD,EsrrgChIP-seq, rep2


title: subject 1, inf, 14W

description:
Library name: IonXpress_009
IonXpress_009


title: Thalamus_Pcoat-MB-_Replicate2


title: B6, Liver,HFD,EsrrgChIP-seq, rep1


title: subject 1, non-inf, 0W

description:
Library name: IonXpress_005
IonXpress_005


title: S129,liver,NC,EsrrgChIP-seq, rep1


title: Liver_Pcoat+MB+_17mg per kg_Replicate3

value: Pcoat+MB+

value: 17mg/kg methylene blue


title: B6, Liver,NC,EsrrgChIP-seq, rep1


title: subject 1, inf, 0W

description:
Library name: IonXpress_001
IonXpress_001


title: B6, Liver,NC,EsrrgChIP-seq, rep2


title: Liver_Pcoat+MB+_17mg per kg_Replicate2


title: B6, Liver,NC,EsrrgChIP-seq, rep3


title: input from flow-sorted chromosome 22

city: Kansas City

first: Tamara

last: Potapova

state: Missouri

institute: Stowers Institute for Medical Research

description: Library name: chr22

data_processing:
Reads were trimmed for adapters and low quality using trim galore with auto-detect adapters and a quality score cutoff of 20.
Alignment to chm13 (v2.0) using the bisulfite sequencing aligner Bismark (v0.24.0). End to end alignment was required (default).
Alignments were deduplicated and extracted into bed coverage format to give a C and T count at each reference position. Complementary CpGs on the two strands were merged into a single site. Positions were filtered by requiring coverage of 5 or more reads.
To assess rDNA alone, reads were aligned to the reference sequence KY962518.1 instead of chm13. Methylation extraction was performed to give bed coverage files with CpGs merged from both strands. Positions were later filtered by requiring coverage of 10 or more reads.
Assembly: chm13 v2.0
Supplementary files format and content: Bed coverage files for whole genome and KY962518.1 alignments
Supplementary files format and content: The columns are unlabelled but have the format of: chromosome, start position, end position, methylation percentage, count methylated, count unmethylated.
Library strategy: EM-seq

source_name: hydatidiform mole

extract_protocol:
Chromosomes were mitotically arrested then isolated with flow cytometry. Two rounds of sorting were used to increase purity. Flow cytometry data were collected and analyzed with FACSDiva (BD Bioscience) and FlowJo software (BD Bioscience).
Genomic DNA from sorted chromosomes was isolated using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s protocol. Methyl-seq libraries were generated from 2.4–6.6ng of DNA isolated from sorted chromosomes, as assessed using the Qubit Fluorometer (Life Technologies). Libraries were made according to the manufacturer’s directions for the NEBNext Enzymatic Methyl-seq kit (New England Biolabs, Cat. No. E7120S) using the S220 Focused-ultrasonicator (Covaris) to shear to 300bp and 9 cycles of library PCR amplification. Resulting short fragment libraries were checked for quality and quantity using the Bioanalyzer (Agilent) and Qubit Fluorometer (Life Technologies). Libraries were pooled, requantified and sequenced as 150bp paired reads on a mid-output flow cell using the Illumina NextSeq 500 instrument, utilizing RTA and instrument software versions current at the time of processing.

value: hydatidiform mole

tag: cell line

tag: cell type

value: extraembryonic tissues


title: Liver_Pcoat+MB+_17mg per kg_Replicate1


title: input from flow-sorted chromosome 21

description: Library name: chr21


title: S129,liver,NC,EsrrgChIP-seq, rep2


title: input from flow-sorted chromosome 15

description: Library name: chr15


title: input from flow-sorted chromosome 14

description: Library name: chr14


title: Liver_Pcoat+MB+_10mg per kg_Replicate3

value: 10mg/kg methylene blue


title: CITED under IL6 bio rep 3

city: Chicago

first: Zongmin

last: Zhao

state: Illinois

address: 833 S Wood St

department: Department of Pharmaceutical Sciences

institute: University of Illinois Chicago

description: Library name: e3

data_processing:
Raw sequencing reads were processed using Salmon (v1.4.0) for pseudoalignment and transcript quantification.
Decoy sequences, including the genome assembly and other non-transcribed regions, were added to the index using the --decoys option in Salmon
The transcriptome reference and decoy sequences were obtained from GENCODE (Genome sequence, primary assembly (GRCm39) )
Quantified transcript level abundances were imported, aggregated to gene level counts and analyzed in R(v4.3.2)
Assembly: mm39
Supplementary files format and content: raw_counts.txt includes raw counts per sample in tab-delimited format

source_name: MDSCs

extract_protocol:
RNA was harvested using Rneasy Mini Kit.
Novogene Genomics Services performed the quality check and processed the RNA for library preparation for RNA-seq  using Abclonal Fast RNA-seq Lib Prep Kit V2.

value: CNS

value: myeloid derived supressor cells

value: WT


title: CITED under IL6 bio rep 4

description: Library name: e4


title: input from flow-sorted chromosome 13

description: Library name: chr13


title: CITED under IL6 bio rep 2

description: Library name: e2


title: GSK-3484862 treated cells

description: Library name: dnmt_chm13

data_processing:
Chm13 untreated reads were trimmed for adapters and low quality using trim galore with auto-detect adapters and a quality score cutoff of 20. GSK-3484862 treated reads were not trimmed.
Alignment to chm13 (v2.0) using the bisulfite sequencing aligner Bismark (v0.24.0). End to end alignment was used for untreated but local alignment was used for GSK-3484862 treated.
Alignments were deduplicated and extracted into bed coverage format to give a C and T count at each reference position. Complementary CpGs on the two strands were merged into a single site. Positions were later filtered by requiring coverage of 5 or more reads.
To assess rDNA alone, reads were aligned to the reference sequence KY962518.1 instead of chm13. The bismark local alignment option was used but reads were not deduplicated. This is due to the high copy number of the rDNA gene which creates many real duplicate alignments that are useful for methylation quantificaiton. Methylation extraction was performed to give bed coverage files with CpGs merged from both strands. Positions were filtered by requiring coverage of 10 or more reads.
Assembly: chm13 v2.0
Supplementary files format and content: Bed coverage files for whole genome and KY962518.1 alignments
Supplementary files format and content: The columns are unlabelled but have the format of: chromosome, start position, end position, methylation percentage, count methylated, count unmethylated.
Library strategy: EM-seq

extract_protocol:
Genomic DNA from untreated and 5uM GSK-3484862 treated cells was isolated using QIAamp DNA Micro Kit (Qiagen).
The library was made according to the manufacturer’s directions for the NEBNext Enzymatic Methyl-seq kit (New England Biolabs, Cat. No. E7120S) using the S220 Focused-ultrasonicator (Covaris) to shear to 300bp and 4 cycles of library PCR amplification.

value: genomic DNA

value: paired-end

tag: batch


title: Liver_Pcoat+MB+_10mg per kg_Replicate2

value: Liver


title: Thalamus_Pcoat-MB-_Replicate1


title: CITED under IL6 bio rep 1

description: Library name: e1


title: MDSCs-Np-Blk under IL6 bio rep 4

description: Library name: b4


title: Liver_Pcoat+MB+_6mg per kg_Replicate3

value: 6mg/kg methylene blue


title: MDSCs-Np-Blk under IL6 bio rep 3

description: Library name: b3


title: Liver_Pcoat+MB+_6mg per kg_Replicate2


title: ZM_Project_YKSZ_ERT_221212_01X_03_0065

city: beijing

first: Tao

last: Tao

state: Beijing city

address: No. 1 Shuaifuyuan, Dongcheng District, Beijing

department: Department of Obstetrics and Gynecology

country: China

institute: Peking Union Medical College Hospital

description: Library name: EIN59

data_processing:
RNA seq
The raw datas were filtered to exclude low-quality reads
The  reads were alternative alignment with RNASeQC
Qualified reads were mapped to the human reference genome(GRCh38) by using STAR
Caculate  the count of each gene
Estimate the RNA expression level by FPKM
Assembly: Ensembl primary assembly, version GRCh38
Supplementary files format and content: tab-delimited text files include raw counts for each Sample
Supplementary files format and content: tab.deimited text file includes FPKM values for each Sampe

source_name: endometrium

growth_protocol: The tip of the endometrial sampler was placed into the uterine fundus, and 5–10 mm3 of endometrial tissues were aspirated into the sampler.

extract_protocol:
Total RNA extraction was performed using the RNeasy Micro Kit (74004; Qiagen, Germantown, MD, USA) and followed by quantification with a Qubit HS RNA Kit.
RNA reverse transcription was conducted using the MALBAC® Platinum Single Cell RNA Amplification Kit.Library construction was accomplished using a gene sequencing and library preparation kit (XY045; Yikon Genomics, Suzhou, Jiangsu, China)

treatment_protocol: The collected endometrial tissues were immediately placed into 1.5 mL RNA-later buffer for RNA stabilization, sealed, and cryopreserved at -20 °C.

value: endometrium

tag: condition

value: EIN


title: ZSY_Project_YKSZ_ERT_230224_01X_02_0133

description: Library name: EIN60


title: ZL_Project_YKSZ_ERT_230319_01X_01_0161

description: Library name: EIN58


title: ZXQ_Project_YKSZ_ERT_240823_01X_0001_0394

description: Library name: EIN62


title: ZXP_Project_YKSZ_ERT_221024_01X_01_0020

description: Library name: EIN61


title: ZYT_Project_YKSZ_ERT_230303_01X_01_0144

description: Library name: EIN64


title: MDSCs-Np-Blk under IL6 bio rep 2

description: Library name: b2


title: ZHN_Project_YKSZ_ERT_230511_01X_01_0237

description: Library name: EIN56


title: Liver_Pcoat+MB+_6mg per kg_Replicate1


title: MDSCs-Np-Blk under IL6 bio rep 1

description: Library name: b1


title: ZF_Project_YKSZ_ERT_230707_01X_02_0312

description: Library name: EIN55


title: Liver_Pcoat-MB-_Replicate4


title: Liver_Pcoat-MB-_Replicate3


title: ZXY_Project_YKSZ_ERTRD2000_220330_01X_01_0910

description: Library name: EIN63


title: YKL_Project_YKSZ_ERT_231123_01X_01_0453

description: Library name: EIN52


title: MDSCs under IL6 bio rep 4

description: Library name: a4


title: Liver_Pcoat+MB+_10mg per kg_Replicate1


title: ZC_Project_YKSZ_ERT_240729_01X_0001_0375

description: Library name: EIN54


title: Liver_Pcoat-MB-_Replicate2


title: XMR_Project_YKSZ_ERT_240517_01X_0002_0296

description: Library name: EIN50


title: MDSCs under IL6 bio rep 3

description: Library name: a3


title: XXT_Project_YKSZ_ERT_240413_01X_0001_2646

description: Library name: EIN51


title: MDSCs under IL6 bio rep 2

description: Library name: a2


title: YXF_Project_YKSZ_ERT_240820_01X_0002_0592

description: Library name: EIN53


title: XMG_Project_YKSZ_ERT_220527_01X_02_0960

description: Library name: EIN49


title: XL_Project_YKSZ_ERT_220601_01X_01_0580

description: Library name: EIN48


title: MDSCs under IL6 bio rep 1

description: Library name: a1


title: ZLJ_Project_YKSZ_ERT_241003_01X_0001_0626

description: Library name: EIN57


title: Normal Chow, B6, ZT6, Rep1

description:
NC.B6.ZT6.Rep1
RNA-seq.Diet.B6and129.quantileNormalized.txt

data_processing:
RNA-seq reads were aligned to mm10 using STAR. The tag directories were established and quantile normalized counts were measured with RefSeq genes using Homer.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include quantile normalized tag counts for each Sample .

