title: SRA01-04, RORα-OE, 24-h selenite treatment, rep 2

status: Public on Oct 05 2025

city: Beijing

first: Jing

last: Tao

address: NO.1 Dongjiaominxiang Street

institute: Beijing Tongren Hospital, Beijing Ophthalmology and Visual Science Key Lab, Capital Medical University

description:
Library name: Sample 7
FPKM.B2

data_processing:
Illumina Sequencing
Sequence reads were trimmed for adaptor sequence/low-quality sequence using Fastp v0.23.1
Trimmed sequence reads were mapped to GRCh38.109 using Hisat2 v2.2.1 with default settings
Read count extraction was performed using Htseq
Assembly: Homo_sapiens.GRCh38.dna.primary_assembly.fa;Homo_sapiens.GRCh38.109.gtf
Supplementary files format and content: fpkm and count

extract_protocol:
RNA was extracted by using TRIzol reagent.
Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified. After the library construction is completed, preliminary quantification is carried out using Qubit, and the library is diluted to 1.5 ng/ul. Subsequently, the inserted fragments of the library are detected. Once the inserted fragments meet expectations, the effective concentration of the library is accurately quantified using qRT-PCR to ensure the quality of the library.

value: lens epithelial cells

value: RORA overexpression

value: Selenite


title: SRA01-04, RORα-OE, 24-h selenite treatment, rep 3

type: SRA

country: China

description:
Library name: Sample 8
FPKM.B3

library_source: transcriptomic

molecule: total RNA

organism: Homo sapiens

tag: cell line

tag: cell type

tag: genotype

tag: treatment


title: SRA01-04, RORα-OE, 24-h selenite treatment, rep 1

description:
Library name: Sample 6
FPKM.B1


title: SRA01-04, EV, 24-h selenite treatment, rep 3

description:
Library name: Sample 3
FPKM.A3

value: Empty vector


title: SRA01-04, RORα-OE, 24-h selenite treatment, rep 4

description:
Library name: Sample 9
FPKM.B4


title: SRA01-04, EV, 24-h selenite treatment, rep 2

description:
Library name: Sample 2
FPKM.A2


title: SRA01-04, EV, 24-h selenite treatment, rep 4

description:
Library name: Sample 4
FPKM.A4


title: SRA01-04, EV, 24-h selenite treatment, rep 1

description:
Library name: Sample 1
FPKM.A1


title: 5637 cells, shCBX7_2

city: Changsha

first: Huaiyuan

last: Liang

address: 183 Renmin Middle Road

institute: Institute of Urological Cancer, Central South University

data_processing:
Total RNA qualification
mRNA enrichment
Double-strand cDNA synthesis
End repair, ploy-A & adaptor addition
Fragment selection and PCR
Library qualty assessement
illumina sequencing
Assembly: mm10

Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

source_name: bladder

growth_protocol: 786-O cells and 5637 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in 5% CO2.

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® following manufacturer's protocols.

tag: tissue

value: bladder

value: 5637 cells

value: bladder cancer cells

value: case


title: 5637 cells, shCBX7_1


title: scATACseq_Multiome_CAR_T_in_vitro

status: Public on Oct 06 2025

city: Dallas

first: Chen

last: Yao

state: Texas

address: 5901 FOREST PARK RD, NS6 218

country: USA

institute: UT Southwestern Medical Center

description:
Library name: Sample 2
10x Genomics ATAC

library_source: genomic

data_processing:
ScRNAseq: Fastq files of gene expression and Hashtag libraries were aligned to mm10 using cellranger count (Cell Ranger v6.0.0), which perform filtering, barcode counting, and UMI counting at the same time.
Multiome: Cell versus gene matrix of UMI counts for gene expression assay and cell versus fragment matrix for ATAC assay were generated from fastq files aligned to mm10 using cellranger-arc (v 2.0.0).
Assembly: mm10
Supplementary files format and content: ScRNA-Seq: matrix H5 files
Supplementary files format and content: SCATAC-Seq: tab-separated values files

molecule: genomic DNA

organism: Mus musculus

source_name: Spleen

extract_protocol:
CAR T cells isolated from spleen were sorted for scRNA-Seq or multiome anallysis.
We used Chromium Next GEM Single Cell 5' Kit (10x Genomics) to generate scRNA-Seq libraries according to the manufacturer’s protocol. We used Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits (10x Genomics) to generate scRNA-Seq and scATAC-Seq libraries according to the manufacturer’s protocol.

value: Spleen

value: in vitro

value: CD8+ CAR T cells

value: WT

tag: batch


title: 5637 cells, shCBX7_3


title: 5638 cells, sh control_1

value: control


title: C1SAN/CSB/WT cell line biological rep 3

city: Durham

first: Hana

middle: Isadora

last: Wasserman

state: NC

address: 3308 Old Chapel Hill Road

institute: Duke University

description: Library name: C1SAN/CSB/WT G2946 bio rep 3

data_processing:
Raw ATAC-seq reads were processed using the ENCODE ATAC-seq-pipeline (v2.2.3) and hg19 reference genome for irreproducibility detection rate (IDR) analysis of the four biological replicates. Per ENCODE pipeline recommendations, conservative IDR peakspeaks were called with MACS2 narrowPeak settings using a threshold p-value<0.01 and subsequent IDR threshold of 0.05.
Assembly: hg19
Supplementary files format and content: narrowPeak files containing IDR conservative peak calls from the four biological replicates

growth_protocol: Human skin fibroblast cells were grown in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) containing 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. Prior to processing, cells were resuspended in growth media (containing 10% DMSO) and frozen for −1°C/min for 24 hours.

extract_protocol:
Four biological replicates of the human skin fibroblasts CS1AN/CSB/WT cell line were prepared with the Omni ATAC-seq protocol, using approximately 50,000 cells per library. Omni ATAC-seq protocol was performed as described in Corces et al. Nature Methods 2017.Oct;14(10):959-962 "An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues".
ATAC-seq

value: Skin fibroblast


title: 786-O cells, shCBX7_3

source_name: kidney

value: kidney

value: 786-O cells

value: renal cancer cells


title: 5639 cells, sh control_1


title: 786-O cells, shCBX7_1


title: 786-O cells, sh control_3


title: 786-O cells, shCBX7_2


title: 786-O cells, sh control_1


title: 786-O cells, sh control_2


title: SRA01-04, RORα-OE, 24-h selenite treatment, rep 5

description:
Library name: Sample 10
FPKM.B5

value: lens epithelial cells


title: Control 4

city: Chongqing

first: Gege

last: Li

address: No.76, Linjiang Road, Yuzhong District

department: Cardiovascular Medicine

institute: The Second Hospital of Chongqing Medical University

description: circRNA

data_processing:
Basecalls performed using Sanger/Illumina version 1.9
Trimmomatic 0.38 used for removing adaptor and low-quality sequence.
The reads were mapped  to genome using the STAR 2.6.1a.
DCC 0.5.0 was used to identify the cirRNA and to estimate the circRNA expression.
Differentially expressed genes were identified using the edgeR 3.34.0 program.
R was used to generated the figure.
Genome_build: GRCH37(hg19)
Supplementary_files_format_and_content: Matrix table include TMM values for each Sample.

source_name: Plasma

growth_protocol: Samples are packed in  blood collection tubes containing  protective solution and stored at minus 80°C until RNA extraction.

extract_protocol:
From blood samples containing protective fluid, total RNA was extracted using Magen Hipure PX Blood RNA kit and according to the kit instructions. Then the total RNA extracted was sampled for quality control. RNA concentration was measured with NanoDrop and RIN values were detected with the Agilent 2100 Bioanalyzer.
After the samples passed quality control, libraries were constructed using the KAPA RNA Hyperprep Kit with RiboErase (HMR) for illumina library building kit, where the designed DNA probes were first hybridized with the RNA samples, thus removing rRNA from the total RNA; linear RNA was removed by RNase R, and then the RNA was fragmented as the first strand of cDNA,  and the strand-specific method was used to label the second strand cDNA by dUTP, and the end repair was completed in this step; followed by the steps of adding-A tail, ligation junction, ligation product purification and fragment size sorting, and library amplification, etc. After amplification, the RNA-Seq library was purified and recovered by magnetic beads.

value: Plasma

tag: Sex

value: female

tag: hypertension

value: negative

tag: diabetes

tag: dyslipidemia

value: positive

tag: copd

tag: renal dysfunction

tag: stroke

tag: smoking

tag: alcohol drinking

tag: coronary lesion

value: normal


title: Control 3

description: circRNA


title: CTO Patient 3

description: circRNA

value: male

value: RCA CTO


title: Control 2

description: circRNA


title: CTO Patient 2

description: circRNA


title: Control 1

description: circRNA


title: CTO Patient 1

description: circRNA

value: LAD CTO


title: CTO Patient 4

description: circRNA


title: Jurkat, Control, replicate 3

city: Boston

first: Ryan

middle: A.

last: Flynn

state: MA

address: 1 Blackfan Circle

department: Department of Pediatrics

institute: Boston Children’s Hospital

description:
Library name: 35h
count_matrix_homo.csv
counts_long.csv

library_source: other

data_processing:
FASTQ files were demultiplexed by Illumina software. Reads were trimmed with cutadapt (v4.9) to remove UMIs, adapters, poly(A/G) tails, and low-quality bases, discarding reads <15 nt. Trimmed reads were aligned with bowtie2 (v2.5.4) to curated small ncRNA transcriptomes (human, mouse, or dog), retaining multimappers. Multi-mapped reads were resolved by an EM-based probabilistic assignment scheme adapted from salmon/alevin, yielding transcript-level counts. After EM hard-assignment, per-base pileups were generated from BAM files using samtools (v1.21) mpileup and processed with cpup (https://github.com/y9c/cpup) to extract per-position base counts (reference vs. non-reference bases, insertions, deletions, skips, mismatches). Count matrices were annotated with sample metadata (Input, IP, Control) and deposited as processed data. Software: cutadapt v4.9; bowtie2 v2.5.4; samtools v1.21; cpup v0.1; R v4.3.3
Assembly: Curated small ncRNA transcriptomes (see GitHub: www.github.com/FlynnLab/rPAL-seq)
Supplementary files format and content: count_matrix_homo.csv, count_matrix_mus.csv, count_matrix_canis.csv: Species-specific, EM-assigned transcript-level raw read counts (rounded to nearest integer). Each row corresponds to a transcript. Each column corresponds to a library. Values are raw counts used for DESeq2 analysis.
Supplementary files format and content: counts_long.csv: Per-base variant count matrix in long format. Each row represents one genomic position within a transcript. Columns include transcript/position identifiers (chr_pos, chr, pos, ref_base), sample metadata (super_group, group_name, replicates, pair_id, sample_id, sample, condition), sequencing depth (depth_raw), reference counts (REF), variant subcategories (SGID, A_star, C_star, G_star, T_star), and summary totals (mismatches, total_variant, nonvariant). Values are raw base counts used as input for variant enrichment analyses.