extract_protocol:
Total RNA was extracted from liver tissue or isolated endothelial cells and kupffer cells using Trizol reagent followed by RNeasy Mini Kit (#74104).
Libraries were prepared according to Illumina's instructions accompanying the TruSeq RNA Sample Preparation. The purified cDNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

tag: time

value: Wild Type

value: Normal Chow

value: male


title: Kidney_Pcoat+MB+_17mg per kg_Replicate2

source_name: Kidney

value: Kidney

value: Pcoat+MB+


title: WX_Project_YKSZ_ERTRD2000_220325_01X_01_0906

description: Library name: EIN45


title: Wk17_Protein_HTO

city: La Jolla

first: Shane

last: Crotty

state: California

address: 9420 Athena

institute: La Jolla Institute For Immunology

description:
Library name: Wk17_Protein_HTO
HTO 5'mRNA library
Wk17_Protein_barcodes.tsv
Wk17_Protein_features.tsv
Wk17_Protein_matrix.mtx

library_source: other

data_processing:
Demultiplexing, barcode processing, gene counting, and aggregation were performed using Cell Ranger v3.0 or higher (10X Genomics). VDJ reads were assembled de novo in Cell Ranger, and GEX sequencing libraries were aligned to the Macaca mulatta reference genome.
Assembly: Mmul10
Supplementary files format and content: Fasta files, Tab-separated values files and matrix files
Library strategy: CITE-seq

molecule: protein

source_name: PBMC

extract_protocol:
Apex-binding B cells were isolated from lymph node and peripheral blood samples collected from rhesus macaques immunized with the Apex-GT6 immunogen. Two groups of animals were used: one group received 100 µg of soluble Apex-GT6 protein formulated with the SMNP adjuvant, and the other received membrane-bound Apex-GT6 protein. Fine needle aspirates (FNAs) were collected from lymph nodes at weeks 2, 4, 8, and 12 post-immunization to isolate germinal center (GC) B cells. Peripheral blood mononuclear cells (PBMCs) were collected at week 10 for both mRNA and protein analysis, and again at week 17 for protein analysis only. To identify Apex-binding B cells, samples were stained with tetramerized Apex-GT6 probes conjugated to fluorescent streptavidin (SA) and TotalSeq-C oligo-tagged SA. Surface staining included a panel of fluorescently labeled antibodies, along with one of ten different TotalSeq-C oligo-tagged antibodies per sample. These oligo-tagged antibodies targeted β2M and CD298 and served as sample-specific hashtags to enable multiplexing across animals. Cells were sorted on a FACSymphony S6 (BD Biosciences). Apex-binding GC and memory B cells were collected into 1.5 mL tubes containing RPMI 1640 medium supplemented with 50% fetal bovine serum (FBS) and kept on ice for immediate downstream processing.
Lymph Node Libraries were constructed according to the manufacturer's protocol using the Chromium Next GEM Single Cell 5’ Reagent Kit v1.1. Lymph Node Libraries were constructed according to the manufacturer's protocol using the Chromium Next GEM Single Cell 5’ Reagent Kit v2

value: PBMC

value: B cells

value: Protein immunized

tag: library type

value: HTO

tag: hto

value: TotalSeq-C0251 anti-Human Hashtag1 (RPb18), TotalSeq-C0253 anti-Human Hashtag3 RJz19, TotalSeq-C0257 anti-Human Hashtag7 (RPz18), TotalSeq-C0258 anti-Human Hashtag8 (REt18), TotalSeq-C0259 anti-Human Hashtag9 (RIo18), TotalSeq-C0260 anti-Human Hashtag10 (RGp18), TotalSeq-C0953 PE Streptavidin (Apex-GT6 knock out probe)


title: WDH_Project_YKSZ_ERT_240820_01X_0003_0592

description: Library name: EIN44


title: TZZ_Project_YKSZ_ERT_231022_01X_01_0088

description: Library name: EIN43


title: Kidney_Pcoat+MB+_17mg per kg_Replicate1

value: 17mg/kg methylene blue


title: Wk17_Protein_VDJ

description:
Library name: Wk17_Protein_VDJ
VDJ 5' mRNA library
Wk17_Protein_all_contig.fasta

data_processing:
*library strategy: BCR-seq
Demultiplexing, barcode processing, gene counting, and aggregation were performed using Cell Ranger v3.0 or higher (10X Genomics). VDJ reads were assembled de novo in Cell Ranger, and GEX sequencing libraries were aligned to the Macaca mulatta reference genome.
Assembly: Mmul10
Supplementary files format and content: Fasta files, Tab-separated values files and matrix files

molecule: polyA RNA

value: VDJ


title: Wk17_Protein_GEX

description:
Library name: Wk17_Protein_GEX
GEX 5' mRNA library
Wk17_Protein_barcodes.tsv
Wk17_Protein_features.tsv
Wk17_Protein_matrix.mtx

library_source: transcriptomic single cell

data_processing:
Demultiplexing, barcode processing, gene counting, and aggregation were performed using Cell Ranger v3.0 or higher (10X Genomics). VDJ reads were assembled de novo in Cell Ranger, and GEX sequencing libraries were aligned to the Macaca mulatta reference genome.
Assembly: Mmul10
Supplementary files format and content: Fasta files, Tab-separated values files and matrix files

value: mRNA


title: Kidney_Pcoat+MB+_10mg per kg_Replicate3

value: 10mg/kg methylene blue


title: TL_Project_YKSZ_ERT_230312_01X_01_0152

description: Library name: EIN41


title: SX_Project_YKSZ_ERT_240722_01X_0001_0369

description: Library name: EIN40


title: Wk12_ApexPos_HTO

description:
Library name: Wk12_ApexPos_HTO
HTO 5'mRNA library
Wk12P_barcodes.tsv
Wk12P_features.tsv
Wk12P_matrix.mtx

source_name: Lymph node

value: Lymph node

value: mixed (protein and mRNA immunized)

value: TotalSeq-C0251 anti-Human Hashtag1 (RGp18), TotalSeq-C0252 anti-Human Hashtag2 (RJz18), TotalSeq-C0253 anti-Human Hashtag3 (RPz18), TotalSeq-C0254 anti-Human Hashtag4 (REt18), TotalSeq-C0255 anti-Human Hashtag5 (RIo18), TotalSeq-C0256 anti-Human Hashtag6 (RGu18), TotalSeq-C0258 anti-Human Hashtag8 (RJr18), TotalSeq-C0259 anti-Human Hashtag9 (RYu18), TotalSeq-C0260 anti-Human Hashtag10 (RWj18), TotalSeq-C0953 PE Streptavidin (Apex-GT6 knock out probe)


title: Kidney_Pcoat+MB+_10mg per kg_Replicate2


title: SW_Project_YKSZ_ERT_240611_01X_0001_0325

description: Library name: EIN39

value: endometrium

value: EIN


title: Liver_Pcoat-MB-_Replicate1

value: Liver

value: Pcoat-MB-


title: Wk12_ApexPos_VDJ

description:
Library name: Wk12_ApexPos_VDJ
VDJ 5' mRNA library
Wk12P_all_contig.fasta


title: WZL_Project_YKSZ_ERTRD2000_220422_01X_03_0930

description: Library name: EIN46


title: Kidney_Pcoat+MB+_10mg per kg_Replicate1

value: Kidney

value: Pcoat+MB+


title: subject 3, inf, 0W

description:
Library name: IonXpress_084
IonXpress_084

value: colonic mucosa


title: SQ_Project_YKSZ_ERT_240919_01X_0001_0610

description: Library name: EIN38


title: SLN_Project_YKSZ_ERT_230614_01X_01_0320

description: Library name: EIN37


title: QBX_Project_YKSZ_ERT_231022_01X_02_0088

description: Library name: EIN36


title: Kidney_Pcoat+MB+_17mg per kg_Replicate3


title: XJY_Project_YKSZ_ERT_240531_01X_0001_0312

description: Library name: EIN47


title: Kidney_Pcoat+MB+_6mg per kg_Replicate3

value: 6mg/kg methylene blue


title: Wk12_ApexPos_GEX

description:
Library name: Wk12_ApexPos_GEX
GEX 5' mRNA library
Wk12P_barcodes.tsv
Wk12P_features.tsv
Wk12P_matrix.mtx

value: B cells


title: Wk12_ApexNeg_HTO

description:
Library name: Wk12_ApexNeg_HTO
HTO 5'mRNA library
Wk12N_barcodes.tsv
Wk12N_features.tsv
Wk12N_matrix.mtx

value: HTO


title: PTT_Project_YKSZ_ERT_230620_01X_01_0295

description: Library name: EIN34


title: TX_Project_YKSZ_ERT_231015_01X_01_0422

description: Library name: EIN42


title: MBY_Project_YKSZ_ERT_240517_01X_0001_0296

description: Library name: EIN33


title: Wk12_ApexNeg_VDJ

description:
Library name: Wk12_ApexNeg_VDJ
VDJ 5' mRNA library
Wk12N_all_contig.fasta


title: LZL_Project_YKSZ_ERT_241121_01X_0001_0683

description: Library name: EIN32


title: High-fat diet, B6, ZT6, Rep1

description:
HFD.B6.ZT6.Rep1
RNA-seq.Diet.B6and129.quantileNormalized.txt

value: Wild Type

value: high fat and sucrose diet, D12492i

value: male


title: Wk12_ApexNeg_GEX

description:
Library name: Wk12_ApexNeg_GEX
GEX 5' mRNA library
Wk12N_barcodes.tsv
Wk12N_features.tsv
Wk12N_matrix.mtx

value: mRNA


title: LY_Project_YKSZ_ERT_240413_01X_0002_2646

description: Library name: EIN30


title: LW_Project_YKSZ_ERT_230224_01X_01_0133

description: Library name: EIN28


title: LXL_Project_YKSZ_ERT_240301_01X_0001_0239

description: Library name: EIN29


title: Kidney_Pcoat+MB-_Replicate5

value: Pcoat+MB-


title: Kidney_Pcoat+MB-_Replicate4


title: Wk10_Protein_VDJ

description:
Library name: Wk10_Protein_VDJ
VDJ 5' mRNA library
Wk10_Protein_all_contig.fasta

value: PBMC

value: Protein immunized

value: VDJ


title: LPX_Project_YKSZ_ERT_240704_01X_0001_0348

description: Library name: EIN26

value: endometrium

value: EIN


title: LSH_Project_YKSZ_ERT_231101_01X_01_0104

description: Library name: EIN27


title: LL_Project_YKSZ_ERT_230329_01X_01_0175

description: Library name: EIN24


title: Wk10_Protein_GEX

description:
Library name: Wk10_Protein_GEX
GEX 5' mRNA library
Wk10_Protein_barcodes.tsv
Wk10_Protein_features.tsv
Wk10_Protein_matrix.mtx


title: Kidney_Pcoat+MB-_Replicate3


title: Kidney_Pcoat+MB-_Replicate2


title: LJY_Project_YKSZ_ERT_231021_01X_01_0088

description: Library name: EIN23


title: Wk10_mRNA_HTO

description:
Library name: Wk10_mRNA_HTO
HTO 5'mRNA library
Wk10_mRNA_barcodes.tsv
Wk10_mRNA_features.tsv
Wk10_mRNA_matrix.mtx

value: mRNA immunized

value: TotalSeq-C0251 anti-Human Hashtag1 (RGu18), TotalSeq-C0253 anti-Human Hashtag3 (RPb18), TotalSeq-C0257 anti-Human Hashtag7 (RJr18), TotalSeq-C0258 anti-Human Hashtag8 (RYu18), TotalSeq-C0259 anti-Human Hashtag9 (RWj18), TotalSeq-C0260 anti-Human Hashtag10 (RAu18), TotalSeq-C0953 PE Streptavidin (Apex-GT6 knock out probe)


title: LHJ_Project_YKSZ_ERT_240806_01X_0001_0580

description: Library name: EIN22


title: LN_Project_YKSZ_ERT_240704_01X_0002_0348

description: Library name: EIN25


title: PY_Project_YKSZ_ERT_241003_01X_0002_0626

description: Library name: EIN35


title: Kidney_Pcoat+MB+_6mg per kg_Replicate2

value: Kidney

value: Pcoat+MB+


title: Kidney_Pcoat+MB+_6mg per kg_Replicate1

value: 6mg/kg methylene blue


title: Kidney_Pcoat-MB-_Replicate4

value: Pcoat-MB-


title: LF_Project_YKSZ_ERTRD2000_220422_01X_02_0930

description: Library name: EIN21


title: LY_Project_YKSZ_ERT_240502_01X_0001_0540

description: Library name: EIN31


title: Wk10_mRNA_VDJ

description:
Library name: Wk10_mRNA_VDJ
VDJ 5' mRNA library
Wk10_mRNA_all_contig.fasta

value: B cells


title: Kidney_Pcoat-MB-_Replicate3


title: Wk10_mRNA_GEX

description:
Library name: Wk10_mRNA_GEX
GEX 5' mRNA library
Wk10_mRNA_barcodes.tsv
Wk10_mRNA_features.tsv
Wk10_mRNA_matrix.mtx


title: Wk10_Protein_HTO

description:
Library name: Wk10_Protein_HTO
HTO 5'mRNA library
Wk10_Protein_barcodes.tsv
Wk10_Protein_features.tsv
Wk10_Protein_matrix.mtx

value: HTO

value: TotalSeq-C0251 anti-Human Hashtag1 (RPb18), TotalSeq-C0252 anti-Human Hashtag2 (RJz18), TotalSeq-C0253 anti-Human Hashtag3 (RPz18), TotalSeq-C0254 anti-Human Hashtag4 (REt18), TotalSeq-C0255 anti-Human Hashtag5 (RIo18), TotalSeq-C0256 anti-Human Hashtag6 (RGp18), TotalSeq-C0953 PE Streptavidin (Apex-GT6 knock out probe)


title: JB_Project_YKSZ_ERT_230716_01X_01_0323

description: Library name: EIN17


title: Kidney_Pcoat-MB-_Replicate2


title: Wk8P2_ApexPos_FNA_HTO

description:
Library name: Wk8P2_ApexPos_FNA_HTO
HTO 5'mRNA library
Wk8P2_barcodes.tsv
Wk8P2_features.tsv
Wk8P2_matrix.mtx

value: Lymph node

value: mixed (protein and mRNA immunized)

value: TotalSeq-C0251 anti-Human Hashtag1 (RPb18), TotalSeq-C0252 anti-Human Hashtag2 (RJz18), TotalSeq-C0253 anti-Human Hashtag3 (RPz18), TotalSeq-C0254 anti-Human Hashtag4 (REt18), TotalSeq-C0255 anti-Human Hashtag5 (RIo18), TotalSeq-C0256 anti-Human Hashtag6 (RGu18), TotalSeq-C0257 anti-Human Hashtag7 (RPb18), TotalSeq-C0258 anti-Human Hashtag8 (RJr18), TotalSeq-C0259 anti-Human Hashtag9 (RYu18), TotalSeq-C0260 anti-Human Hashtag10 (RWj18), TotalSeq-C0953 PE Streptavidin (Apex-GT6 knock out probe)


title: HSS_Project_YKSZ_ERT_221018_01X_01_0014

description: Library name: EIN15


title: Kidney_Pcoat-MB-_Replicate1


title: HYP_Project_YKSZ_ERT_240812_01X_0002_0585

description: Library name: EIN16


title: Wk8P2_ApexPos_FNA_VDJ

description:
Library name: Wk8P2_ApexPos_FNA_VDJ
VDJ 5' mRNA library
Wk8P2_all_contig.fasta


title: Heart_Pcoat+MB+_17mg per kg_Replicate3

source_name: Heart

value: Heart

value: 17mg/kg methylene blue


title: HNN_Project_YKSZ_ERT_220920_01X_01_1064

description: Library name: EIN14


title: GYX_Project_YKSZ_ERT_220712_01X_01_1000

description: Library name: EIN13


title: GX_Project_YKSZ_ERT_240204_01X_0001_0214

description: Library name: EIN12


title: Wk8P2_ApexPos_FNA_GEX

description:
Library name: Wk8P2_ApexPos_FNA_GEX
GEX 5' mRNA library
Wk8P2_barcodes.tsv
Wk8P2_features.tsv
Wk8P2_matrix.mtx

value: mRNA


title: GTL_Project_YKSZ_ERT_240924_01X_0001_0616

description: Library name: EIN10

value: endometrium


title: GXJ_Project_YKSZ_ERT_230424_01X_02_0214

description: Library name: EIN11

value: EIN


title: Heart_Pcoat+MB+_17mg per kg_Replicate2


title: Wk8P1_ApexPos_FNA_HTO

description:
Library name: Wk8P1_ApexPos_FNA_HTO
HTO 5'mRNA library
Wk8P1_barcodes.tsv
Wk8P1_features.tsv
Wk8P1_matrix.mtx


title: Heart_Pcoat+MB+_17mg per kg_Replicate1


title: LAN_Project_YKSZ_ERT_241015_01X_0002_0639

description: Library name: EIN19


title: LBQ_Project_YKSZ_ERT_241015_01X_0001_0639

description: Library name: EIN20


title: GLL_Project_YKSZ_ERT_240510_01X_0001_0287

description: Library name: EIN09


title: Wk8P1_ApexPos_FNA_VDJ

description:
Library name: Wk8P1_ApexPos_FNA_VDJ
VDJ 5' mRNA library
Wk8P1_all_contig.fasta

value: VDJ


title: FH_Project_YKSZ_ERT_220823_01X_01_1037

description: Library name: EIN07


title: JYY_Project_YKSZ_ERT_220927_01X_01_0007

description: Library name: EIN18


title: FH_Project_YKSZ_ERTRD2000_220422_01X_01_0930

description: Library name: EIN06


title: Wk8P1_ApexPos_FNA_GEX

description:
Library name: Wk8P1_ApexPos_FNA_GEX
GEX 5' mRNA library
Wk8P1_barcodes.tsv
Wk8P1_features.tsv
Wk8P1_matrix.mtx


title: FY_Project_YKSZ_ERT_221129_01X_01_0051

description: Library name: EIN08


title: Heart_Pcoat+MB+_10mg per kg_Replicate3

value: 10mg/kg methylene blue


title: CY_Project_YKSZ_ERT_230707_01X_01_0312

description: Library name: EIN04


title: Heart_Pcoat+MB+_10mg per kg_Replicate2


title: CYY_Project_YKSZ_ERT_240806_01X_0002_0580

description: Library name: EIN03


title: Heart_Pcoat+MB+_10mg per kg_Replicate1


title: CH_Project_YKSZ_ERT_221024_01X_02_0020

description: Library name: EIN02


title: Wk8_ApexNeg_FNA_VDJ

description:
Library name: Wk8_ApexNeg_FNA_VDJ
VDJ 5' mRNA library
Wk8N_all_contig.fasta


title: Heart_Pcoat+MB+_6mg per kg_Replicate3


title: BY_Project_YKSZ_ERTRD2000_220426_01X_01_0932

description: Library name: EIN01


title: ZZ_Project_YKSZ_ERT_230911_01X_01_0393

description: Library name: RIF25

value: RIF


title: Heart_Pcoat+MB+_6mg per kg_Replicate2

value: Pcoat+MB+


title: Wk8_ApexNeg_FNA_GEX

description:
Library name: Wk8_ApexNeg_FNA_GEX
GEX 5' mRNA library
Wk8N_barcodes.tsv
Wk8N_features.tsv
Wk8N_matrix.mtx


title: ZXS_Project_YKSZ_ERT_240820_01X_0004_0592

description: Library name: RIF24


title: Wk4_ApexPos_FNA_HTO

description:
Library name: Wk4_ApexPos_FNA_HTO
HTO 5'mRNA library
Wk4P_barcodes.tsv
Wk4P_features.tsv
Wk4P_matrix.mtx

value: TotalSeq-C0251 anti-Human Hashtag1 (RPb18), TotalSeq-C0252 anti-Human Hashtag2 (RJz18), TotalSeq-C0253 anti-Human Hashtag3 (RPz18), TotalSeq-C0254 anti-Human Hashtag4 (REt18), TotalSeq-C0255 anti-Human Hashtag5 (RIo18), TotalSeq-C0256 anti-Human Hashtag6 (RPb18), TotalSeq-C0257 anti-Human Hashtag7 (RJr18), TotalSeq-C0258 anti-Human Hashtag8 (RGu18), TotalSeq-C0259 anti-Human Hashtag9 (RYu18), TotalSeq-C0260 anti-Human Hashtag10 (RWj18), TotalSeq-C0953 PE Streptavidin (Apex-GT6 knock out probe)


title: ZP_Project_YKSZ_ERT_240104_01X_0001_0167

description: Library name: RIF22


title: YWC_Project_YKSZ_ERT_240502_01X_0003_0540

description: Library name: RIF21


title: Wk4_ApexPos_FNA_VDJ

description:
Library name: Wk4_ApexPos_FNA_VDJ
VDJ 5' mRNA library
Wk4P_all_contig.fasta


title: Heart_Pcoat+MB-_Replicate5

value: Pcoat+MB-


title: YHQ_Project_YKSZ_ERT_230424_01X_01_0214

description: Library name: RIF19


title: YL_Project_YKSZ_ERT_240413_01X_0003_2646

description: Library name: RIF20


title: DY_Project_YKSZ_ERT_221212_01X_02_0065

description: Library name: EIN05


title: Heart_Pcoat+MB-_Replicate4


title: Wk4_ApexPos_FNA_GEX

description:
Library name: Wk4_ApexPos_FNA_GEX
GEX 5' mRNA library
Wk4P_barcodes.tsv
Wk4P_features.tsv
Wk4P_matrix.mtx

value: B cells


title: Wk4_ApexNeg_FNA_HTO

description:
Library name: Wk4_ApexNeg_FNA_HTO
HTO 5'mRNA library
Wk4N_barcodes.tsv
Wk4N_features.tsv
Wk4N_matrix.mtx

value: HTO


title: YHL_Project_YKSZ_ERT_240419_01X_0001_0531

description: Library name: RIF18


title: XJS_Project_YKSZ_ERT_240522_01X_0002_0301

description: Library name: RIF17


title: Wk8_ApexNeg_FNA_HTO

description:
Library name: Wk8_ApexNeg_FNA_HTO
HTO 5'mRNA library
Wk8N_barcodes.tsv
Wk8N_features.tsv
Wk8N_matrix.mtx


title: Heart_Pcoat+MB-_Replicate3


title: WYM_Project_YKSZ_ERT_220527_01X_01_0960

description: Library name: RIF16


title: WXP_Project_YKSZ_ERT_240320_01X_0001_0253

description: Library name: RIF15


title: Wk4_ApexNeg_FNA_VDJ

description:
Library name: Wk4_ApexNeg_FNA_VDJ
VDJ 5' mRNA library
Wk4N_all_contig.fasta


title: WWQ_Project_YKSZ_ERT_240719_01X_0001_0365

description: Library name: RIF14


title: Heart_Pcoat+MB-_Replicate2


title: Sen1 Yeast, mut-NNS-BS, OD1, rep2

city: Edinburgh

first: Sander

last: Granneman

address: Mayfield Road, Kings Buildings, Waddington building, room 3.06

department: Centre for Synthetic and Systems Biology

country: United Kingdom

institute: University of Edinburgh

description:
Sen1_PIC2-GFP_vs_mut-NNS-BS_min_peak_width_10_noblocks_merged_peaks_numbered_with_padj.gtf
Sen1_PIC2-GFP_vs_mut-NNS-BS_min_peak_width_10_noblocks_merged_hittables_DESeq2_results.txt
Sen1_merged_hittable_reads.txt

data_processing:
Raw sequencing outputs were returned as FASTQ files. Hence, after conversion into FASTA format, files were processed using the paired-end (PE) version of the pyCRAC pipeline (Webb et al., 2014). This package demultiplexes barcoded reads and removes adapter sequences applying the previously developed Flexbar tool (Dodt et al., 2012). Having been filtered based on their length and nucleotide quality values (i.e., PHRED scores), remaining reads were aligned to the provided Saccharomyces_cerevisiae.R64-1-1.75 reference genome with the Novoalign tool (version 2.07). Filtered and aligned reads were gathered into BAM outputs and fed into the newly developed DBPeaks.py peak calling script (https://git.ecdf.ed.ac.uk/sgrannem/dbpeaks). Firstly, the countReadsBam function of the DBPeaks script employs the pyReadCounters tool from the pyCRAC package to generate GTF files with read counts and gene mapping locations. Next, the getPeaks function uses the pyCalculateFDRs script from the pyCRAC package to retrieve peaks enriched in each library relative to randomly distributed reads over the same genes. The resulting peak GTF files are then filtered by the filterPeaks function, to remove peaks comprising less reads than the mean coverage of all the intervals in the dataset. Although we used mean, median or mean with one standard deviation can be selected as thresholds. Peak widths were normalised to a minimum length of 10 nucleotides with the adjustPeakWidths function. Afterwards, the mergeGTFintervals applied BEDTools (Quinlan and Hall, 2010) to concatenate intervals or peaks that are present in all replicates. Subsequently, the numberPeaks function assigns a number to each filtered peak to prevent misidentification in downstream steps. The software then performs a pyReadCounters analysis on the BAM files using the new GTF file encompassing the numbered filtered peaks. We then gathered the hit tables outputted by pyReadCounters and combined them using the mergeHittables function. Finally, the runDESeq function automatically performs the statistical analysis to identify differential peaks using the DESeq2 pipeline (Love et al., 2014). By default, DESeq returns results in a TXT file, so we developed two additional functions to extract the calculated fold change and incorporate it into the original peak file. Finally, the getSignificantPeaks function generates a new GTF file containing solely the peaks that were called significant by the DESeq.
Assembly: Saccharomyces_cerevisiae.R64-1-1.75 (see supplementary files)
Supplementary files format and content: Bedgraph files include aligned single-collapsed reads
Supplementary files format and content: GTF files contain peaks (called and filtered with DBPeaks.py) and an adjusted p-value resulting from the statistical comparison of their coverage in the parental referrence and the mutant strain.
Library strategy: CRAC

organism: Saccharomyces cerevisiae

growth_protocol: Cells were grown to OD600 1 in 0.5 L-cultures of SC-Ura containing 1% (w/v) raffinose