molecule: other

source_name: Jurkat

growth_protocol: OCI-AML2, OCI-AML3, Molm13, Jurkat, Jeko1, Nalm6, and EMT6 cells were cultured in RPMI-1640 medium (Gibco, cat. no. 61870036); HeLa, A549, 293T, Huh7, LPS853 and LN308 were cultured in DMEM medium (Gibco, cat. no. 10566024). DiFi and MDCK cells were cultured in DMEM medium supplemented with 1% L-glutamine (Gibco, cat. no. 25030081) and 1% non-essential amino acids (Gibco, cat. no. 11140050). All media were supplemented with 10% FBS (R&D Systems, cat. no. S11150) and 1% penicillin-streptomycin (Gibco, cat. no. 15140122). Cells were maintained at 37 °C, 5% CO2. All cell lines tested negative for mycoplasma contamination (Universal mycoplasma detection kit; ATCC, cat. no. 30-1012K).

extract_protocol:
Cells were washed with PBS and lysed directly in RNAzol (Molecular Research Center) without trypsinization (1 mL per 10 cm plate of adherent cells or per 5 × 10^6 suspension cells). Lysates were homogenized, clarified by centrifugation, and crude small RNA was precipitated with isopropanol and water. RNA was purified on Zymo Research spin columns using guanidine/ethanol washes, on-column Proteinase K digestion, and water elution. Purified RNA was further treated with StcE mucinase (Sigma) followed by an additional Zymo column cleanup. Small RNAs were fractionated by mixing RNA with adjusted RNA binding buffer (Zymo RNA binding buffer mixed with one volume of ethanol) and recovered from the flow-through on a second Zymo column._x000B_To obtain secreted fractions, cells were cultured to ~80% confluence, washed with PBS, and incubated in serum-free medium for 48 h. Conditioned medium was cleared by centrifugation and filtration, concentrated with a 100 kDa cutoff filter, and ultracentrifuged to obtain small EVs (sEVs). Exomeres were isolated from the supernatant by additional ultracentrifugation, and supermeres were recovered from the exomere supernatant by higher-speed ultracentrifugation. Crude sEVs were further purified by iodixanol (Sigma) density-gradient centrifugation, and pooled fractions containing sEVs were washed and pelleted in PBS.
Total or small RNA (50 ng–2 µg) was 3' polyadenylated with end blocking and purified. Polyadenylated RNA was subjected to enhanced rPAL labeling, biotin enrichment on neutravidin beads, and sialidase-mediated release of sialoglycoRNA. Released RNA was reverse-transcribed using anchored oligo-dT primers with template switching, followed by PCR amplification with dual-indexed primers. Amplicons (200–500 bp) were purified by gel extraction and bead clean-up. Libraries were assessed by TapeStation, pooled by concentration, and sequenced on an Illumina NovaSeq X (single-end 100 bp, dual 8 bp indexes).

treatment_protocol: For production of extracellular vesicles (EVs) and non-vesicular extracellular nanoparticles (NVEPs), cells were cultured until 80% confluent. The cells were then washed three times with PBS and cultured in serum-free medium for 48 h.

value: Jurkat

value: hematopoietic

tag: sample group

value: Jurkat, Control


title: Jurkat, Input, replicate 4

description:
Library name: 36i
count_matrix_homo.csv
counts_long.csv

tag: cell line

value: Jurkat, Input


title: SRA01-04, EV, 24-h selenite treatment, rep 5

description:
Library name: Sample 5
FPKM.A5

tag: cell type

value: Empty vector

value: Selenite


title: AMs_Cg_D_7

city: klong luang

first: Siranart

last: Jeerawattanawart

state: Not US or Canada

address: 99 Moo 18, Paholyothin Road

department: Department of Medical Technology,Faculty of Allied Health Sciences

country: Thailand

institute: Thammasat University

data_processing:
The raw sequence read was assessed for quality using an in-house Perl script quality control, which is demonstrated by the per-base quality Phred Q score30, sequencing error rate distribution, and per-read GC content
raw sequence reads were aligned to the mouse genome (Mus musculus, GRCm38) using TopHat2 v2.0.12/Bowtie v2.2.3.
Read data were annotated using NCBI BLAST v2.2.28 based on known mouse genes with an expected value of 1.0 x 10-10
To estimate gene expression levels, HTSeq v0.6.1.was used to count the read numbers mapped of each gene and calculated the fragments per kilobase of transcript per million (FPKM) of base pairs sequenced which is a normalized measure of relative abundance of transcripts according to the length and reads count mapped of the gene.
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes RPKM values for each sample

source_name: Lung

extract_protocol:
Total RNA was extracted from isolated alveolar macrophages from C. gattii-infected mice (6-10 mice/group) using a RNeasy plus mini kit (Qiagen) .  The pooled total RNA at 600 ng was used for cDNA library preparation and sequencing.
The paired-end cDNA library was prepared using the NEBNext Ultra RNA library prep kit for Illumina (New England BioLabs, Ipswich, MA).

treatment_protocol: BALB/c mice were anesthetized and infected with C. gattii R265 (5 × 104 cells/mouse) via the intranasal route. After 7 days of infection, mice were sacrificed through CO₂ inhalation, alveolar macrophages were extracted from lung tissue. The alveolar macrophage population (Gr.1−CD11c+CD11bintSiglec F+) was isolated by using  the FACSAria III sorter.

value: Lung

value: Alveolar macrophages

tag: strain

value: BALB/c

tag: agent

value: Cryptococcus gattii

tag: time

value: Day 7


title: Jurkat, IP, replicate 4

description:
Library name: 36p
count_matrix_homo.csv
counts_long.csv

value: Jurkat, IP


title: Jurkat, Input, replicate 2

description:
Library name: 34i
count_matrix_homo.csv
counts_long.csv


title: 5637 cells, sh control_1

tag: tissue

value: bladder

value: 5637 cells

value: bladder cancer cells

tag: genotype

value: control

tag: treatment


title: Jurkat, IP, replicate 2

description:
Library name: 34p
count_matrix_homo.csv
counts_long.csv


title: Jurkat, Input, replicate 3

description:
Library name: 35i
count_matrix_homo.csv
counts_long.csv


title: Jurkat, IP, replicate 3

description:
Library name: 35p
count_matrix_homo.csv
counts_long.csv


title: Jurkat, Control, replicate 2

description:
Library name: 34h
count_matrix_homo.csv
counts_long.csv


title: Jurkat, Control, replicate 4

description:
Library name: 36h
count_matrix_homo.csv
counts_long.csv


title: Jurkat, IP, replicate 1

description:
Library name: 33p
count_matrix_homo.csv
counts_long.csv


title: Jurkat, Control, replicate 1

description:
Library name: 33h
count_matrix_homo.csv
counts_long.csv


title: 231/Ctrl rep1

city: wuhan

first: Wei

last: Yan

state: Hubei

address: Bayi Street 229 in Wuchang district

department: College of life science

institute: Wuhan University

description: Library name: Sample 1

data_processing:
The raw paired end reads were trimmed and quality controlled by SeqPrep  and Sickle with default parameters.
Then clean reads were separately aligned to reference genome with orientation mode using HISAT2 software.
The mapped reads of each sample were assembled by StringTie in a reference-based approach.
The expression level of each transcript was calculated according to the transcripts per million reads (TPM) method.RSEM was used to quantify gene abundances.
Assembly: GRCm39
Supplementary files format and content: tab-delimited text files include TPM values for each Sample

growth_protocol: MDA-MB-231 or MDA-MB-231/DPP3 KO cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37°C

extract_protocol:
RNA-seq transcriptome library was prepared following TruSeq RNA sample preparation Kit from Illumina (San Diego, CA) using 1 μg of total RNA.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: orthotopic injection 2 × 105 MDA-MB-231 cells into the fourth pair of mammary fat pad


title: 231/DPP3 KO rep3

description: Library name: Sample 6


title: 231/DPP3 KO rep2

description: Library name: Sample 5


title: 232/Ctrl rep3

description: Library name: Sample 3


title: 231/DPP3 KO rep1

description: Library name: Sample 4


title: 231/Ctrl rep2

description: Library name: Sample 2


title: F123 nucleolus 50

city: Zurich

first: Raffaella

last: Santoro

address: Winterthurerstrasse 190

department: Dep. of Molecular Mechanisms  of Disease

country: Switzerland

institute: University of Zurich

data_processing:
NoLM-seq reads were aligned to the mouse mm10 reference genome using Bowtie2 (version 2.5.0; (Langmead & Salzberg, 2012)) with default parameters. PCR duplicates were removed using samtools (Danecek et al, 2021). Positive NAD bins were called using SICER2 (Zang et al, 2020), using the sequenced gDNA file as the control file and the parameters w=100000, g=100000, fdr=0.0001. Bigwig files were created from the bam files using bamCoverage from deeptools (Ramirez et al, 2014). For F123-ESCs, N-masked genome was created using the mm10 reference genome and the Variant Call Format (VCF) file from the mouse genome project containing the SNP information for all strains. The SNPsplit package (Krueger & Andrews, 2016) was used to read the VCF file and extract the SNP information for the S129/SvJae and CAST strains and N-mask for the mm10 genome. The reads were aligned using Bowtie2 to this N-masked reference genome using default parameters. SNPsplit was then used to tag the SNPs for S129/SvJae and CAST alleles in the bam file and sort the haplotype-specific bam files for both the alleles. bamCoverage and Sicer2 positive NAD calling was performed independently on the allele-specific bam files. Integrative Genome Viewer (IGV, version 2.15.4) (Robinson et al, 2011) was used to visualize and extract representative single nucleolus NoLMseq bigwig and bed tracks.
Assembly: mm10
Supplementary files format and content: BED

source_name: F123 hyrbid ESC

growth_protocol: 129 mouse embryonic stem cells (E14 line) were cultured in serum medium containing Dulbecco’s modified Eagle’s medium + Glutamax (Life Technologies), 15% FCS (Life Technologies; Cat no. 10270106 FBS South American), 1× MEM NEAA (Life Technologies), 100 μM β-mercaptoethanol, recombinant leukemia inhibitory factor, LIF (Polygene, 1000 U/ml), 1× penicillin/streptomycin (Life Technologies). ESCs were seeded at a density of 50,000 cells/cm2 in culture dishes (Corning CellBIND surface) coated with 0.1% gelatin without a feeder layer. Propagation of cells was carried out every 2 days using enzymatic cell dissociation. ESCs were treated with Actinomycin D (10ng/ml) for 24h before downstream processing. Neural progenitor cells were generated from ESCs, according to a previously established protocol (Joshi et al, 2015). In brief, differentiation used a suspension-based embryoid bodies formation (Bacteriological Petri Dishes, Bio-one with vents, Greiner). The neural differentiation media (DMEM, 10% fetal calf serum, 1× MEM NEAA, 2 mM Pen/Strep, β-mercaptoethanol, and sodium pyruvate) was filtered through 0.22 μm filters and stored at 4 °C. During the 8-day differentiation procedure, media was exchanged every 2 days. In the last 4 days of differentiation, the media was supplemented with 2 μM retinoic acid to generate neural precursors that are Pax-6- positive radial glial cells. F123-ESCs (mouse male, hybrid cell line, F1 S129/Jae and Cast mouse cross) (Gribnau et al, 2003) were cultured on a layer of mitotically inactivated feeder murine embryonic fibroblasts under standard conditions (DMEM, supplemented with 15 % KSR, 1x Glutamax, 10 mM non-essential amino acids, 50 µM beta-mercaptoethanol, 1,000 U/ml LIF). Media was changed every 24h. Before harvesting, mESCs were passaged onto feeder-free ESGRO-gelatin (EMD Millipore SF008). coated plates for at least 2 passages to remove feeder cells.

extract_protocol:
For laser-microdissection, cells were grown on ESGRO-gelatin coated UV- irradiated PET membrane slides (1.4um thick, RNAse-free, MMI, 50102) for 24h. Cells were washed twice with 1X PBS and fixed with EM-grade freshly-depolymerised 3% PFA in 1X PBS for 30mins. The slides were then placed in 70% ethanol for 1min and air dried. The slides were then washed with filtered ultra-pure H2O (3x 1 min each) and air dried. Single nucleoli were laser microdissected with the MMI Laser Capture Microdissection Nikon microscope (Molecular Machines & Industries) into single 0.2 ml tubes (transparent MMI caps, 50208). Nucleoli were identified by bright-field imaging. A 0.2ml cap filled with transparent adhesive material was lowered onto the cells. A laser was used to cut the PET membrane surrounding each nucleolus and the cut PET membrane section along with the single nucleolus was lifted off by the sticky cap. Each single nucleolus was collected in a separate cap and stored for further processing. Whole-genome amplification (WGA) was carried out using the MALBAC single cell WGA kit (YK001A) following manufacturer’s recommendations with minor modifications. The entire WGA reaction was carried out in the same 0.2ml tube with a single nucleolus. Another tube, with an empty section was used as a negative control whereas 30 pg of whole cell DNA were used as a positive control for the WGA reaction. The cell lysis reaction was first carried out by using a heated lid at 50 °C for 50 mins and then samples were collected by briefly spinning and proceeding with the cell lysis reaction again at 50 °C for 50 mins, followed by inactivation at 80 °C for 10mins. The reaction is then cooled at 4 °C. The lysis step is followed by a pre-amplification step which involves the quasilinear amplification using random primers at 20 °C progressively with 10 °C steps till 70 °C. After cooling, the samples were subjected to a final amplification step by 20 cycles of PCR. WGA-amplified DNA was purified using a Qiagen MinElute PCR Purification Kit and eluted in 140 μl ultrapure library-grade H2O. The amplified nucleolar DNA was checked on a 2% agarose gel to select positive samples showing smears corresponding to amplified DNA similar to the positive control. The positive samples were further checked for the presence of rDNA and the absence of Tubulin gene by PCR on an agarose gel.
The samples which passed the quality control check were then used for library preparation and sequencing. 120 ml of the reaction was first sonicated in split-cap tubes by Covaris E220. Depending on the initial size of the smear, the durations were altered slightly to get fragments around 300-500nt in length keeping other parameters the same. For most of the samples with fragments under 1500bp, following parameters were used- Peak power- 75, Duty factor- 20, cycle/bursts- 1000, duration- 35s. The samples were then purified and concentrated with AMPure XP beads (Beckman Coulter, A63881) and used for Illumina library using the NEBNext ultra II kit (NEB E7600). Briefly, NoLMseq samples (10-100 ng) were end-repaired and polyadenylated before the ligation of Illumina compatible adapters. The adapters contain the index for multiplexing. Library concentrations were estimated using a Qubit®  4 fluorometer (Thermo Fisher Scientific) and the 4200 TapeStation system and libraries were pooled together in batches averaging 40 samples. Each library pool was sequenced in single end 100bp in one lane of Novaseq6000 to get an average 10 million reads per library.
NoLM-seq

value: F123 hyrbid ESC

value: Untreated


title: F123 scRNAseq

description: Library name: F123_scRNAseq

library_source: transcriptomic single cell

data_processing:
Data processing was done by the 10x cell ranger software
Assembly: mm10
Supplementary files format and content: Matrix and tab separated value files

molecule: polyA RNA

growth_protocol: F123-ESCs (mouse male, hybrid cell line, F1 S129/Jae and Cast mouse cross) 41 were cultured on a layer of mitotically inactivated feeder murine embryonic fibroblasts under standard conditions (DMEM, supplemented with 15 % KSR, 1x Glutamax, 10 mM non-essential amino acids, 50 µM beta-mercaptoethanol, 1,000 U/ml LIF). Media was changed every 24h. Before harvesting, mESCs were passaged onto feeder-free ESGRO-gelatin (EMD Millipore SF008). coated plates for at least 2 passages to remove feeder cells. Cells were harvested after approximately 48 h at 70 – 80 % confluency.

extract_protocol:
A single-cell suspension was prepared by dissociating the cells by treatment with Accutase at 37 °C for 3 minutes. The reaction was quenched with serum-containing medium, and cells were washed twice with PBS supplemented with 0.04% bovine serum albumin (BSA).
10x chromium single 3' reagent kit

treatment_protocol: No treatment


title: F123 nucleolus 47

treatment_protocol: ESCs were treated with Actinomycin D (10ng/ml) for 24h before downstream processing


title: F123 nucleolus 48


title: F123 nucleolus 46


title: Jurkat, Input, replicate 1

description:
Library name: 33i
count_matrix_homo.csv
counts_long.csv

value: Jurkat

value: hematopoietic


title: F123 nucleolus 49


title: F123 nucleolus 44


title: F123 nucleolus 43


title: F123 nucleolus 45


title: F123 nucleolus 41


title: Pair 2 Timepoint T4 Mouse B3 SU369

city: San Francisco

first: Amy

last: Fan

state: CA

address: 769 25th Ave

department: Pathology

institute: UCSF

description: Library name: C2T4_B3_369

data_processing:
FASTQ sequencing reads were trimmed and quality checked using fastp (--qualified_quality_phred 15 --length_required 20) and quantified using kallisto pseudoalignment to a cDNA index built using gene annotations downloaded from Ensembl and the reference human genome GRCh38.
The kallisto output was imported into R using tximport using gene mode.
Assembly: GRCh38
Supplementary files format and content: tab-delimited file containing counts per sample