extract_protocol:
Following optimised crosslinking (500 mJ UV 254 nm), UV-irradiated pellets were resuspended in a volume of lysis buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40, 1 EDTA-free protease inhibitor cocktail (Roche) per 50 mL and 5 mM β-mercaptoethanol) corresponding to twice their mass. Lysates were then transferred to a fresh 50 mL tubes, where they were mixed with 2 volumes of 0.5 mm zirconia beads (Thistle Scientific), and vortexed at maximum speed for five 1-minute intervals alternated with 1-minute incubations on ice. An additional volume of lysis buffer was pipetted into each sample before partitioning the cell debris by centrifugation at 4ºC (4000 rpm, 15 minutes) and spinning down lighter impurities in an additional centrifugation step (10000 rpm, 20 minutes) at 4ºC. Immunoprecipitation of the labelled protein from the supernatant was achieved during a two-hour incubation of the sample with 75 µL of anti-FLAG® M2 magnetic beads (Sigma-Aldrich, M8823-5ML) which had been previously washed three times with 1 mL TN150 buffer. Immobilised beads underwent three 5-minute washes with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and three additional 5-minute washes in 2 mL TN150 buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol). Afterwards, beads were resuspended in 550 µL of TN150 and incubated with 1 µL of a 1:100 dilution of RNace-It™ for exactly 5 minutes at 37 °C. To prevent overdigestion of co-immunoprecipitated transcripts and elute the bait proteins from the anti-FLAG beads, the solution was mixed with 0.4 g of guanidium hydrochloride (Gu-HCl) and the necessary volume of NaCl and imidazole (pH 8.0) to reach a final concentration of 300 mM and 10 mM respectively. After removing the beads, samples were pipetted onto 50 µL of Ni-NTA agarose resin (Qiagen) equilibrated with wash buffer I (6 M Gu-HCl, 10 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and incubated at 4 °C for two hours while rotating at 12 rpm.
Upon Ni-NTA binding, beads were transferred to Pierce columns (Thermo Fisher Scientific) and washed twice with 500 µL of wash buffer I before undergoing three additional washes of 500 µL with NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40, 5 mM β-mercaptoethanol). Alkaline phosphatase treatment was performed by resuspending immunoprecipitated RNA-protein conjugates in 80 µL of NP-PNK buffer containing 4 units of FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and 80 units of RNasin® ribonuclease inhibitor (Promega) and incubating the samples at 37 °C for 15 minutes. Dephosphorylation was stopped upon addition of 500 µL of wash buffer. Having equilibrated the Ni-NTA agarose with three washes of 500 µL with NP-PNK, we performed adapter ligation by resuspending the beads 80 µL of reaction mix containing 0.6 µM of the applicable App-PE 3′ adapter (5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’), 30 units of T4 RNA ligase 2 truncated K227Q (New England Biolabs), 60 units RNasin® (Promega) and 10% (w/v) polyethylene glycol 8000 (PEG 8000). The reaction took place at 25 °C for three hours.  After washing once with 500 µL of wash buffer I and three times with 500 µL of NP-PNK buffer, we 5’end radiolabelled co-immunoprecipitated RNAs by incubating the Ni-NTA resin with 60 µL of NP-PNK buffer containing 30 µCi 32P-γATP (PerkinElmer) and 30 units of T4 polynucleotide kinase (New England Biolabs). After a 40-minute incubation of the reaction at 37ºC, ATP (Roche) was supplemented to a final concentration of 1 mM and the reaction continued for another 20 minutes. The reaction was stopped by washing three times with 500 µL of wash buffer I and three 500 µL washes of NP-PNK buffer followed before the addition of the 5′ linker ligation mix. The reaction took place in a volume of 80 µL of NP-PNK buffer supplemented with 1.25 µM of the fitting adapter, 40 units of T4 RNA ligase 1 (New England Biolabs), 80 units of RNasin® and 10 mM ATP (Roche).  5’ adapter ligation was left to proceed overnight at 16 °C for all bait proteins. The adapter sequences were 5’-ACACGACGCTCTTCCGATCTNNNCGCTTAGCN-3’ for PIC2-GFP_Rep1 and 5’-ACACGACGCTCTTCCGATCTNNNAGAGCN-3’,  5’-ACACGACGCTCTTCCGATCTNNNATTAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNGCGCAGCN-3’ for PIC2-GFP_Rep2; 5’-ACACGACGCTCTTCCGATCTNNNTGGAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ for mut-Nab3-BS_Rep1 and  5’-ACACGACGCTCTTCCGATCTNNNGACTTAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNATCACGN-3’ for mut-Nab3-BS_Rep2;  and 5’-ACACGACGCTCTTCCGATCTNNNCACTGTN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNACTCAGCN-3’ for mut-NNS-BS_Rep1 and 5’-ACACGACGCTCTTCCGATCTNNNGTGACAN-3’ for the second mut-NNS-BS library. After pooling suitable libraries together, the samples and its corresponding traces were submitted to Novogene, where paired-end sequencing of 150 bp-reads was performed in a NovaSeq 6000 system (Illumina).

treatment_protocol: When SC-Ura cultures containing 1% (w/v) raffinose reached an OD600 of 1, cells were UV cross-linked (254 nm; 500 mJ/cm2) in the Vari-X-linker (UVO3) and rapidly filtered on 0.8 μm membranes (Merck) using a filtration device connected to a vacuum pump. Membranes were collected in 50 mL tubes, flash frozen by submersion into liquid nitrogen and stored at -80ºC until the day of the experiment.

value: mut-NNS-BS

value: mutant

value: mut-NNS-BS, SEN1-HTF

value: 500 mJ UV 254 nm


title: Heart_Pcoat-MB-_Replicate4

value: Heart

value: Pcoat-MB-


title: Wk4_ApexNeg_FNA_GEX

description:
Library name: Wk4_ApexNeg_FNA_GEX
GEX 5' mRNA library
Wk4N_barcodes.tsv
Wk4N_features.tsv
Wk4N_matrix.mtx

value: Lymph node

value: mixed (protein and mRNA immunized)

value: mRNA


title: WLL_Project_YKSZ_ERT_221129_01X_02_0051

description: Library name: RIF13

value: endometrium


title: ZXL_Project_YKSZ_ERT_230511_01X_02_0237

description: Library name: RIF23


title: Heart_Pcoat+MB+_6mg per kg_Replicate1

value: 6mg/kg methylene blue


title: Sen1 Yeast, mut-Nab3-BS, OD1, rep2

description:
Sen1_PIC2-GFP_vs_mut-Nab3-BS_min_peak_width_10_noblocks_merged_peaks_numbered_with_padj.gtf
Sen1_PIC2-GFP_vs_mut-Nab3-BS_min_peak_width_10_noblocks_merged_hittables_DESeq2_results.txt
Sen1_merged_hittable_reads.txt

value: mut-Nab3-BS, SEN1-HTF


title: Wk2_ApexPos_FNA_HTO

description:
Library name: Wk2_ApexPos_FNA_HTO
HTO 5'mRNA library
Wk2P_barcodes.tsv
Wk2P_features.tsv
Wk2P_matrix.mtx


title: Wk2_ApexPos_FNA_VDJ

description:
Library name: Wk2_ApexPos_FNA_VDJ
VDJ 5' mRNA library
Wk2P_all_contig.fasta

value: VDJ


title: QM_Project_YKSZ_ERT_230402_01X_01_0181

description: Library name: RIF11

value: RIF


title: Heart_Pcoat-MB-_Replicate2


title: Sen1 Yeast, mut-NNS-BS, OD1, rep1


title: LZ_Project_YKSZ_ERT_230402_01X_02_0181

description: Library name: RIF10


title: Sen1 Yeast, PIC2-GFP, OD1, rep2

value: parental

value: PIC2-GFP, SEN1-HTF


title: Sen1 Yeast, mut-Nab3-BS, OD1, rep1


title: Cerebellum_Pcoat+MB+_17mg per kg_Replicate3

source_name: Cerebellum

value: Cerebellum

value: Pcoat+MB+

value: 17mg/kg methylene blue


title: Wk2_ApexPos_FNA_GEX

description:
Library name: Wk2_ApexPos_FNA_GEX
GEX 5' mRNA library
Wk2P_barcodes.tsv
Wk2P_features.tsv
Wk2P_matrix.mtx

value: B cells


title: Cerebellum_Pcoat+MB+_17mg per kg_Replicate2


title: Sen1 Yeast, PIC2-GFP, OD1, rep1

value: 500 mJ UV 254 nm


title: LX_Project_YKSZ_ERT_231202_01X_01_0465

description: Library name: RIF09


title: Wk2_ApexNeg_FNA_VDJ

description:
Library name: Wk2_ApexNeg_FNA_VDJ
VDJ 5' mRNA library
Wk2N_all_contig.fasta


title: LQ_Project_YKSZ_ERT_240309_01X_0001_0521

description: Library name: RIF08


title: Wk2_ApexNeg_FNA_GEX

description:
Library name: Wk2_ApexNeg_FNA_GEX
GEX 5' mRNA library
Wk2N_barcodes.tsv
Wk2N_features.tsv
Wk2N_matrix.mtx


title: Wk2_ApexNeg_FNA_HTO

description:
Library name: Wk2_ApexNeg_FNA_HTO
HTO 5'mRNA library
Wk2N_barcodes.tsv
Wk2N_features.tsv
Wk2N_matrix.mtx

value: HTO

value: TotalSeq-C0251 anti-Human Hashtag1 (RPb18), TotalSeq-C0252 anti-Human Hashtag2 (RJz18), TotalSeq-C0253 anti-Human Hashtag3 (RPz18), TotalSeq-C0254 anti-Human Hashtag4 (REt18), TotalSeq-C0255 anti-Human Hashtag5 (RIo18), TotalSeq-C0256 anti-Human Hashtag6 (RPb18), TotalSeq-C0257 anti-Human Hashtag7 (RJr18), TotalSeq-C0258 anti-Human Hashtag8 (RGu18), TotalSeq-C0259 anti-Human Hashtag9 (RYu18), TotalSeq-C0260 anti-Human Hashtag10 (RWj18), TotalSeq-C0953 PE Streptavidin (Apex-GT6 knock out probe),


title: LJ_Project_YKSZ_ERT_240820_01X_0001_0592

description: Library name: RIF07


title: Heart_Pcoat-MB-_Replicate1

value: Heart

value: Pcoat-MB-


title: Heart_Pcoat-MB-_Replicate3


title: SB_Project_YKSZ_ERT_240522_01X_0001_0301

description: Library name: RIF12


title: LJH_Project_YKSZ_ERT_240425_01X_0001_0272

description: Library name: RIF06


title: RNA Yeast, mut-NNS-BS, OD 0.5, rep3

description:
mut-NNS-BS_merged_forward.bedgraph
mut-NNS-BS_merged_reverse.bedgraph
RNA-seq_merged_count_table.txt

data_processing:
Raw sequencing outputs were returned as FASTQ files. Hence, after conversion into FASTA format, files were processed using the paired-end (PE) version of the pyCRAC pipeline (Webb et al., 2014). This package demultiplexes barcoded reads and removes adapter sequences applying the previously developed Flexbar tool (Dodt et al., 2012). The resulting reads undergo two quality control steps. Firstly, sequences are filtered based on their length and nucleotide quality values (i.e., PHRED scores). Secondly, since PCR artifacts will display identical random prefixes, they can easily be pinpointed and excluded from downstream analysis (collapsed). The remaining unique reads were subsequently aligned to the corresponding regions of the provided Saccharomyces_cerevisiae.R64-1-1.75 reference genome with the Novoalign tool (version 2.07). The pyReadCounters function of the pyCRAC package (version 1.5.1; Webb et al., 2014) was used to compute the number of reads that map to genomic features. The resulting tables for all the samples were merged into a single TXT file that was analysed by the DESeq2 R package (Love et al., 2014). The pipeline corrects for size variability by computing a scaling factor for each sample. To obtain such normalisation factor, the software calculates the ratio of the raw count of a given gene in one of the samples to the geometric mean of the counts for that same gene across the compared samples. The median of all the ratios calculated within a sample is defined as the scaling factor for that replicate. Having adjusted the raw reads, the package then performs a logarithmic transformation of the data and calculates a fold change and an FDR for each entry. Statistical significance was assessed for entries which exceeded or were lower than log2 fold change o 1 and -1 respectively and had an FDR value of 0.05 or less.
To perform statistical comparisons, all repeats for each strain were merged and processed through pyCRAC as described in the previous step. Resulting coverage files (_merged.bedgraph) were statistically compared using DESeq2. Output DESeq TXT files are also included.
Assembly: Saccharomyces_cerevisiae.R64-1-1.75 (see supplementary files)
Supplementary files format and content: Bedgraph files include aligned double-collapsed reads

growth_protocol: Cells were grown to OD 0.5 in 25 mL-cultures of SC-Ura containing 1% (w/v) raffinose

extract_protocol:
Cell lysis was performed by adding 400 μL of glass beads to each sample and vortexing all tubes at full speed for five 1-minute intervals separated by four 1-minute incubations on ice. An additional 1.5 mL of GTC phenol mix was added to the samples before incubating them at 65ºC for 10 minutes. Samples were then placed on ice for another 10 min. 800 μL of sodium acetate mix (3.3 mL 3 M NaOAc pH5.2, 0.2 mL 0.5M EDTA pH 8, 1mL 1M Tris-HCl pH 8, water to 100 mL) and 1.5 mL of chloroform were pipetted into each extract before vortexing at maximum speed for 5 seconds. Lysates were centrifuged at 4ºC (10000 rpm, 30 minutes) and the resulting aqueous phase was transferred into a new tube containing an equivalent volume of Phenol:Chloroform:Isoamylalcohol mix. The mixtures were vortexed vigorously and centrifuged at 10000 rpm for 5 minutes. Once more, the aqueous phase of each sample was mixed with an equal volume of Chloroform:Isoamylalcohol and vortexed at maximum speed before centrifugation at 10000 rpm for 5 minutes. Finally, the aqueous phase of each sample was relocated to tubes containing 3 volumes of 96% ethanol. The mix was vortexed vigorously and preserved at -80°C for at least 30 minutes or at -20°C overnight. Following ethanol precipitation, samples were spun at 1000 rpm for 30 minutes at 4ºC. After discarding the resulting supernatants, 70% ethanol was pipetted into each tube. After centrifuging samples at 10000 rpm for 5 minutes at 4ºC, ethanol was removed, and pellets were left to air-dry for approximately 5 minutes before resuspension in DEPC water. Finally, RNA concentrations were measured in the Qubit™ 4 fluorometer (Thermo Fisher Scientific) using the Qubit™ RNA HS assay kit (Thermo Fisher Scientific).
RNA extracts were treated with RQ1 RNase-Free DNase as per manufacturer’s guidelines. The quality of the RNA samples was then inspected in an Agilent 2100 bioanalyzer (Agilent Technologies) using an RNA 6000 pico assay (Agilent Technologies) according to the manufacturer’s instructions. Samples with RNA integrity number (RIN) scores greater than 7 were submitted to Novogene for paired-end sequencing of 150 bp-reads in a NovaSeq 6000 system (Illumina). Before sequencing, samples underwent a rRNA depletion step and libraries were generated using the TruSeq preparation protocol (Illumina).