extract_protocol:
Qiagen miRNeasy micro kit
SMART-Seq v4

value: AML

value: Chemotherapy-recovery timepoint 2


title: F123 nucleolus 40


title: F123 nucleolus 42


title: Pair 2 Timepoint T4 Mouse B2 SU369

description: Library name: C2T4_B2_369


title: F123 nucleolus 37


title: Pair 2 Timepoint T4 Mouse B1 SU266

description: Library name: C2T4_B1_266


title: F123 nucleolus 36


title: Pair 2 Timepoint T4 Mouse B3 SU266

description: Library name: C2T4_B3_266


title: F123 nucleolus 35


title: F123 nucleolus 34


title: Pair 2 Timepoint T4 Mouse A4 SU369

description: Library name: C2T4_A4_369


title: F123 nucleolus 38


title: Pair 2 Timepoint T4 Mouse A4 SU266

description: Library name: C2T4_A4_266


title: Pair 2 Timepoint T4 Mouse B2 SU266

description: Library name: C2T4_B2_266


title: F123 nucleolus 33


title: Pair 2 Timepoint T3 Mouse B2 SU369

description: Library name: C2T3_B2_369

value: Chemotherapy-recovery timepoint 1


title: Pair 2 Timepoint T3 Mouse B3 SU266

description: Library name: C2T3_B3_266


title: Pair 2 Timepoint T3 Mouse B2 SU266

description: Library name: C2T3_B2_266


title: F123 nucleolus 27


title: Pair 2 Timepoint T3 Mouse B1 SU369

description: Library name: C2T3_B1_369


title: F123 nucleolus 26


title: F123 nucleolus 32


title: Pair 2 Timepoint T4 Mouse A2 SU369

description: Library name: C2T4_A2_369


title: Pair 2 Timepoint T4 Mouse A2 SU266

description: Library name: C2T4_A2_266


title: Pair 2 Timepoint T3 Mouse B1 SU266

description: Library name: C2T3_B1_266


title: F123 nucleolus 31


title: F123 nucleolus 39


title: F123 nucleolus 30


title: Pair 2 Timepoint T4 Mouse B1 SU369

description: Library name: C2T4_B1_369


title: Pair 2 Timepoint T3 Mouse A4 SU369

description: Library name: C2T3_A4_369


title: Pair 2 Timepoint T3 Mouse B3 SU369

description: Library name: C2T3_B3_369


title: F123 nucleolus 24


title: Pair 2 Timepoint T3 Mouse A2 SU369

description: Library name: C2T3_A2_369


title: F123 nucleolus 23


title: Pair 2 Timepoint T2 Mouse B3 SU369

description: Library name: C2T2_B3_369

value: Chemotherapy-post-chemotherapy nadir


title: Pair 2 Timepoint T2 Mouse B3 SU266

description: Library name: C2T2_B3_266


title: F123 nucleolus 29


title: F123 nucleolus 28


title: F123 nucleolus 18


title: F123 nucleolus 17


title: Pair 2 Timepoint T2 Mouse A4 SU369

description: Library name: C2T2_A4_369


title: F123 nucleolus 25


title: F123 nucleolus 16


title: Pair 2 Timepoint T2 Mouse A2 SU369

description: Library name: C2T2_A2_369


title: Pair 2 Timepoint T2 Mouse A2 SU266

description: Library name: C2T2_A2_266


title: Pair 2 Timepoint T3 Mouse A4 SU266

description: Library name: C2T3_A4_266


title: F123 nucleolus 22


title: F123 nucleolus 21


title: Pair 2 Timepoint T1 Mouse B3 SU369

description: Library name: C2T1_B3_369

value: Chemotherapy-pre-chemotherapy


title: F123 nucleolus 12


title: Pair 2 Timepoint T2 Mouse B2 SU266

description: Library name: C2T2_B2_266


title: Pair 2 Timepoint T1 Mouse B2 SU369

description: Library name: C2T1_B2_369


title: F123 nucleolus 19


title: Pair 2 Timepoint T2 Mouse B1 SU369

description: Library name: C2T2_B1_369


title: Pair 2 Timepoint T1 Mouse B1 SU369

description: Library name: C2T1_B1_369


title: F123 nucleolus 8


title: Pair 2 Timepoint T2 Mouse B1 SU266

description: Library name: C2T2_B1_266


title: Pair 2 Timepoint T1 Mouse B1 SU266

description: Library name: C2T1_B1_266


title: F123 nucleolus 6


title: F123 nucleolus 13


title: F123 nucleolus 15


title: Pair 2 Timepoint T2 Mouse A4 SU266

description: Library name: C2T2_A4_266


title: Pair 2 Timepoint T1 Mouse B3 SU266

description: Library name: C2T1_B3_266


title: F123 nucleolus 14


title: F123 nucleolus 5


title: F123 nucleolus 4


title: Pair 2 Timepoint T1 Mouse A2 SU369

description: Library name: C2T1_A2_369


title: F123 nucleolus 10


title: Pair 1 Timepoint T4 Mouse H4 SU540

description: Library name: C1T4_H4_540


title: F123 nucleolus 3


title: Pair 1 Timepoint T4 Mouse H3 SU540

description: Library name: C1T4_H3_540


title: Pair 2 Timepoint T1 Mouse B2 SU266

description: Library name: C2T1_B2_266


title: Pair 1 Timepoint T4 Mouse H3 SU416

description: Library name: C1T4_H3_416


title: NPC nucleolus 23

source_name: NPC

value: NPC

value: WT


title: F123 nucleolus 9


title: F123 nucleolus 7


title: Pair 1 Timepoint T4 Mouse H2 SU540

description: Library name: C1T4_H2_540


title: Pair 1 Timepoint T4 Mouse H4 SU416

description: Library name: C1T4_H4_416


title: F123 nucleolus 11


title: Pair 2 Timepoint T1 Mouse A4 SU369

description: Library name: C2T1_A4_369


title: NPC nucleolus 22


title: Pair 1 Timepoint T4 Mouse H2 SU416

description: Library name: C1T4_H2_416


title: Pair 1 Timepoint T4 Mouse H1 SU540

description: Library name: C1T4_H1_540


title: F123 nucleolus 2


title: NPC nucleolus 21


title: Pair 2 Timepoint T1 Mouse A4 SU266

description: Library name: C2T1_A4_266


title: F123 nucleolus 1


title: NPC nucleolus 20


title: Pair 1 Timepoint T4 Mouse H1 SU416

description: Library name: C1T4_H1_416


title: NPC nucleolus 19


title: Pair 1 Timepoint T4 Mouse B5 SU540

description: Library name: C1T4_B5_540


title: Pair 1 Timepoint T4 Mouse B5 SU416

description: Library name: C1T4_B5_416


title: NPC nucleolus 16


title: NPC nucleolus 15


title: Pair 1 Timepoint T4 Mouse A4 SU416

description: Library name: C1T4_A4_416


title: NPC nucleolus 17


title: NPC nucleolus 18


title: Pair 1 Timepoint T4 Mouse B1 SU416

description: Library name: C1T4_B1_416


title: F123 nucleolus 20


title: Pair 1 Timepoint T4 Mouse A4 SU540

description: Library name: C1T4_A4_540


title: NPC nucleolus 14


title: NPC nucleolus 13


title: NPC nucleolus 10


title: Pair 1 Timepoint T4 Mouse A3 SU540

description: Library name: C1T4_A3_540


title: Pair 1 Timepoint T3 Mouse H3 SU540

description: Library name: C1T3_H3_540


title: NPC nucleolus 9


title: NPC nucleolus 7


title: Pair 1 Timepoint T3 Mouse H2 SU540

description: Library name: C1T3_H2_540


title: Pair 1 Timepoint T4 Mouse B1 SU540

description: Library name: C1T4_B1_540


title: Pair 1 Timepoint T4 Mouse A3 SU416

description: Library name: C1T4_A3_416


title: Pair 1 Timepoint T3 Mouse H4 SU540

description: Library name: C1T3_H4_540


title: NPC nucleolus 6


title: Pair 1 Timepoint T3 Mouse H4 SU416

description: Library name: C1T3_H4_416


title: NPC nucleolus 8


title: Pair 1 Timepoint T3 Mouse H3 SU416

description: Library name: C1T3_H3_416


title: Pair 1 Timepoint T3 Mouse H1 SU540

description: Library name: C1T3_H1_540


title: NPC nucleolus 5


title: Pair 1 Timepoint T3 Mouse H1 SU416

description: Library name: C1T3_H1_416


title: Pair 1 Timepoint T3 Mouse H2 SU416

description: Library name: C1T3_H2_416


title: NPC nucleolus 1


title: NPC nucleolus 11


title: Pair 1 Timepoint T3 Mouse B1 SU540

description: Library name: C1T3_B1_540


title: ActD treated ESC nucleolus 33

source_name: E14 ESC

value: E14 ESC

value: ActD treated


title: NPC nucleolus 3


title: Pair 1 Timepoint T3 Mouse B5 SU540

description: Library name: C1T3_B5_540


title: NPC nucleolus 12


title: NPC nucleolus 2


title: Pair 1 Timepoint T3 Mouse B5 SU416

description: Library name: C1T3_B5_416


title: Pair 1 Timepoint T3 Mouse A4 SU416

description: Library name: C1T3_A4_416


title: ActD treated ESC nucleolus 32


title: Pair 1 Timepoint T3 Mouse A3 SU540

description: Library name: C1T3_A3_540


title: ActD treated ESC nucleolus 31


title: Pair 1 Timepoint T3 Mouse B1 SU416

description: Library name: C1T3_B1_416


title: ActD treated ESC nucleolus 28


title: ActD treated ESC nucleolus 29


title: Pair 1 Timepoint T2 Mouse H3 SU540

description: Library name: C1T2_H3_540


title: ActD treated ESC nucleolus 27


title: Pair 1 Timepoint T2 Mouse H4 SU416

description: Library name: C1T2_H4_416


title: ActD treated ESC nucleolus 30


title: ActD treated ESC nucleolus 26


title: NPC nucleolus 4


title: ActD treated ESC nucleolus 24


title: Pair 1 Timepoint T2 Mouse H2 SU540

description: Library name: C1T2_H2_540


title: ActD treated ESC nucleolus 34


title: Pair 1 Timepoint T2 Mouse H2 SU416

description: Library name: C1T2_H2_416


title: Pair 1 Timepoint T2 Mouse H3 SU416

description: Library name: C1T2_H3_416


title: Pair 1 Timepoint T2 Mouse H1 SU416

description: Library name: C1T2_H1_416


title: ActD treated ESC nucleolus 23


title: ActD treated ESC nucleolus 22


title: Pair 1 Timepoint T3 Mouse A4 SU540

description: Library name: C1T3_A4_540


title: ActD treated ESC nucleolus 21


title: Pair 1 Timepoint T2 Mouse B5 SU416

description: Library name: C1T2_B5_416


title: Pair 1 Timepoint T2 Mouse B5 SU540

description: Library name: C1T2_B5_540


title: ActD treated ESC nucleolus 19


title: ActD treated ESC nucleolus 17


title: Pair 1 Timepoint T2 Mouse B1 SU540

description: Library name: C1T2_B1_540


title: Pair 1 Timepoint T2 Mouse B1 SU416

description: Library name: C1T2_B1_416


title: Pair 1 Timepoint T2 Mouse H4 SU540

description: Library name: C1T2_H4_540


title: Pair 1 Timepoint T3 Mouse A3 SU416

description: Library name: C1T3_A3_416


title: Pair 1 Timepoint T2 Mouse A4 SU416

description: Library name: C1T2_A4_416


title: Pair 1 Timepoint T2 Mouse A4 SU540

description: Library name: C1T2_A4_540


title: ActD treated ESC nucleolus 25


title: ActD treated ESC nucleolus 16


title: ActD treated ESC nucleolus 18


title: ActD treated ESC nucleolus 14


title: ActD treated ESC nucleolus 15


title: PDX, YKL-2-124 rep2

city: Oviedo

first: Francisco

last: Hermida-Prado

address: Av del Hospital Universitario s/n

department: Head and Neck Lab

country: Spain

institute: Instituto de Investigación Sanitaria del Principado de Asturias

description:
Library name: Sample 6
PDX_YKL2

data_processing:
HISAT2 was used to align paired-end clean reads to the reference genome.
featureCounts (2.0.6) was used to count the reads numbers mapped to each gene.
Assembly: GRCh38
Supplementary files format and content: gene counts

source_name: HNSCC PDX

growth_protocol: 7-8 weeks-old female AthymicNude-Fox1nu mice were subcutaneously implanted with resected, and previously expanded in successive groups of mice, head and neck squamous cell carcinoma tumors directly derived from patients.