value: mut-NNS-BS

value: mutant


title: Cerebellum_Pcoat+MB+_10mg per kg_Replicate3

value: 10mg/kg methylene blue


title: RNA Yeast, mut-NNS-BS, OD 0.5, rep2


title: RNA Yeast, mut-NNS-BS, OD 0.5, rep1


title: KYY_Project_YKSZ_ERT_240502_01X_0002_0540

description: Library name: RIF05

value: endometrium


title: R11 (1 µM) 10 min post-treatment, BR 2

city: Trondheim

first: Amanda

middle: Holstad

last: Singleton

address: Erling Skjalgsons gate 1

department: Department of Clinical and Molecular Medicine

country: Norway

institute: Norwegian University of Science and Technology

description: High-dose and R11 experiment

data_processing:
FASTQ files were generated using bcl2fastq2 Conversion Software v 2.20.0.422. Processing of the sequence data was done with the ProkSeq v 2.0 program using Docker, with the  E. coli K-12 substr. MG1655 reference genome ASM584v2 (RefSeq: GCF_000005845.2) and corresponding reference transcriptome. ProkSeq is a docker based, full RNA-Seq pipeline for prokaryotes, which includes quality assessment, alignment, gene counting, and analysis. The count tables countFile.csv, countFileNucleotideAvgCount.csv and countFile_TPM_CPM.tsv were exported from ProkSeq and used for further analysis.
The sample counts were were analyzed in R using the quasi-likelihood test in EdgeR v 3.36.0 from Bioconductor. Poorly expressed genes were filtered out, and library size was normalized using trimmed mean of M-value (TMM). Differentially expressed genes were defined with FDR < 0.05 and presented as LFC compared to the control.
Assembly: ASM584v2 (RefSeq: GCF_000005845.2)
Supplementary files format and content: The processed data file (countFile.csv) contains the count for each sample of each identified gene.

organism: Escherichia coli

growth_protocol: The E. coli pre-culture was diluted to OD600 = 0.0125. E. coli was grown in cation-adjusted Mueller-Hinton broth in 1 1L Applikon bioreactors as batch culitvation. Stirring was adjusted between 200 and 600 rpm to maintain a dissolved oxygen concentration above 40%. The aeration rate was kept at 500 mL min-1. pH 7 was maintained by automatic titration of 3 M HCl and 4 M NaOH.

extract_protocol:
Transcriptomics was sampled according to the protocol provided with RNeasy Mini kit (Qiagen). The sample volume was based on OD600 at each sampling timepoint to ensure 3.35 x 10^8 cells per sample. The sample was immediately added to 2 volumes of RNA protect and vortexed. After minimum 5 min, the samples were centrifuged (10 min, max rcf). The supernatant was discarded, and the pellet was frozen in N2 (l). RNA was isolated using RNeasy Mini Kit (Qiagen), according to manufacturer's protocol 4 (Enzymatic lysis and Proteinase K digestion of bacteria) and protocol 7 (Purification of total RNA) with on-column Dnase (Qiagen) digestion. The RNA concentration in each sample was measured using NanoDrop-1000 spectrophotometer. Isolated RNA was sent to the Genomics Core Facility at NTNU for further processing and sequencing.
RNA sequencing libraries were prepared using the QIAseq FastSelect 5S/16S/23S kit (Qiagen) for rRNA removal and the QIAseq stranded RNA Lib kit (Qiagen) for library construction, according to the manufacturer's instructions.  Briefly, 500 ng total RNA was used as starting material. Removal of ribosomal RNA (rRNA) was conducted by a combined heat fragmentation (89°C for 7 min) and FastSelect hybridization protocol (75-4°C ramping process) where the FastSelect reagent inhibited reverse transcription of bacterial rRNA. Next, purification was conducted using QIAseq Beads followed by a first-strand synthesis using a RNase H- Reverse Transcriptase (RT) in combination with random primers, a second-strand synthesis, end-repair, A-addition, and adapter ligation. The second-strand synthesis was performed using 5’phosphorylated random primers which enable subsequent strand-specific ligation. DNA fragments were further enriched by CleanStart library amplification (15 cycles of PCR reaction). Finally, the libraries were purified using the QIAseq Beads, quantitated by qPCR using Collibri Library Quantification Kit (Thermo Fisher Scientific), and validated using Perkin Elmer DNA 1K/12K/Hi Sensitivity Assay LabChip on a Labchip GX instrument (Perkin Elmer, USA). The size range of the DNA fragments were measured to be in the range of 270 to 570 bp and peaked around 355 bp. Prior to sequencing, libraries were normalized and pooled to 2.3 pM and subjected to clustering on three NextSeq 500 HO flowcells (Illumina, USA).

treatment_protocol: At OD600 = 0.2, E. coli was treated with either BTP-001 (1µM), BTP-001 (3 µM), or R11 (1 µM)

value: Wild type


title: RNA Yeast, mut-Nab3-BS, OD 0.5, rep3

description:
mut-Nab3-BS_merged_forward.bedgraph
mut-Nab3-BS_merged_reverse.bedgraph
RNA-seq_merged_count_table.txt


title: Cerebellum_Pcoat+MB+_10mg per kg_Replicate2


title: HZZ_Project_YKSZ_ERT_240130_01X_0001_0207

description: Library name: RIF04

value: RIF


title: Untreated control 10 min post-treatment, BR 2 (for BTP-001 3 µM and R11)

description: High-dose and R11 experiment


title: Cerebellum_Pcoat+MB+_10mg per kg_Replicate1


title: RNA Yeast, mut-Nab3-BS, OD 0.5, rep2


title: HY_Project_YKSZ_ERT_240412_01X_0001_2646

description: Library name: RIF03


title: R11 (1 µM) 10 min post-treatment, BR 3

description: High-dose and R11 experiment


title: RNA Yeast, mut-Nab3-BS, OD 0.5, rep1


title: GWY_Project_YKSZ_ERTRD2000_220430_01X_01_0936

description: Library name: RIF02


title: BTP-001 (3 µM) 10 min post-treatment; BR 2

description: High-dose and R11 experiment


title: RNA Yeast, PIC2-GFP, OD 0.5, rep2

description:
PIC2-GFP_merged_forward.bedgraph
PIC2-GFP_merged_reverse.bedgraph
RNA-seq_merged_count_table.txt

value: parental


title: Cerebellum_Pcoat+MB+_6mg per kg_Replicate2

value: 6mg/kg methylene blue


title: RNA Yeast, PIC2-GFP, OD 0.5, rep3


title: BTP-001 (3 µM) 10 min post-treatment; BR 1

description: High-dose and R11 experiment


title: BTP-001 (3 µM) 10 min post-treatment; BR 3

description: High-dose and R11 experiment


title: R11 (1 µM) 10 min post-treatment, BR 1

description: High-dose and R11 experiment


title: Cerebellum_Pcoat+MB+_6mg per kg_Replicate1

value: Cerebellum


title: RNA Yeast, PIC2-GFP, OD 0.5, rep1


title: SM_Project_YKSZ_ERT_221212_01X_01_0065

description: Library name: control02

value: control


title: Cerebellum_Pcoat+MB+_6mg per kg_Replicate3

value: Pcoat+MB+


title: Cerebellum_Pcoat+MB+_17mg per kg_Replicate1

value: 17mg/kg methylene blue


title: WYB_Project_YKSZ_ERT_230706_01X_01_0328

description: Library name: control03


title: Cerebellum_Pcoat+MB-_Replicate5

value: Pcoat+MB-


title: LM_Project_YKSZ_ERT_220719_01X_001_1004

description: Library name: control01


title: Cerebellum_Pcoat+MB-_Replicate4


title: Nrd1 Yeast, mut-NNS-BS, OD1, rep2

description:
Nrd1_PIC2-GFP_vs_mut-NNS-BS_min_peak_width_10_noblocks_merged_peaks_numbered_with_padj.gtf
Nrd1_PIC2-GFP_vs_mut-NNS-BS_min_peak_width_10_noblocks_merged_hittables_DESeq2_results.txt
Nrd1_merged_hittable_reads.txt

extract_protocol:
Following optimised crosslinking (500 mJ UV 254 nm), UV-irradiated pellets were resuspended in a volume of lysis buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40, 1 EDTA-free protease inhibitor cocktail (Roche) per 50 mL and 5 mM β-mercaptoethanol) corresponding to twice their mass. Lysates were then transferred to a fresh 50 mL tubes, where they were mixed with 2 volumes of 0.5 mm zirconia beads (Thistle Scientific), and vortexed at maximum speed for five 1-minute intervals alternated with 1-minute incubations on ice. An additional volume of lysis buffer was pipetted into each sample before partitioning the cell debris by centrifugation at 4ºC (4000 rpm, 15 minutes) and spinning down lighter impurities in an additional centrifugation step (10000 rpm, 20 minutes) at 4ºC. Immunoprecipitation of the labelled protein from the supernatant was achieved during a two-hour incubation of the sample with 75 µL of anti-FLAG® M2 magnetic beads (Sigma-Aldrich, M8823-5ML) which had been previously washed three times with 1 mL TN150 buffer. Immobilised beads underwent three 5-minute washes with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and three additional 5-minute washes in 2 mL TN150 buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol). Afterwards, beads were resuspended in 550 µL of TN150 and incubated with 1 µL of a 1:100 dilution of RNace-It™ for exactly 5 minutes at 37 °C. To prevent overdigestion of co-immunoprecipitated transcripts and elute the bait proteins from the anti-FLAG beads, the solution was mixed with 0.4 g of guanidium hydrochloride (Gu-HCl) and the necessary volume of NaCl and imidazole (pH 8.0) to reach a final concentration of 300 mM and 10 mM respectively. After removing the beads, samples were pipetted onto 50 µL of Ni-NTA agarose resin (Qiagen) equilibrated with wash buffer I (6 M Gu-HCl, 10 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and incubated at 4 °C overnight while rotating at 12 rpm.
Upon Ni-NTA binding, beads were transferred to Pierce columns (Thermo Fisher Scientific) and washed twice with 500 µL of wash buffer I before undergoing three additional washes of 500 µL with NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40, 5 mM β-mercaptoethanol). Alkaline phosphatase treatment was performed by resuspending immunoprecipitated RNA-protein conjugates in 80 µL of NP-PNK buffer containing 4 units of FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and 80 units of RNasin® ribonuclease inhibitor (Promega) and incubating the samples at 37 °C for 1 hour in the case of Nab3 and Nrd1, or 15 minutes for Sen1. Dephosphorylation was stopped upon addition of 500 µL of wash buffer. Having equilibrated the Ni-NTA agarose with three washes of 500 µL with NP-PNK, we performed adapter ligation by resuspending the beads 80 µL of reaction mix containing 0.6 µM of the applicable App-PE 3′ adapter (5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’), 30 units of T4 RNA ligase 2 truncated K227Q (New England Biolabs), 60 units RNasin® (Promega) and 10% (w/v) polyethylene glycol 8000 (PEG 8000). The reaction took place at 25 °C for six hours.  After washing once with 500 µL of wash buffer I and three times with 500 µL of NP-PNK buffer, we 5’end radiolabelled co-immunoprecipitated RNAs by incubating the Ni-NTA resin with 60 µL of NP-PNK buffer containing 30 µCi 32P-γATP (PerkinElmer) and 30 units of T4 polynucleotide kinase (New England Biolabs). After a 40-minute incubation of the reaction at 37ºC, ATP (Roche) was supplemented to a final concentration of 1 mM and the reaction continued for another 20 minutes. The reaction was stopped by washing three times with 500 µL of wash buffer I and three 500 µL washes of NP-PNK buffer followed before the addition of the 5′ linker ligation mix. The reaction took place in a volume of 80 µL of NP-PNK buffer supplemented with 1.25 µM of the fitting adapter, 40 units of T4 RNA ligase 1 (New England Biolabs), 80 units of RNasin® and 10 mM ATP (Roche).  5’ adapter ligation was left to proceed overnight at 16 °C for all bait proteins. The adapter sequences were 5’-ACACGACGCTCTTCCGATCTNNNTAAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ for PIC2-GFP_Rep1 and 5’-ACACGACGCTCTTCCGATCTNNNTCTCTAGCN-3’ for PIC2-GFP_Rep2; 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ and 5’-ACACGACGCTCTTCCGATCTNNNGACTTAGCN-3’ for mut-Nab3-BS libraries 1 and 2 respectively;  and 5’-ACACGACGCTCTTCCGATCTNNNTGGAGCN-3’,  5’-ACACGACGCTCTTCCGATCTNNNACTCAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNACAGTGN-3’ for mut-NNS-BS_Rep1  and 5’-ACACGACGCTCTTCCGATCTNNNGTGACAN-3’ for mut-NNS-BS_Rep2. After pooling suitable libraries together, the samples and its corresponding traces were submitted to Novogene, where paired-end sequencing of 150 bp-reads was performed in a NovaSeq 6000 system (Illumina).