extract_protocol:
RNA was extracted from frozen tumors with RNeasy Mini Kit (Qiagen), following the manufacturer’s instructions
Novogene NGS RNA Library Prep Set (PT042)

treatment_protocol: Mice with tumors between 200-400 mm3 were randomized into three treatment groups (six mice per group): (i) Intraperitoneal/oral vehicle (in alternate days); (ii) YKL-5-124 (10 mg/kg, 5 doses/week, intraperitoneal) and (iii) samuraciclib (70 mg/kg, 5 doses/week, orally)

value: HNSCC PDX

value: HNSCC

tag: batch


title: Pair 1 Timepoint T2 Mouse H1 SU540

description: Library name: C1T2_H1_540


title: PDX, YKL-2-124 rep1

description:
Library name: Sample 5
PDX_YKL1


title: ActD treated ESC nucleolus 12


title: Pair 1 Timepoint T1 Mouse H4 SU540

description: Library name: C1T1_H4_540


title: ActD treated ESC nucleolus 13


title: ActD treated ESC nucleolus 20


title: PDX, Samuraciclib rep2

description:
Library name: Sample 4
PDX_Sam2

value: Samuraciclib


title: Pair 1 Timepoint T1 Mouse H2 SU540

description: Library name: C1T1_H2_540


title: PDX, Control rep2

description:
Library name: Sample 2
PDX_Control2

value: Vehicle


title: PDX, Control rep1

description:
Library name: Sample 1
PDX_Control1


title: FaDu cells, YKL-5-124 rep3

description:
Library name: Sample 9
YKL_3

source_name: Cell line

growth_protocol: FaDu cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 200 mg/mL streptomycin, 2 mmol/L L-glutamine, 20 mmol/L HEPES (pH 7.3), and 100 mmol/L non-essential amino acids in humidified atmosphere with 5% CO2 at 37C.

extract_protocol:
Total RNA isolation was performed using GeneJET RNA Purification Kit (Thermo Scientific). DNA was degraded on columns with DNase I (Qiagen).
Novogene NGS RNA Library Prep Set (PT042)

treatment_protocol: Cells were treated with vehicle DMSO, Samuraciclib or YKL-5-124 for 48h.

value: Cell line

value: FaDu

value: Wt


title: PDX, Samuraciclib rep1

description:
Library name: Sample 3
PDX_Sam1


title: Pair 1 Timepoint T1 Mouse H3 SU416

description: Library name: C1T1_H3_416


title: ActD treated ESC nucleolus 11


title: Pair 1 Timepoint T1 Mouse H4 SU416

description: Library name: C1T1_H4_416


title: FaDu cells, YKL-5-124 rep1

description:
Library name: Sample 7
YKL_1


title: Pair 1 Timepoint T1 Mouse H1 SU540

description: Library name: C1T1_H1_540


title: ActD treated ESC nucleolus 9


title: Pair 1 Timepoint T1 Mouse H2 SU416

description: Library name: C1T1_H2_416


title: ActD treated ESC nucleolus 10


title: Pair 1 Timepoint T1 Mouse H1 SU416

description: Library name: C1T1_H1_416


title: FaDu cells, Samuraciclib rep2

description:
Library name: Sample 5
Sam_2


title: FaDu cells, YKL-5-124 rep2

description:
Library name: Sample 8
YKL_2


title: FaDu cells, DMSO rep3

description:
Library name: Sample 3
Control_3


title: ActD treated ESC nucleolus 6


title: FaDu cells, Samuraciclib rep3

description:
Library name: Sample 6
Sam_3


title: FaDu cells, DMSO rep2

description:
Library name: Sample 2
Control_2


title: Pair 1 Timepoint T1 Mouse H3 SU540

description: Library name: C1T1_H3_540


title: ActD treated ESC nucleolus 7


title: ActD treated ESC nucleolus 5


title: C4-2, EPZ6438, RiboLace, rep 3

city: Chicago

first: Yang

last: Yi

state: IL

address: 303 E Superior St

department: Department of Urology

institute: Northwestern University

description:
Library name: EPZ6438_ribo_rep3
E3
Ribo_Lace_counts.txt

data_processing:
Nanopore electrical signals were converted to FASTQ using the guppy base caller (v6.0.1) and reads were aligned to the human transcriptome using minimap2  and direct RNA-seq parameters (“-ax splice -uf -k14”, v2.24) with the GENCODE v42 annotation
RNA-seq read processing and quality control were performed using the nf-core/rnaseq pipeline (v3.15.0). Reads were aligned to the human reference genome hg38 using STAR (v2.7.10a) and raw read counts were obtained using featureCounts (v2.0.3) using GENCODE v42 annotation.
RiboLace raw reads were first trimmed using Cutadapt (v4.2) to remove LACE-seq 3’ linker sequences, retaining sequences longer than the minimum ribosome protected fragment length (20 nts) plus UMI sequence length. UMI sequences were then extracted using umi_tools (v1.1.5). Sequences of rRNA, tRNA and other ncRNA were obtained from RNAcentral and RiboLace reads were aligned to these ncRNAs using Bowtie2 (v2.5.4). Unaligned reads were then mapped to the human reference genome hg38 using STAR (v2.7.10a) and deduplicated based on UMI sequences using umi_tools. Raw read counts were obtained using featureCounts (v2.0.3) using GENCODE v42 annotation.
Assembly: hg38
Supplementary files format and content: Processed bigwig files were obtained from aligned BAM files using bedGraphToBigWig.
Supplementary files format and content: Processed count data were obtained using featureCounts from the GENCODE v42 annotation

growth_protocol: C4-2 cells were grown in RPMI 1640 medium supplemented with 10% FBS

extract_protocol:
The total RNA was first extracted using RNeasy Plus Mini Kit (Qiagen), followed by mRNA purification using PolyATtract mRNA Isolation System (Promega).
500 ng mRNA was subjected to library construction using an Oxford Nanopore direct RNA sequencing kit (SQK-RNA002) according to the manufacturer’s manual.

treatment_protocol: For drug treatment, C4-2 cells were treated with EPZ6438 at the concentration of 5 μM for 3 days until further use. For YTHDF1 siRNA transfection, Lipofectamine RNAiMAX (Invitrogen) were utilized according to the procedure of manufacturer.


title: Pair 1 Timepoint T1 Mouse B5 SU416

description: Library name: C1T1_B5_416


title: Pair 1 Timepoint T1 Mouse B1 SU540

description: Library name: C1T1_B1_540


title: FaDu cells, Samuraciclib rep1

description:
Library name: Sample 4
Sam_1


title: FaDu cells, DMSO rep1

description:
Library name: Sample 1
Control_1


title: Pair 1 Timepoint T1 Mouse B1 SU416

description: Library name: C1T1_B1_416


title: ActD treated ESC nucleolus 4


title: ActD treated ESC nucleolus 8


title: C4-2, DMSO, RiboLace, rep 3

description:
Library name: DMSO_ribo_rep3
D3
Ribo_Lace_counts.txt

value: DMSO


title: C4-2, DMSO, RiboLace, rep 2

description:
Library name: DMSO_ribo_rep2
D2
Ribo_Lace_counts.txt


title: Pair 1 Timepoint T1 Mouse A4 SU540

description: Library name: C1T1_A4_540

value: AML


title: C4-2, DMSO, RiboLace, rep 1

description:
Library name: DMSO_ribo_rep1
D1
Ribo_Lace_counts.txt


title: ActD treated ESC nucleolus 2


title: C4-2, siYTHDF1, RiboLace, rep 3

description:
Library name: siYTHDF1_ribo_rep3
Y3
Ribo_Lace_counts.txt


title: Pair 1 Timepoint T1 Mouse A4 SU416

description: Library name: C1T1_A4_416


title: C4-2, siYTHDF1, RiboLace, rep 1

description:
Library name: siYTHDF1_ribo_rep1
Y1
Ribo_Lace_counts.txt


title: ESC E14 nucleolus 53

value: Untreated


title: C4-2, siCTRL, RiboLace, rep 3

description:
Library name: siCTRL_ribo_rep3
C3
Ribo_Lace_counts.txt

value: siCTRL


title: C4-2, EPZ6438, RiboLace, rep 2

description:
Library name: EPZ6438_ribo_rep2
E2
Ribo_Lace_counts.txt


title: C4-2, siCTRL, RiboLace, rep 2

description:
Library name: siCTRL_ribo_rep2
C2
Ribo_Lace_counts.txt


title: ESC E14 nucleolus 52


title: C4-2, EPZ6438, rep 3

description:
Library name: EPZ6438_rep3
E3
RNA_counts.txt


title: C4-2, EPZ6438, rep 2

description:
Library name: EPZ6438_rep2
E2
RNA_counts.txt


title: ESC E14 nucleolus 51


title: Pair 1 Timepoint T1 Mouse B5 SU540

description: Library name: C1T1_B5_540


title: C4-2, EPZ6438, rep 1

description:
Library name: EPZ6438_rep1
E1
RNA_counts.txt


title: Pair 3 mouse C4 SU369 Supp

description: Library name: P3_C4_369

value: Competition


title: ESC E14 nucleolus 50


title: C4-2, DMSO, rep 1

description:
Library name: DMSO_rep1
D1
RNA_counts.txt


title: ESC E14 nucleolus 49


title: Pair 3 mouse C2 SU369 Supp

description: Library name: P3_C2_369


title: C4-2, EPZ6438, RiboLace, rep 1

description:
Library name: EPZ6438_ribo_rep1
E1
Ribo_Lace_counts.txt


title: ESC E14 nucleolus 48


title: C4-2, siYTHDF1, rep 2

description:
Library name: siYTHDF1_rep2
Y2
RNA_counts.txt


title: ActD treated ESC nucleolus 3


title: C4-2, siYTHDF1, RiboLace, rep 2

description:
Library name: siYTHDF1_ribo_rep2
Y2
Ribo_Lace_counts.txt


title: C4-2, siYTHDF1, rep 1

description:
Library name: siYTHDF1_rep1
Y1
RNA_counts.txt


title: ESC E14 nucleolus 47


title: C4-2, DMSO, rep 3

description:
Library name: DMSO_rep3
D3
RNA_counts.txt


title: C4-2, siCTRL, rep 2

description:
Library name: siCTRL_rep2
C2
RNA_counts.txt


title: ActD treated ESC nucleolus 1


title: C4-2, siCTRL, rep 1

description:
Library name: siCTRL_rep1
C1
RNA_counts.txt


title: TCR_tfh day 36 post-infection

city: Brisbane

first: Zuleima

last: Pava Imitola

state: Queensland

address: 300 Herston road

country: Australia

institute: QIMR Berghofer

description: 10x Genomics

data_processing:
Single cell sequencing data was demultiplexed, aligned and quantified using Cell Ranger software v6.0.1  (10x Genomics) against the human reference genome, with default parameters. (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Quality control of the data, normalization, integration, clustering and sub-clustering was done using Seurat version 4.0.2.
Gene set enrichment analysis software was used to identify significantly enriched gene ontology terms
the HOMER v4.9 package was used to identify significantly overrepresented upstream regulators
Assembly: GRCh38 v 2.7.1a
Supplementary files format and content: Tab-separated values files and matrix files
Library strategy: TCR-seq