value: mut-NNS-BS, NRD1-HTF

value: 500 mJ UV 254 nm


title: Cerebellum_Pcoat+MB-_Replicate3


title: Nrd1 Yeast, mut-Nab3-BS, OD1, rep2

description:
Nrd1_PIC2-GFP_vs_mut-Nab3-BS_min_peak_width_10_noblocks_merged_peaks_numbered_with_padj.gtf
Nrd1_PIC2-GFP_vs_mut-Nab3-BS_min_peak_width_10_noblocks_merged_hittables_DESeq2_results.txt
Nrd1_merged_hittable_reads.txt

value: mut-Nab3-BS, NRD1-HTF


title: BTP-001 (1 µM) 120 min post-treatment; BR 2

description: Time-series experiment


title: ChIP-nexus, mouse IgG, THP1, untreated, exp2

city: Melbourne

first: Jennifer

middle: Rose

last: Devlin

state: Victoria

address: Peter MacCallum Cancer Centre, 300 Grattan Street

department: The Sir Peter MacCallum Department of Oncology at Peter MacCallum Cancer Centre

country: Australia

institute: University of Melbourne

data_processing:
Adaptor sequences were removed using CutAdapt (v.1.9)  or trimgalore (v.0.4.4) and trimmed DNA-seq files were aligned to the human hg19 or hg38 genome using bowtie2 (v.2.3.4.1) or STAR (v.2.5.3a)
Bam files were generared using samtools (v.1.9) and bigWig files were generated using Deeptools (v.3.0.0) bamCompare (normaliseUsing CPM, smoothLength 150 extend 200, binSize 50)
Average profiles and metagene occupancy heatmaps were generated using Deeptools computeMatrix scale-regions and reference-point, and plotHeatmap functions
Peak calling was performed using MACS2 (v.2.2.7.1) callpeak function or GEM (v.3.4)
Peak annotation was performed using ChIPseeker
Assembly: hg19, hg38
Supplementary files format and content: bigWig
Supplementary files format and content: narrowPeak (except for input)

source_name: Blood

growth_protocol: THP1 and THP1 FLAG-CDK11-mAID cells were maintained in RPMI-16410 supplemented with 10% FBS, 2mM GlutaMax, 100U/mL penicillin and 100ug/mL streptomycin at 37C and 5% CO2

extract_protocol:
Cells were crosslinked with 11% formaldehyde for 30 minutes and quenched with 125mM glycine for 5 minutes prior to isolation of nuclei
Nuclei were sonicated with a Covaris S220 instrument for 18 x 1 minute cycles, and sonicated material was incubated overnight with antibodies and protein A/G dynabeads
Antibody-enriched DNA was isolated from chromatin-antibody-protein A/G complexes using proteinase K and DNA was purified using Zymo Research ChIP DNA Clean and Concentrator kit
For ChIP-nexus, end-repair, dA-tailing, adaptor ligation, barcode extension and lamda exonuclease digestion were performed on the chromatin-antibody-protein A/G complexes.
For ChIP-nexus lamda exonuclease digested DNA was isolated using proteinaise K digestion followed by phenol:chloroform:IAA extraction and ethanol precipitation. Single-strand DNA circularisation was peformed using CircLigase prior to library amplification.
For ChIP-seq, libraries were constructed using the NEBNext Ultra II DNA for Illumina kit as per manufacturer's instructions and size selection was performed using a PippenPrep

treatment_protocol:
THP1 cells were treated with DMSO, OTS964 (156nM), or AZ5576 (170nM) for 2 hours. LPS (1ug/mL) treatment was for 3 hours.
THP1 FLAG-CDK11-mAID cells were treated with 0.5mM 3-IAA (Auxin) or equivalent concentration of 100% ethanol for 4 hours

value: Blood

value: Acute myeloid leukeamia

value: IgG


title: Cerebellum_Pcoat-MB-_Replicate4

value: Pcoat-MB-


title: Nrd1 Yeast, PIC2-GFP, OD1, rep2

value: PIC2-GFP, NRD1-HTF


title: BTP-001 (1 µM) 75 min post-treatment; BR 2

description: Time-series experiment

value: Wild type


title: Untreated control 10 min post-treatment, BR 3 (for BTP-001 3 µM and R11)

description: High-dose and R11 experiment


title: GJ_Project_YKSZ_ERT_240812_01X_0001_0585

description: Library name: RIF01

value: endometrium

value: RIF


title: Cerebellum_Pcoat-MB-_Replicate3


title: ChIP-nexus, Pol II CTD4H8, THP1, AZ5576 170nM 2 hours, exp2, rep2

value: Pol II CTD4H8


title: Nrd1 Yeast, mut-NNS-BS, OD1, rep1

value: mut-NNS-BS

value: mutant


title: ChIP-nexus, Pol II CTD4H8, THP1, AZ5576 170nM 2 hours, exp2, rep1


title: BTP-001 (1 µM) 50 min post-treatment; BR 2

description: Time-series experiment


title: Cerebellum_Pcoat-MB-_Replicate2

value: Cerebellum


title: Nrd1 Yeast, mut-Nab3-BS, OD1, rep1

description:
Nrd1_PIC2-GFP_vs_mut-Nab3-BS_min_peak_width_10_noblocks_merged_peaks_numbered_with_padj.gtf
Nrd1_PIC2-GFP_vs_mut-Nab3-BS_min_peak_width_10_noblocks_merged_hittables_DESeq2_resultss.txt
Nrd1_merged_hittable_reads.txt


title: Untreated control 10 min post-treatment, BR1 (for BTP-001 3 µM and R11)

description: High-dose and R11 experiment


title: BTP-001 (1 µM) 25 min post-treatment; BR 2

description: Time-series experiment


title: Brainstem_Pcoat+MB+_17mg per kg_Replicate3

source_name: Brainstem

value: Brainstem

value: Pcoat+MB+

value: 17mg/kg methylene blue


title: ChIP-nexus, Pol II CTD4H8, THP1, OTS964 156nM 2 hours, exp2 rep1


title: BTP-001 (1 µM) 10 min post-treatment; BR 2

description: Time-series experiment


title: Nab3 Yeast, mut-NNS-BS, OD1, rep2

description:
Nab3_PIC2-GFP_vs_mut-NNS-BS_min_peak_width_10_noblocks_merged_peaks_numbered_with_padj.gtf
Nab3_PIC2-GFP_vs_mut-NNS-BS_min_peak_width_10_noblocks_merged_hittables_DESeq2_results.txt
Nab3_merged_hittable_reads.txt

extract_protocol:
Following optimised crosslinking (500 mJ UV 254 nm), UV-irradiated pellets were resuspended in a volume of lysis buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40, 1 EDTA-free protease inhibitor cocktail (Roche) per 50 mL and 5 mM β-mercaptoethanol) corresponding to twice their mass. Lysates were then transferred to a fresh 50 mL tubes, where they were mixed with 2 volumes of 0.5 mm zirconia beads (Thistle Scientific), and vortexed at maximum speed for five 1-minute intervals alternated with 1-minute incubations on ice. An additional volume of lysis buffer was pipetted into each sample before partitioning the cell debris by centrifugation at 4ºC (4000 rpm, 15 minutes) and spinning down lighter impurities in an additional centrifugation step (10000 rpm, 20 minutes) at 4ºC. Immunoprecipitation of the labelled protein from the supernatant was achieved during a two-hour incubation of the sample with 75 µL of anti-FLAG® M2 magnetic beads (Sigma-Aldrich, M8823-5ML) which had been previously washed three times with 1 mL TN150 buffer. Immobilised beads underwent three 5-minute washes with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and three additional 5-minute washes in 2 mL TN150 buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol). Afterwards, beads were resuspended in 550 µL of TN150 and incubated with 1 µL of a 1:100 dilution of RNace-It™ for exactly 5 minutes at 37 °C. To prevent overdigestion of co-immunoprecipitated transcripts and elute the bait proteins from the anti-FLAG beads, the solution was mixed with 0.4 g of guanidium hydrochloride (Gu-HCl) and the necessary volume of NaCl and imidazole (pH 8.0) to reach a final concentration of 300 mM and 10 mM respectively. After removing the beads, samples were pipetted onto 50 µL of Ni-NTA agarose resin (Qiagen) equilibrated with wash buffer I (6 M Gu-HCl, 10 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and incubated at 4 °C overnight while rotating at 12 rpm.
Upon Ni-NTA binding, beads were transferred to Pierce columns (Thermo Fisher Scientific) and washed twice with 500 µL of wash buffer I before undergoing three additional washes of 500 µL with NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40, 5 mM β-mercaptoethanol). Alkaline phosphatase treatment was performed by resuspending immunoprecipitated RNA-protein conjugates in 80 µL of NP-PNK buffer containing 4 units of FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and 80 units of RNasin® ribonuclease inhibitor (Promega) and incubating the samples at 37 °C for 1 hour in the case of Nab3 and Nrd1, or 15 minutes for Sen1. Dephosphorylation was stopped upon addition of 500 µL of wash buffer. Having equilibrated the Ni-NTA agarose with three washes of 500 µL with NP-PNK, we performed adapter ligation by resuspending the beads 80 µL of reaction mix containing 0.6 µM of the applicable App-PE 3′ adapter (5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’), 30 units of T4 RNA ligase 2 truncated K227Q (New England Biolabs), 60 units RNasin® (Promega) and 10% (w/v) polyethylene glycol 8000 (PEG 8000). The reaction took place at 25 °C for six hours.  After washing once with 500 µL of wash buffer I and three times with 500 µL of NP-PNK buffer, we 5’end radiolabelled co-immunoprecipitated RNAs by incubating the Ni-NTA resin with 60 µL of NP-PNK buffer containing 30 µCi 32P-γATP (PerkinElmer) and 30 units of T4 polynucleotide kinase (New England Biolabs). After a 40-minute incubation of the reaction at 37ºC, ATP (Roche) was supplemented to a final concentration of 1 mM and the reaction continued for another 20 minutes. The reaction was stopped by washing three times with 500 µL of wash buffer I and three 500 µL washes of NP-PNK buffer followed before the addition of the 5′ linker ligation mix. The reaction took place in a volume of 80 µL of NP-PNK buffer supplemented with 1.25 µM of the fitting adapter, 40 units of T4 RNA ligase 1 (New England Biolabs), 80 units of RNasin® and 10 mM ATP (Roche).  5’ adapter ligation was left to proceed overnight at 16 °C for all bait proteins. The adapter sequences were 5’-ACACGACGCTCTTCCGATCTNNNCGCTTAGCN-3’ and 5’-ACACGACGCTCTTCCGATCTNNNGACTTAGCN-3’ or 5’-ACACGACGCTCTTCCGATCTNNNTAAGCN-3’ for PIC2-GFP repeats 1 and 2 respectively; 5’-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3’ and 5’-ACACGACGCTCTTCCGATCTNNNACTCAGCN-3’ for mut-Nab3-BS replicates 1 and 2 respectively;  and 5’-ACACGACGCTCTTCCGATCTNNNGTGACAN-3’ for both mut-NNS-BS libraries. After pooling suitable libraries together, the samples and its corresponding traces were submitted to Novogene, where paired-end sequencing of 150 bp-reads was performed in a NovaSeq 6000 system (Illumina).