source_name: blood

extract_protocol:
PBMCs were isolated from whole blood via density centrifugation with Ficoll-Paque prior to cryopreservation. PBMC samples were thawed in RPMI 1640 (Gibco) containing 10% FCS and 0.02% Benzonase. 1E6 PBMCs were stained for viability with Propidium iodide (PI) and live cells were sorted on BD FACSAria™ III Cell Sorter into 2% FBS/PBS and counted on hemocytometer
Up to 10 000 cells were loaded into each lane of Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 and Gel Bead-in-Emulsion (GEMs) generated in Chromium Controller. 3’ Gene Expression Libraries were then generated according to manufacturer’s instructions. Generated libraries were sequenced in a NextSeq 550 System using High Output Kit (150 Cycles) with the following parameters read 1: 26 cycles, i7 index: 8 cycles, i5 index: 0 cycles, read 2: 98 cycles

value: blood

tag: infected with

value: P. falciparum

value: tfh

value: day 36

tag: library type

value: TCR


title: ESC E14 nucleolus 45

value: E14 ESC

value: WT


title: Pair 1 Timepoint T1 Mouse A3 SU416

description: Library name: C1T1_A3_416

value: Chemotherapy-pre-chemotherapy


title: Pair 1 Timepoint T1 Mouse A3 SU540

description: Library name: C1T1_A3_540


title: gd day 36 post-infection

description: 10x Genomics

data_processing:
Single cell sequencing data was demultiplexed, aligned and quantified using Cell Ranger software v6.0.1  (10x Genomics) against the human reference genome, with default parameters. (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Quality control of the data, normalization, integration, clustering and sub-clustering was done using Seurat version 4.0.2.
Gene set enrichment analysis software was used to identify significantly enriched gene ontology terms
the HOMER v4.9 package was used to identify significantly overrepresented upstream regulators
Assembly: GRCh38 v 2.7.1a
Supplementary files format and content: Tab-separated values files and matrix files

value: gd-tcells


title: TCR_tfh day 0 pre-infection

description: 10x Genomics

value: none (pre-infection)

value: day 0


title: C4-2, DMSO, rep 2

description:
Library name: DMSO_rep2
D2
RNA_counts.txt


title: C4-2, siCTRL, RiboLace, rep 1

description:
Library name: siCTRL_ribo_rep1
C1
Ribo_Lace_counts.txt


title: Pair 3 mouse C4 SU575 Dom

description: Library name: P3_C4_575


title: Pair 3 mouse C2 SU575 Dom

description: Library name: P3_C2_575


title: C4-2, siYTHDF1, rep 3

description:
Library name: siYTHDF1_rep3
Y3
RNA_counts.txt


title: Pair 3 mouse C1 SU369 Supp

description: Library name: P3_C1_369


title: Pair 2 mouse C4 SU369 Dom

description: Library name: P2_C4_SU369


title: gd day 8 post-infection

description: 10x Genomics


title: Pair 2 mouse C1 SU369 Dom

description: Library name: P2_C1_SU369


title: gd day 0 pre-infection

description: 10x Genomics


title: TCR_tfh day 8 post-infection

description: 10x Genomics

value: day 8


title: Pair 3 mouse C1 SU575 Dom

description: Library name: P3_C1_575


title: C4-2, siCTRL, rep 3

description:
Library name: siCTRL_rep3
C3
RNA_counts.txt


title: ESC E14 nucleolus 46


title: Pair 2 mouse B5 SU369 Dom

description: Library name: P2_B5_SU369


title: Pair 3 mouse A5 SU369 Non-Comp

description: Library name: P3_A5_369

value: Non-Competitive


title: tfh day 0 pre-infection

description: 10x Genomics


title: TCR_tfh day 16 post-infection

description: 10x Genomics

value: day 16


title: monocyte day 8 post-infection

description: 10x Genomics

value: monocyte


title: ESC E14 nucleolus 39

value: Untreated


title: Pair 2 mouse C1 SU266 Supp

description: Library name: P2_C1_SU266

value: AML

value: Competition


title: Pair 3 mouse A3 SU369 Non-Comp

description: Library name: P3_A3_369


title: monocyte day 0 pre-infection

description: 10x Genomics

value: blood

value: P. falciparum


title: ESC E14 nucleolus 44


title: Pair 3 mouse A2 SU369 Non-Comp

description: Library name: P3_A2_369


title: ESC E14 nucleolus 38


title: Pair 2 mouse B5 SU266 Supp

description: Library name: P2_B5_SU266


title: gd day 16 post-infection

description: 10x Genomics


title: Pair 2 mouse B1 SU266 Non-Comp

description: Library name: P2_B1_SU266


title: ESC E14 nucleolus 43


title: VASA-WEE11-DMSO-r3

city: Utrecht

first: Michael

last: VanInsberghe

state: Utrecht

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

description: Library name: VASA-WEE11-DMSO-r3

data_processing:
See github repository https://github.com/mvanins/scRiboSeq_manuscript/tree/main/data_processing for code associated with raw data processing. See github repository https://github.com/mvanins/WEE1_manuscript for scripts used to create associated figures.  RPF: Basecalls were performed on the Illumina NextSeq 2000. Reads from each plate were demultiplexed using bcl2fastq (version 2.20.0.422) with --use-bases-mask Y*,I*,Y* --no-lane-splitting --mask-short-adapter-reads 0 --minimum-trimmed-read-length 0. Read 1 contains the insert and Read 2 contains the cell barcode. Next, the UMI was extracted from the first 10 bases of read 1 and concatenated to the start of the cell barcode. Adapter sequences were then trimmed from read 1 using cutadapt (version 2.7) with -m 15: -a TGGAATTCTCGGGT. Trimmed reads were aligned to the reference genome using STARSolo (version 2.7.3a_2020-05-22) with a 50-base overhang (--sjdbOverhang 50) with the following parameters: --seedSearchStartLmax 10 –alignIntronMax 1000000 --outFilterScoreMin 0 --outFilterMultimapNmax 1 --chimScoreSeparation 10 --chimScoreMin 20 --chimSegmentMin 15 --outFilterMismatchNmax 5. Aligned reads were deduplicated with UMI-tools (version 1.0.1) using --spliced-is-unique --per-cell --read-length --no-sort-output, and sorted using sambamba (version 0.7.1). Finally, aligned and deduplicated reads were parsed to extract the mapping coordinates and the reference sequence contexts around both the 5’ and 3’ ends of the reads. Count tables report the number of unique RPFs aligning within any annotated coding sequence for each gene. Reads were only counted if they aligned to the correct strand, if the trimmed length was between 30 and 45 nt long, and if the trimmed length was equal to the mapped length. Additionally, for these counts, the coding sequence was expanded to include 25 nt upstream and downstream of the start and stop codons, respectively. A random forest model was trained to predict the A-site location within an RPF read based on the footprint length and the sequence context around the 5’ and 3’ ends. The model was implemented in R (version 3.6.3) using ranger (version 0.12.1) with mlr (version 2.17.1) wrappers for training, tuning, assessment, and prediction. The model was trained on reads spanning a stop codon that satisfied the counting requirements listed above. The number of nucleotides between the 5’ end of these reads and the annotated stop codon was used for training.  VASA: Basecalls were performed on the Illumina NextSeq 2000. Reads from each plate were demultiplexed using bcl2fastq (version 2.20.0.422). Read 1 contains the UMI (fist 6 cycles), barcode (next 8 cycles), and a poly(T) stretch. Read 2 contains the insert. The UMI (first 6 cycles) and barcode (next 8 cycles) were extracted from read 1 and added to the read name. Adapter sequences were then trimmed from read 2 using cutadapt (version 2.7) with -U 6 -U -1 --times 5 -m :15  -A GTTCAGAGTTCTACA -A AAAAAAAAAAAAAAA -A TTTTTTTTTTTTTTT -A CCCCCCCCCCCCCCC -A GGGGGGGGGGGGGGG. Trimmed reads were aligned to the reference genome using STARSolo (version 2.7.3a_2020-05-22) with a 50-base overhang (--sjdbOverhang 50) with the following parameters: --seedSearchStartLmax 10 –alignIntronMax 1000000 --outFilterScoreMin 0 --outFilterMultimapNmax 1 --chimScoreSeparation 10 --chimScoreMin 20 --chimSegmentMin 15 --outFilterMismatchNmax 5. Aligned reads were deduplicated with UMI-tools (version 1.0.1) using --spliced-is-unique --per-cell –per-gene --read-length --no-sort-output, and sorted using sambamba (version 0.7.1). Finally, aligned and deduplicated reads were parsed to extract the mapping coordinates and the reference sequence contexts around both the 5’ and 3’ ends of the reads. Count tables report the number of unique reads aligning within any annotated transcript for each gene. Reads were only counted if they aligned to the correct strand.
Assembly: hg38
Supplementary files format and content: Metadata file: Sample-level metadata. Column Headers: CB: Sample barcode. well: Sample well. barcode_number: barcode number. plate: Plate ID. cds: Number of unique reads aligning to coding sequences. utr5: Number of unique reads aligning to 5' UTRs. utr3: Number of unique reads aligning to 3' UTRs. cell_total: Total number of unique reads aligning to protein-coding genes. cds_frac: Fraction of unique total reads aligning to CDS. replicate: Replicate number: num_genes: number of genes detected per sample. type: Library type. treatment: Culture condition prior to harvesting.  Count file: Sample-level count tables denoting the number of unique reads aligned to CDS regions for each gene.