value: mut-NNS-BS, NAB3-HTF

value: 500 mJ UV 254 nm


title: ChIP-nexus, Pol II CTD4H8, THP1, OTS964 156nM 2 hours, exp2 rep2

value: Blood


title: Brainstem_Pcoat+MB+_17mg per kg_Replicate2


title: Cerebellum_Pcoat-MB-_Replicate1


title: Nrd1 Yeast, PIC2-GFP, OD1, rep1

value: parental


title: Nab3 Yeast, mut-Nab3-BS, OD1, rep2

description:
Nab3_PIC2-GFP_vs_mut-Nab3-BS_min_peak_width_10_noblocks_merged_peaks_numbered_with_padj.gtf
Nab3_PIC2-GFP_vs_mut-Nab3-BS_min_peak_width_10_noblocks_merged_hittables_DESeq2_results.txt
Nab3_merged_hittable_reads.txt

value: mut-Nab3-BS, NAB3-HTF


title: BTP-001 (1 µM) 1 min pre-treatment; BR 2

description: Time-series experiment


title: ChIP-nexus, Pol II CTD4H8, THP1, DMSO 2 hours, exp2, rep2

value: Acute myeloid leukeamia


title: BTP-001 (1 µM) 1 min post-treatment; BR 2

description: Time-series experiment


title: ChIP-nexus, Pol II CTD4H8, THP1, DMSO 2 hours, exp2, rep1


title: Nab3 Yeast, PIC2-GFP, OD1, rep2

value: PIC2-GFP, NAB3-HTF


title: Untreated control 120 min post-treatment; BR 2

description: Time-series experiment

value: Wild type


title: ChIP-nexus, Pol II CTD4H8, THP1, AZ5576 170nM 2 hours, exp1


title: Brainstem_Pcoat+MB+_10mg per kg_Replicate3

value: 10mg/kg methylene blue


title: Nab3 Yeast, mut-NNS-BS, OD1, rep1


title: Untreated control 75 min post-treatment; BR 2

description: Time-series experiment


title: ChIP-nexus, Pol II CTD4H8, THP1, OTS964 156nM 2 hours, exp1


title: Nab3 Yeast, mut-Nab3-BS, OD1, rep1


title: Untreated control 50 min post-treatment; BR 2

description: Time-series experiment


title: Brainstem_Pcoat+MB+_10mg per kg_Replicate1


title: ChIP-nexus, Pol II CTD4H8, THP1, DMSO 2 hours, exp1


title: Nab3 Yeast, PIC2-GFP, OD1, rep1


title: Untreated control 25 min post-treatment; BR 2

description: Time-series experiment


title: ChIP-seq, CDK11, THP1, LPS 3 hours and OTS964 2 hours


title: Brainstem_Pcoat+MB+_6mg per kg_Replicate3

value: 6mg/kg methylene blue


title: Untreated control 10 min post-treatment; BR 2

description: Time-series experiment


title: ChIP-seq, CDK11,THP1, OTS964 2 hours


title: Untreated control 1 min post-treatment; BR 2

description: Time-series experiment


title: ChIP-seq, CDK11, THP1, LPS 3 hours


title: Brainstem_Pcoat+MB+_6mg per kg_Replicate2


title: MEF, H3K9me3, Rep1. CUT&Run

city: Rochester

first: Patrick

middle: J

last: Murphy

state: New York

address: 601 Elmwood Avenue

department: Biomedical Genetics and Genomics

institute: University of Rochester

description:
Library name: H3K9me3_rep1_CUTRUN
MEF, H3K9me3, Rep1

data_processing:
cutAdapt was used on all raw fastq files, and then each was mapped with bowtie2 using mm10.
PICARD MergeSamFiles was used to convert files to BAM files, and PICARD MarkDuplicates was used to remove duplicate reads.
bigWig files were made with deeptools BAMcoverage  --normalizeUsing RPKM --binSize 10 --extendReads 100.
Assembly: mm10
Supplementary files format and content: bigWig
Library strategy: CUT&Run

source_name: mouse embyronic fibroblasts

growth_protocol: Primary MEFs were obtained from embryonic day 13.5 mouse embryos and grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin.  Cells were cultured at 37°C.

extract_protocol:
MEFs were used in the Epicyper CUT&RUN (https://www.epicypher.com/wp-content/uploads/2024/10/cutana-cutrun-protocol-2.1.pdf.) protocols.
Libraries were prepared for sequencing using the Epicyper CUT&RUN (https://www.epicypher.com/wp-content/uploads/2024/10/cutana-cutrun-protocol-2.1.pdf.) protocols. i5 and i7 sequencing adaptors were added to isolated DNA via PCR.

value: mouse embyronic fibroblasts

value: WT


title: Brainstem_Pcoat+MB-_Replicate5

value: Pcoat+MB-


title: Brainstem_Pcoat+MB+_17mg per kg_Replicate1


title: ChIP-seq, CDK11, THP1, DMSO only 2 hours,


title: Brainstem_Pcoat+MB+_6mg per kg_Replicate1


title: Untreated control 1 min pre-treatment; BR 2

description: Time-series experiment


title: Sample 6378 leukemic blasts

city: Philadelphia

first: Robert

middle: L

last: Bowman

state: PA

address: 421 Curie Blvd

department: Cancer Biology

institute: University of Pennsylvania School of Medicine

description: Library name: 6378

data_processing:
FASTQ files were aligned to hg19 using STAR Align version 2.7.8a
Genes were counted using HTSeq-Count version 2.0.3
Assembly: hg19
Supplementary files format and content: Raw sequencing counts are provided as a txt file.

source_name: leukemic blast

extract_protocol:
Patient samples were thawed and washed with PBS supplemented with 5% FBS (Corning) supplemented with DNase I (Sigma) at 50mg/mL. Cells were incubated with a fixable viability stain 510 (FVS510, BD Biosciences) for 15 min at room temperature, washed, and then stained with antibodies against human CD45 (BD Horizon BV786), CD33 (BD Horizon BV421), CD3 (BD Horizon APCR700), and CD19 (BD Horizon BB515) for 20 min at room temperature. Cells were then washed and resuspended in PBS supplemented with 2% FBS and flow sorted to isolate viable (FVS510−) CD3−/CD19−/CD45+ / CD33+ cells using a BD FACSAria III cell sorter. Following flow sorting, primary samples were washed in PBS and then extraction was performed using the Qiagen AllPrep DNA/RNA kit. The purity and concentration of the extracted DNA and RNA were assessed using a Nanodrop before downstream applications.
RNA was sent to a commercial provider, Tempus, for library preparation and sequencing on an Illumina HiSeq. RNA libraries were prepared from PolyA selected mRNA species

value: leukemic blast

tag: stratification group

tag: disease type

value: Under 2 Year Survival


title: MEF, H3K9me3, Rep2. CUT&Run

description:
Library name: H3K9me3_rep2_CUTRUN
MEF, H3K9me3, Rep2


title: BTP-001 (1 µM) 120 min post-treatment; BR 3

description: Time-series experiment


title: ChIP-seq, Pol II CTD4H8, THP1, LPS 3 hours and OTS964 2 hours

tag: tissue

tag: antibody

value: Pol II CTD4H8


title: Sample 935 leukemic blasts

description: Library name: 935

value: Good

tag: genotype

value: WILDTYPE

value: 9+ Year Survival


title: Brainstem_Pcoat+MB-_Replicate4

value: Brainstem


title: BTP-001 (1 µM) 75 min post-treatment; BR 3

description: Time-series experiment


title: ChIP-seq, Pol II CTD4H8, THP1, LPS 3 hours

value: Blood


title: Brainstem_Pcoat+MB+_10mg per kg_Replicate2

value: Pcoat+MB+


title: Sample 6063 leukemic blasts

description: Library name: 6063


title: Brainstem_Pcoat+MB-_Replicate3


title: Sample 5653 leukemic blasts

description: Library name: 5653


title: BTP-001 (1 µM) 50 min post-treatment; BR 3

description: Time-series experiment


title: Sample 5402 leukemic blasts

description: Library name: 5402


title: BTP-001 (1 µM) 25 min post-treatment; BR 3

description: Time-series experiment


title: ChIP-seq, Pol II CTD4H8,THP1, OTS964 2 hours

value: Acute myeloid leukeamia


title: Brainstem_Pcoat+MB-_Replicate2


title: ChIP-seq, Pol II CTD4H8, THP1, DMSO only 2 hours,


title: Brainstem_Pcoat+MB-_Replicate1


title: ChIP-seq, CDK11, THP1 FLAG-CDK11-AID, Auxin 0.5mM 4 hours


title: BTP-001 (1 µM) 10 min post-treatment; BR 3

description: Time-series experiment


title: ChIP-seq, CDK11, THP1 FLAG-CDK11-AID, Ethanol 4 hours


title: BTP-001 (1 µM) 1 min post-treatment; BR 3

description: Time-series experiment


title: Brainstem_Pcoat-MB-_Replicate3

value: Pcoat-MB-


title: Brainstem_Pcoat-MB-_Replicate4


title: Sample 4748 leukemic blasts

description: Library name: 4748


title: Untreated control 120 min post-treatment; BR 3

description: Time-series experiment

value: Wild type


title: Brainstem_Pcoat-MB-_Replicate2


title: Sample 5052 leukemic blasts

description: Library name: 5052


title: Brainstem_Pcoat-MB-_Replicate1


title: ChIP-seq, CDK11, THP1, OTS964 156nM 2 hours


title: ChIP-seq, CDK11, THP1, AZ5576 170nM 2 hours


title: BTP-001 (1 µM) 1 min pre-treatment; BR 3

description: Time-series experiment


title: Sample 4454 leukemic blasts

description: Library name: 4454


title: Untreated control 75 min post-treatment; BR 3

description: Time-series experiment


title: ChIP-seq, CDK11, THP1, DMSO 2 hours


title: WT_10uM_iCB_rep3

city: Valencia

first: Pedro

middle: L.

last: Rodriguez

state: Valencia

address: C/ Ingeniero Fausto Elio, s/n

country: Spain

institute: IBMCP

data_processing:
After quality analysis with FastQC and read cleaning and adaptor trimming with cutadapt, clean reads were mapped to reference genome of Arabodpsis thaliana TAIR10 using STAR. Read counts table produced by STAR was used to perform the differential expression analysis with DESeq2.
Assembly: Arabidopsis thaliana TAIR10
Supplementary files format and content: tab-delimited text fille includes raw counts for each sample

organism: Arabidopsis thaliana

source_name: whole seedlings

growth_protocol: Arabidopsis thaliana seedlings (3 d.a.g.) were transferred from plates to flashes with liquid media during 7 days

extract_protocol:
RNA was harvested using E.Z.N.A.® Plant RNA Kit (Omega Biotek).
The library was constructed using the DNBSEQ Eukaryotic Strand-specific mRNA library kit (BGI Genomics, Shenzhen, China)

treatment_protocol: the medium was refreshed, and it was supplemented with 0.1% DMSO (mock) or 10 μM ABA or 10 μM iCB . Samples were collected after 3 hours of incubation

value: whole seedlings

value: 10uM iCB


title: Sample 432 leukemic blasts

description: Library name: 432

value: 15+ Year Survival


title: Untreated control 50 min post-treatment; BR 3

description: Time-series experiment


title: WT_10uM_iCB_rep2

value: WT


title: ChIP-seq, input, THP1 untreated

value: input control


title: Untreated control 25 min post-treatment; BR 3

description: Time-series experiment


title: ChIP-seq, CDK11, THP1 untreated


title: Sample 4217 leukemic blasts

description: Library name: 4217

value: leukemic blast

value: Bad

value: Primary Refractory, Under 2 Year Survival


title: Sample 5091 leukemic blasts

description: Library name: 5091

value: Under 2 Year Survival


title: Sample 3965 leukemic blasts

description: Library name: 3965

value: WILDTYPE

value: Primary Refractory, Over 2 Year Survival


title: Untreated control 10 min post-treatment; BR 3

description: Time-series experiment


title: Sample 474 leukemic blasts

description: Library name: 474

value: Good


title: ChIP-seq, Pol II CTD4H8, THP1 untreated

value: Blood

value: Pol II CTD4H8


title: WT_10uM_ABA_rep3

value: 10uM ABA


title: ChIP-seq, Pol II CTD4H8, THP1 FLAG-CDK11-AID no OsTIR control, Auxin 0.5mM 4 hours

value: Acute myeloid leukeamia


title: Sample 3875 leukemic blasts

description: Library name: 3875


title: WT_10uM_ABA_rep2


title: Untreated control 1 min post-treatment; BR 3

description: Time-series experiment

value: Wild type


title: WT_10uM_ABA_rep1

value: whole seedlings


title: Sample 354 leukemic blasts

description: Library name: 354

value: 7+ Year Survival, died from relapsed AML


title: WT_DMSO_rep3

value: 0.1% DMSO


title: BTP-001 (1 µM) 120 min post-treatment; BR 1

description: Time-series experiment


title: ChIP-seq, Pol II CTD4H8, THP1 FLAG-CDK11-AID, Auxin 0.5mM 4 hours


title: ChIP-seq, Pol II CTD4H8, THP1 FLAG-CDK11-AID no OsTIR control, Ethanol 4 hours