source_name: Eye

growth_protocol: RPE-1 hTERT TP53-KO cells were cultured in DMEM/F12 with Glutamax (Gibco) supplemented with 10% FBS (Gibco) and 1× Pen-Strep (Gibco) at 37 °C 5% CO2

extract_protocol:
RPF: Cells were collected by trypsinization (TrypLE, Gibco), counted, washed in PBS0, and approximately 2000 were resuspended in 2.5 µL polysome lysis buffer [20 mM Tris pH 7.5, 15 mM MgCl2, 5 mM CaCl2, 150 mM NaCl, 1% Triton X-100, 2 U/µL RNaseIN Plus (Promega), 0.1 mM Cycloheximide (Sigma Aldrich), 0.1 mM Chloramphenicol (Sigma Aldrich)] and incubated on ice. No RNA purification was performed.  VASA: Cells were collected by trypsinization (TrypLE, Gibco), counted, washed in PBS0, and approximately 2000 were resuspended in 2.5 µL of 2.5 µM SORT-seq primer, snap frozen, and stored at -80 °C until processing. No RNA purification was performed.
RPF: To generate ribosome-protected footprints, 2.5 µL of Micrococcal Nuclease (MNase, 62.5 U/µL, New England Biolabs) was added to each sample and incubated at 37 °C for 30 minutes. In order to stop digestion, 2.5 µL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and samples were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.  After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 2.5 µL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 13.2 µL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 2.5 µL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 7.8 µL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 38.5 µL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, libraries were indexed during PCR by first transferring 3.6 µL of 20 µM unique forward index primers (Integrated DNA Technologies) and 78 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to half of the cDNA from each sample. Plates were then incubated at 98 °C for 30 s followed by 8 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until gel purification.  After library construction the samples were purified. Product was cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 6× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 20 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 20 cm 7 % polyacrylamide gel at 300 V until the lower marker ran off of the gel. The ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in LTET8. Finally, eluate was cleaned up using AMPure XP beads that had been diluted 6× in bead binding buffer and resuspended in LTET8.  VASA: Cells were first lysed by adding 2.5 µL of Lysis and Fragmentation brew [3.4× First Strand Buffer (Invitrogen), 0.024 U/µL Thermolabile Proteinase K (New England Biolabs), 0.2 % Triton X-100, 1:125000 diluted ERCC Spike-in] and incubating at 25 °C for 1 hour, then 55 °C for 10 minutes and holding at 4 °C. Next, 2.5  µL of End Repair and Poly(A) tailing brew was added [0.6× First Strand Buffer (Invitrogen), 20 mM DTT (Invitrogen), 7.5 µM ATP (New England Biolabs), 0.075 U/µL E. coli Poly(A) Polymerase (New England Biolabs), 1 U/µL T4 Polynucleotide Kinase (New England Biolabs), 4 U/µL RNase OUT (Invitrogen)] and incubated at 37 °C for 1 hour and held at 4 °C. Then 2.5  µL of Reverse Transcription Brew was added [2 mM each dNTP (Promega), 16 U/µL SuperScript III (Invitrogen)] and incubated at 50 °C for 1 hour and held at 4 °C. Finally, 55 µL of Second Strand brew [1.14× Second Strand Buffer (Invitrogen), 0.23 mM each dNTP (Promega), 0.32 U/µL E. coli DNA Polymerase I (Invitrogen), 0.018 U/µL RNase H (Invitrogen)] was added and samples were incubated at 16 °C, 85 °C for 20 minutes, and held at 4 °C. Double stranded libraries were cleaned up using AMPure XP beads (Beckman) that had been diluted 8× in Bead Binding Buffer [20 % PEG-8000 (Sigma Aldrich), 2.5 M NaCl (Ambion), 0.02 % Sodium Azide (Sigma Aldrich)], resuspended in 12 µL of water, and amplified using in vitro transcription at 37 °C overnight using the MegaScript T7 kit (Invitrogen) in a 30 µL reaction.  In vitro transcription was stopped by adding 12 µL of ExoSAP-IT (Invitrogen) and incubating at 37 °C  for 15 minutes and holding at 4 °C. Resulting amplified RNA (aRNA) was then cleaned up using AMPure XP Beads at a 1.8× ratio and resuspended in 10 µL of water. 600 ng of aRNA was then mixed with 10 µL hybridization brew [100 mM Tris pH 7.5, 200 mM NaCl (Ambion), 5 µM rRNA depletion oligonucleotides], incubated at 95 °C for 2 minutes, then decreased to 45 °C at a rate of 0.1 °C/s. While keeping samples at 45 °C, 10 µL of RNase H mix [100 mM Tris pH 7.5, 200 mM NaCl (Ambion), 40 mM MgCl2, 1 U/µL Hybridase Thermostable RNase H (Epicenter)] was added, mixed by pipetting, and incubated at 45 °C for 30 minutes and held at 4 °C. Next, 30 µL of DNase mix [1.67 mM CaCl2, 0.133 U/µL RQ1 DNase (Promega)] was added and samples were incubated at 37 °C for 30 minutes. The resulting rRNA-depleted samples were cleaned up using AMPure XP beads at a 1.6× ratio and eluted in 5 µL of water.  The cleaned up, rRNA-depleted samples were then each mixed with 1 µL of RA3 adapter (/5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/, 20 µM), denatured at 70 °C for 2 minutes, then snap chilled on ice. 4 µL of ligation brew was added [2.5× T4 RNA Ligase Reaction Buffer (New England Biolabs), 50 U/µL T4 RNA Ligase 2 truncated (New England Biolabs), 10 U/µL RNaseOUT (Invitrogen), 12.5% PEG-8000 (New England Biolabs)], and incubated at 25 °C for 1 hour and held at 4 °C. Ligated RNA was then converted to cDNA by adding 3 µL of dNTP-primer mix [3.33 µM each dNTPs (Promega), 13.33 µM RTP (CAAGCAGAAGACGGCATACGA)], incubating at 65 °C for 5 minutes and snap chilling on ice. 8 µL of RT brew [2.5× First Strand Buffer (Invitrogen), 12.5 mM DTT (Invitrogen), 5 U/µL RNaseOUT (Invitrogen), 25 U/µL SuperScript III (Invitrogen)], was added, incubated at 50 °C for 1 hour, 70 °C for 15 minutes, and held at 4 °C. RNA was next degraded by adding 1 µL RNase A (10 mg/mL, ThermoFisher Scientific) and incubating at 37 °C for 30 minutes. Complementary DNA was cleaned up using AMPure XP beads at a 1× ratio and resuspended in 10 µL of water. Purified cDNA was amplified by adding PCR brew [1.25 × NEBNext PCR mix (New England Biolabs), 0.5 µM RP1 (AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA), and 0.5 µM indexed RPI (CAAGCAGAAGACGGCATACGAGAT[6 bases]GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA)], and incubating at 98 °C for 30 s followed by 8 cycles of 98 °C for 15 s, 60 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Amplified and indexed libraries were cleaned up with two successive AMPure XP bead cleanups with 0.8× ratio and resuspended in LTET8.

treatment_protocol: Subconfluent (50 %) RPE-1 hTERT TP53-KO cells were treated with DMSO or AZD1775 for 24 h.

value: Eye

value: RPE-1 hTERT TP53-KO

value: retinal pigment epithelia

value: hTERT TP53-KO

value: DMSO for 24 h


title: VASA-WEE11-DMSO-r2

description: Library name: VASA-WEE11-DMSO-r2


title: tfh day 16 post-infection

description: 10x Genomics

value: tfh


title: ESC E14 nucleolus 42

value: E14 ESC

value: WT


title: ESC E14 nucleolus 41


title: VASA-WEE11-DMSO-r1

description: Library name: VASA-WEE11-DMSO-r1


title: ESC E14 nucleolus 35


title: RPFv4B-WEE11-WEE1i-r3

description: Library name: RPFv4B-WEE11-WEE1i-r3

value: 1 µM AZD1775 WEE1 inhibitor for 24 h


title: VASA-WEE11-WEE1i-r3

description: Library name: VASA-WEE11-WEE1i-r3


title: tfh day 8 post-infection

description: 10x Genomics


title: WT_OS_2

city: Xiamen

first: Songsong

last: Wang

address: Xiangan South Road

institute: Xiamen university

description:
Library name: Sample8
OS_2

data_processing:
Illumina casava2.19 software used for basecalling.
Assembly: mm9
Supplementary files format and content: gene_fpkm.txt

extract_protocol:
Total RNA was extracted from confluent 786-O and OS-RC-2 cells in both the CDKN2A knockout group and the blank control group, cultured in 60 mm dishes, using TRIzol® reagent (AgRNA ex Pro Regent, China). The extracted RNA samples were subsequently sent to Novogene (Beijing) for whole transcriptome resequencing using the Illumina HiSeq platform.
RNA libraries were trimmed for adaptor sequence

value: wild type


title: WT_OS_1

description:
Library name: Sample7
OS_1


title: monocyte day 36 post-infection

description: 10x Genomics


title: Pair 2 mouse C4 SU266 Supp

description: Library name: P2_C4_SU266


title: ESC E14 nucleolus 40


title: monocyte day 16 post-infection

description: 10x Genomics


title: WT_786_2

description:
Library name: Sample6
786_2


title: tfh day 36 post-infection

description: 10x Genomics


title: ESC E14 nucleolus 32


title: RPFv4B-WEE11-DMSO-r2

description: Library name: RPFv4B-WEE11-DMSO-r2


title: ESC E14 nucleolus 37


title: VASA-WEE11-WEE1i-r2

description: Library name: VASA-WEE11-WEE1i-r2


title: VASA-WEE11-WEE1i-r1

description: Library name: VASA-WEE11-WEE1i-r1


title: Pair 2 mouse A3 SU266 Non-Comp

description: Library name: P2_A3_SU266

value: Non-Competitive


title: KO_OS_412

description:
Library name: Sample3
OS_412

value: CDKN2A knockout

value: knockout


title: Pair 2 mouse A2 SU266 Non-Comp

description: Library name: P2_A2_SU266

value: AML


title: ESC E14 nucleolus 34

value: Untreated


title: ESC E14 nucleolus 29


title: RPFv4B-WEE11-WEE1i-r2

description: Library name: RPFv4B-WEE11-WEE1i-r2


title: ESC E14 nucleolus 33


title: Pair 1 mouse C2 SU416 Non-Comp

description: Library name: P1_C2_SU416


title: ESC E14 nucleolus 28


title: RPFv4B-WEE11-WEE1i-r1

description: Library name: RPFv4B-WEE11-WEE1i-r1


title: EIF2A-KO cells treated with WEE1i labeled with 15 minutes of EdU plate 2

first: Jeroen

last: van den Berg

description:
Library name: JvB-128-TP53NULL-MK1775-15E-F-pl2
2x100bp

data_processing:
see scEdU-seq (Nature Methods, 2024)
Assembly: hg38
Supplementary files format and content: count tables