title: Untreated control 1 min pre-treatment; BR 3

description: Time-series experiment


title: ChIP-seq, Pol II CTD4H8, THP1 FLAG-CDK11-AID, Ethanol 4 hours


title: BTP-001 (1 µM) 75 min post-treatment; BR 1

description: Time-series experiment


title: Sample 3344 leukemic blasts

description: Library name: 3344


title: WT_DMSO_rep2

value: WT


title: Sample 3043 leukemic blasts

description: Library name: 3043


title: WT_10uM_iCB_rep1

value: 10uM iCB


title: BTP-001 (1 µM) 50 min post-treatment; BR 1

description: Time-series experiment


title: WT_DMSO_rep1


title: ChIP-seq, Pol II CTD4H8, THP1, AZ5576 170nM 2 hours


title: Sample 2910 leukemic blasts

description: Library name: 2910

value: leukemic blast


title: BTP-001 (1 µM) 25 min post-treatment; BR 1

description: Time-series experiment


title: ChIP-seq, Pol II CTD4H8, THP1, OTS964 156nM 2 hours


title: BTP-001 (1 µM) 10 min post-treatment; BR 1

description: Time-series experiment


title: Sample 2837 leukemic blasts

description: Library name: 2837

value: Bad


title: Lung-Scaup-5dpi-rep-2

city: Daejeon

first: Woohyun

last: Kwon

address: expo-ro

country: South Korea

institute: Institute for Basic Science

description:
Library name: L-86_S2
10x Genomics

data_processing:
10x genomics Cell Ranger (version 8.0.1)
Assembly: ASM1176430v1.1 combined with H5N1 influenza KR/W811 isolate and H5N1 influenza GA/W22-145E isolate
Supplementary files format and content: matrix files

organism: Mustela putorius furo

source_name: Lung

extract_protocol:
In the initial study, 16-18 weeks old male ferrets were intranasally inoculated under anesthesia with 105.5 TCID50/mL of GA/W22-145E/22 or KR/W811/21 virus (n = 24 per group).For, scRNA-seq, one ferret per group was euthanized at 3 dpi and two ferrets per group at 5 dpi to collect brain, lungs, and cervical lymph node samples. In the second study, 16-18 weeks old male ferrets were intranasally inoculated with 105.5 TCID50/mL of GA/W22-145E/22-PB2478V, GA/W22-145E/22-NP450S, and GA/W22-145E/22-PB2478V/NP450S. At 4 and 6 dpi three per group were euthanized for tissue collection, above them one cervical lymph nodes per group were used for scRNA-seq.
10x Genomics Chromium Single Cell 3′ GEM, Library & Gel Bead kit v3.1 (PN-1000121; 10X Genomics)

value: Lung


title: ChIP-seq, Pol II CTD4H8, THP1, DMSO 2 hours


title: Sample 346 leukemic blasts

description: Library name: 346

value: 15+ Year Survival


title: BTP-001 (1 µM) 1 min post-treatment; BR 1

description: Time-series experiment


title: Lung-Scaup-5dpi-rep-1

description:
Library name: L-81_S1
10x Genomics


title: Sample 2817 leukemic blasts

description: Library name: 2817

value: WILDTYPE

value: Primary Refractory, Under 2 Year Survival


title: TT-seq, THP1, OTS964 156nM 30 minutes, 4sU

data_processing:
Adaptor sequences were removed using CutAdapt (v.1.9) and trimmed reads were aligned to the human hg19 genome or the combined hg19/dm6 genome using hisat2 (v.2.1.0) or bowtie2 (v.2.3.4.1)
Bam files were generared using samtools (v.1.9) and bigWig files were generated using Deeptools (v.3.0.0) bamCompare (normaliseUsing CPM or RPKM, smoothLength 150 or 30, extend 200, binSize 50 or 10). For TT-seq bigWig files were generated using scaleFactor based on D.melanogaster S2 spike-in.
Average profiles and metagene occupancy heatmaps were generated using Deeptools computeMatrix scale-regions and reference-point, and plotHeatmap functions
Assembly: hg19, dm6
Supplementary files format and content: bigWig
Library strategy: PRO-seq

growth_protocol: THP1 cells were maintained in RPMI-16410 supplemented with 10% FBS, 2mM GlutaMax, 100U/mL penicillin and 100ug/mL streptomycin at 37C and 5% CO2

extract_protocol:
For PRO-seq nuclei were isolated and subjected to in vitro transcription reaction (37C, 3min) with 0.05mM biotin-11-CTP. Nuclear RNA was isolated by phenol-chloroform extraction and ethanol precipitation. Nuclear RNA was hydrolysed using 1M NaOH and buffer exchange was performed using BioRad Micro Bio-Spin P30 columns. Biotin-11-CTP labelled RNA was enriched using Streptavidin-linked dynabeads and enriched RNA was isolated by phenol-chloroform extraction and ethanol precipitation.
For TT-seq total RNA was isolated by phenol-chloroform extraction and isopropanoal precipiation. Isolated human RNA was combined with a D.melanogaster S2 4SU-labelled RNA spike-in (5%) and the RNA mixture was fragmented by sonication using the Covaris S220 and AFA Fiber tubes at maximum power for 15 seconds. Fragmented 4sU-labelled RNA was biotinylated using EZ-link HPDP-Biotin and enriched using Miltenyi uMACS streptavidin beads and columns. Enriched RNA was purified using the Qiagen RNeasy MinElute kit as per manufacturer's instructions.
Libraries were generated using the NEBNext Ultra II Directional RNA Library prep kit and NEBNext rRNA Depletion kit as per manufacturer's instructions

treatment_protocol:
For PRO-seq exponentially growing cells were treated with DMSO, OTS964 (156nM) or AZ'5576 (170nM) for 2 hours; cells were treated with DRB (100uM) for 4 hours prior to washout and treatment with DMSO, OTS964 (156nM) or AZ5576 (170nM) for 10 minutes.
For TT-seq exponentially growing cells were treated with DMSO or OTS964 (156nM) for 30 minutes and pulse labelled with 1mM 4sU for the final 5 minutes.


title: BTP-001 (1 µM) 1 min pre-treatment; BR 1

description: Time-series experiment


title: TT-seq, THP1, DMSO 30 minutes, 4sU


title: Untreated control 120 min post-treatment; BR 1

description: Time-series experiment


title: Lung-Scaup-3dpi-rep-1

description:
Library name: L-66_S2
10x Genomics


title: Lung-W811-5dpi-rep-1

description:
Library name: L-71_S3
10x Genomics


title: Untreated control 75 min post-treatment; BR 1

description: Time-series experiment


title: Sample 2320 leukemic blasts

description: Library name: 2320

value: Primary Refractory, Over 2 Year Survival


title: PRO-seq, THP1, OTS964 156nM 2 hours

molecule: nuclear RNA

value: Blood


title: Sample 2293 leukemic blasts

description: Library name: 2293


title: PRO-seq, THP1, DMSO 2 hours


title: Lung-W811-3dpi-rep-1

description:
Library name: L-61_S1
10x Genomics


title: Untreated control 50 min post-treatment; BR 1

description: Time-series experiment


title: Untreated control 25 min post-treatment; BR 1

description: Time-series experiment


title: Mesenteric lymph node-scaup-4dpi-rep-1

description:
Library name: M_LN-Scaup_S1
10x Genomics

source_name: Mesenteric lymph node

value: Mesenteric lymph node


title: UTR-seq, THP1, LPS 3 hours and OTS964 2 hours, rep3

data_processing:
SLAM-seq files were processed using the slamdunk package (v.0.2.4)
SLAM-seq files were mapped to a combined hg38/dm6 genome using NextGenMap as part of the slamdunk package
SLAM-seq files were filtered and counted using slamdunk package
3'UTR-seq files were mapped to a hg19 genome using hisat2 (v.2.1.0), bam files were generared using samtools (v.1.9) and counted using Subread (v.2.0.0) featureCounts package
Assembly: hg19, hg38, dm6
Supplementary files format and content: tab separated values files for total and nascent counts for each SLAM-seq sample
Supplementary files format and content: tab delimited text files for 3'UTR-seq counts for all 3'UTR samples

extract_protocol:
For SLAM-seq total RNA was isolated by phenol-chloroform extraction and isopropanol precipitation - total RNA was incubated with 10mM iodoacetamide for 4sU conversion prior to recovery by ethanol precipitation.
For 3'UTR-seq total RNA was isolated using the Zymo Research Direct-zol RNA isolation kit
Libraries were prepared using the Lexogen QuantSeq 3'mRNA-Seq kit as per manufacturer's instructions

treatment_protocol:
For SLAM-seq THP1 FLAG-CDK11-mAID cells were treated with 0.5mM 3-IAA (Auxin) or equivalent concentration of 100% ethanol for 4 hours, and pulse labelled with 100uM 4sU for the final 1 hour.
For 3'UTR-seq AML cells were treated with OTS964 (156nM) for 2 hours, LPS (1ug/mL) for 3 hours, or both

value: LPS 1ug/mL, OTS964 156nM


title: Sample 2077 leukemic blasts

description: Library name: 2077


title: Untreated control 10 min post-treatment; BR 1

description: Time-series experiment


title: Sample 1756 leukemic blasts

description: Library name: 1756


title: Peripheral lymph node-scaup-4dpi-rep-1

description:
Library name: P_LN-Scaup_S2
10x Genomics

source_name: Axillary lymph node

value: Axillary lymph node


title: Sample 2753 leukemic blasts

description: Library name: 2753

value: Good

value: 8+ Year Survival


title: UTR-seq, THP1, OTS964 2 hours, rep3

value: Acute myeloid leukeamia


title: Sample 2582 leukemic blasts

description: Library name: 2582

value: 5+ Year Survival, died from relapsed AML


title: Sample 1737 leukemic blasts

description: Library name: 1737

value: 10+ Year Survival


title: UTR-seq, THP1, LPS 3 hours, rep3

value: LPS 1ug/mL


title: Lung-W811-5dpi-rep-2

description:
Library name: L-76_S4
10x Genomics


title: Sample 1532 leukemic blasts

description: Library name: 1532

value: leukemic blast

value: 11+ Year Survival


title: Mesenteric lymph node-W811-4dpi-rep-1

description:
Library name: M_LN-W811_S3
10x Genomics


title: Untreated control 1 min post-treatment; BR 1

description: Time-series experiment

value: Wild type


title: PRO-seq, THP1, AZ5576 170nM 2 hours


title: Peripheral lymph node-W811-4dpi-rep-1

description:
Library name: P_LN-W811_S1
10x Genomics


title: Dirty_VM_rep3

city: Ithaca

first: Andrew

last: Grimson

state: NY

address: 445 Biotechnology Building

department: Molecular Biology & Genetics

institute: Cornell University

data_processing:
Mapper.pl in miRDeep2 was used for preprocessing and adapter removal (-d -e -h -k TGGAATTCTCGGGTGCCAAGG -l 18 -m -v). Quantifier.pl in miRDeep2 was used to map sequencing reads to miRNAs (-t mmu -d -e 0 -W). Sequences of precursors and mature miRNAs in mouse were downloaded from miRbase v22.
Assembly: mm10
Supplementary files format and content: Text file includes raw counts for each sample

source_name: spleen

extract_protocol:
Splenic CD8+ T cells were isolated and purified using positive selection with CD8a microbeads (Miltenyi). Purified CD8+ T cells were stained using antibodies against CD4 (Gk1.5), CD44 (IM7), CD122 (TM-B1), CD49d (R1-2), and CD8a (53-6.7). ‘Stamped VMs’ populations were sorted for CD4-CD8+CD49dloGFP+CD44hi, and ‘stamped TN’ populations were sorted for CD4- CD8+CD49dloGFP+CD44lo. Samples were sorted using a BD FACS Aria Fusion II equipped with four lasers. Samples were sorted to >95% cell purity as determined by a post-sort check. 10,000 FACS sorted cells were placed in Trizol and RNA was extracted according to manufacturer’s instructions, with the addition of a chloroform extraction, Glycoblue (ThermoFisher) carrier prior to precipitation (1 hour at 4°C), and a second wash of the pelleted RNA in 70% ethanol. RNA integrity was confirmed on a Fragment Analyzer (AATI).
Libraries were prepared with NEBNext Small RNA Library Prep Kit (New England Biolabs) using 60-100ng total RNA as input, size selected for insert sizes ~18-35bp.
small RNA-seq

value: spleen

value: CD8+ T cells


title: UTR-seq, THP1, DMSO only 2 hours, rep3

value: DMSO


title: Peripheral lymph node-W811-4dpi-rep-2

description:
Library name: P_LN-W811_S2
10x Genomics

source_name: Inguinal lymph node

value: Inguinal lymph node


title: Peripheral lymph node-scaup-4dpi-rep-2

description:
Library name: P_LN-Scaup_S3
10x Genomics


title: Dirty_VM_rep2


title: UTR-seq, THP1, OTS964 2 hours, rep2


title: Dirty_VM_rep1