source_name: eye

growth_protocol: RPE-1 cells were cultured in DMEM/F12 Glutamax and 10% FCS plus PenStep (Gibco)

extract_protocol:
Proteinase K digestion
see scEdU-seq (Nature Methods, 2024)

treatment_protocol: subconfluent (50%) RPE-1 hTERT cells were

value: eye

value: RPE-1 hTERT


title: Pair 1 mouse C1 SU416 Non-Comp

description: Library name: P1_C1_SU416


title: ESC E14 nucleolus 31


title: Pair 1 mouse A3 SU540 Dom

description: Library name: P1_A3_SU540

value: Competition


title: Pair 1 mouse A2 SU416 Supp

description: Library name: P1_A2_SU416


title: ESC E14 nucleolus 36


title: RPFv4B-WEE11-DMSO-r3

description: Library name: RPFv4B-WEE11-DMSO-r3

value: Eye

value: RPE-1 hTERT TP53-KO


title: EIF2A-KO cells treated with WEE1i labeled with 15 minutes of EdU plate 1

description:
Library name: JvB-128-TP53NULL-MK1775-15E-F-pl1
2x100bp

value: retinal pigment epithelia


title: WT_786_1

description:
Library name: Sample5
786_1


title: Pair 1 mouse B5 SU416 Non-Comp

description: Library name: P1_B5_SU416


title: KO_OS_770

description:
Library name: Sample4
S_770


title: ESC E14 nucleolus 30


title: RPFv4B-WEE11-DMSO-r1

description: Library name: RPFv4B-WEE11-DMSO-r1

value: hTERT TP53-KO

value: DMSO for 24 h


title: Pair 1 mouse A3 SU416 Supp

description: Library name: P1_A3_SU416


title: Pair 1 mouse A1 SU416 Supp

description: Library name: P1_A1_SU416


title: MZnps donor to WT host, replicate 1, 12 hpf, hCD4-selected

city: New Haven

first: Scott

middle: Elliot

last: Youlten

state: CONNECTICUT

address: 333 Cedar Street, SHM-I

institute: Yale University

description:
Library name: MZnps_WT_rep1_12hpf
Single-cell RNA-seq library prepared using 10x Genomics Chromium v3 chemistry from MZnps donor cells transplanted into WT host embryos at 12 hpf, enriched using hCD4 selection.

data_processing:
Raw sequencing reads were processed with CellRanger v3.0.2 (10x Genomics) and aligned to the zebrafish GRCz11 genome reference (with additional GFP, DsRed, and hCD4 sequences).
Cells with <1000 UMIs, >25% mitochondrial UMIs, or doublet score >0.4 (Scrublet v0.2.3) were excluded.
Genes expressed in <5 cells, mitochondrial genes, and transgenes (GFP, DsRed, hCD4) were removed.
Normalization, dimensionality reduction (PCA, UMAP), clustering, and annotation were performed in Scanpy v1.9.3 (Python 3.11.5).
Assembly: GRCz11 (Ensembl release 95) + GFP, DsRed, hCD4 sequences.
Supplementary files format and content: Anndata .h5ad file containing normalized expression matrix (19,782 genes × 10,551 cells), cell-level metadata (QC metrics, donor/host identity, replicate ID), gene metadata, clustering assignments, and PHATE embedding coordinates.

organism: Danio rerio

source_name: embryo

growth_protocol: Zebrafish husbandry was conducted according to AAALAC research guidelines under a protocol approved by the Yale University IACUC. TU/AB strain fish were maintained under standard conditions. Maternal-zygotic nanog-/-, pou5f3-/-, sox19b-/- (MZnps) mutants were generated and maintained as described in Giraldez laboratory protocols.

extract_protocol:
Single-cell suspensions were prepared in ice-cold PBE solution (PBS, 0.5% BSA, 2 mM EDTA). Viable hCD4+ cells were collected and immediately loaded onto a 10x Genomics Chromium v3 chip for cDNA and library preparation following manufacturer’s instructions.
Single-cell RNA libraries were generated using the 10x Genomics Chromium Single Cell 3′ v3 kit according to the manufacturer’s protocol, and sequenced on an Illumina NovaSeq 6000 platform (paired-end).

treatment_protocol: Embryos were dechorionated at the 1-cell stage. Donor embryos (WT or MZnps) were injected with hCD4 and fluorescent reporter mRNAs, and transplanted into WT host embryos. At 11–12 hpf (3–6 somite stage), host embryos were deyolked and dissociated. hCD4+ cells were enriched using magnetic microbeads (Miltenyi Biotec).

value: embryo

value: mixed embryonic cell types

value: donor:MZnps (nanog-/-, pou5f3-/-, sox19b-/-); host:WT

value: hCD4 mRNA injection + transplantation


title: ESC E14 nucleolus 25

value: E14 ESC

value: WT


title: EIF2A-KO cells treated with DMSO labeled with 15 minutes of EdU plate 3

description:
Library name: JvB-128-TP53NULL-DMSO-15E-F-pl3
2x100bp

value: DMSO


title: iPSC mouse E5 BFP Non-Comp

description: Library name: iPSC_E5_BFP

source_name: BFP

value: BFP

value: iAML

value: Non-Competitive


title: EIF2A-KO cells treated with DMSO labeled with 15 minutes of EdU plate 2

description:
Library name: JvB-128-TP53NULL-DMSO-15E-F-pl2
2x100bp

value: eye

value: RPE-1 hTERT


title: ESC E14 nucleolus 24

value: Untreated


title: EIF2A-KO cells treated with WEE1i labeled with 15 minutes of EdU plate 3

description:
Library name: JvB-128-TP53NULL-MK1775-15E-F-pl3
2x100bp


title: KO_786_770

description:
Library name: Sample2
786_770

value: CDKN2A knockout

value: knockout


title: Pair 1 mouse A2 SU540 Dom

description: Library name: P1_A2_SU540

value: AML

value: Competition


title: WT donor to WT host, 12 hpf, hCD4-selected

description:
Library name: WT_WT_12hpf
Single-cell RNA-seq library prepared using 10x Genomics Chromium v3 chemistry from WT donor cells transplanted into WT host embryos at 12 hpf, enriched using hCD4 selection.

value: embryo

value: donor:WT; host:WT


title: KO_786_412

description:
Library name: Sample1
786_412


title: ESC E14 nucleolus 23


title: ESC E14 nucleolus 27


title: TP53-KO cells treated with WEE1i labeled with 15 minutes of EdU plate 3

description:
Library name: JvB-128-EIF2A-NULL-MK1775-15E-F-pl3
2x100bp

value: retinal pigment epithelia


title: ESC E14 nucleolus 26


title: Pair 1 mouse A1 SU540 Dom

description: Library name: P1_A1_SU540


title: 711GRKO-2

city: Heidelberg

first: Chunxuan

last: Shao

address: Im Neuenheimer Feld 581

country: Germany

institute: Deutsches Krebsforschungszentrum

data_processing:
Raw reads from RNA-seq experiments of NCH601 and NCH711 BTSC cell cultures under different treatment conditions were aligned to hg19 (ensemble version 75) by STAR (2.5.3a) with default parameters. The quality of alignments were examined by QoRTs  and read counts were quantified by “featureCounts” in the R package Rsubread4. DESeq2 was employed to identify the differentially regulated genes with the option “cooksCutoff = FALSE”.
Assembly: hg19
Supplementary files format and content: tab-delimited text files include raw counts values for each Sample

growth_protocol: NCH601/711 patient-derived glioblastoma stem cells were established at the Heidelberg University Hospital. NCH BTSC cells were cultured in DMEM/Ham F-12 medium (1-26F09-I, Bioconcept) supplemented with 20 % BIT supplement (Pelo Biotech), 1 % PenStrep (Thermo Fisher), GlutaMAX Supplement (0.5 mM, 35050061, ThermoFisher), Epidermal growth factor (20 ng/ml, E4127-5X.1MG, Sigma) and Fibroblast growth factor 2 (20 ng/ml, F0291-25UG, Sigma).

extract_protocol:
Total RNA was isolated using the RNeasy Micro kit (Cat.74004, Qiagen) according to the manufacturer's instruction. 1000 ng of total RNA was reverse transcribed with QuantiTect Rev transcription Kit (205311, Qiagen).
Total RNA  were sent in house facility for Illumina Hiseq sequencing according to related protocols

treatment_protocol: CRISPR/Cas9 mediated system was used21. sgRNA targeting GR/GPD1 which were design based on the online tools (https://chopchop.cbu.uib.no/) are listed below. The cells were transfected by electroporation according to the manufacturer’s protocol (Voltage:1300V, Duration:10ms, Pulse:2). Puromycin (2 μg/μL final concentration, A1113802, Thermo) was added to the culture medium 24h post transfection and maintained for one week. Fresh medium and puromycin was replaced every two days. Then cells were counted and seeded into 96-well plate to achieve single cell per well.

value: cancer stem cells

value: GR knock out

value: no treatment


title: EIF2A-KO cells treated with DMSO labeled with 15 minutes of EdU plate 1

description:
Library name: JvB-128-TP53NULL-DMSO-15E-F-pl1
2x100bp

value: DMSO


title: TP53-KO cells treated with WEE1i labeled with 15 minutes of EdU plate 2

description:
Library name: JvB-128-EIF2A-NULL-MK1775-15E-F-pl2
2x100bp


title: iPSC mouse E4 BFP Non-Comp

description: Library name: iPSC_E4_BFP

value: BFP


title: TP53-KO cells treated with WEE1i labeled with 15 minutes of EdU plate 1

description:
Library name: JvB-128-EIF2A-NULL-MK1775-15E-F-pl1
2x100bp


title: 711GRKO-3


title: iPSC mouse E3 BFP Non-Comp

description: Library name: iPSC_E3_BFP

value: iAML


title: ESC E14 nucleolus 22

value: E14 ESC

value: WT


title: iPSC mouse C5 mCherry Dom

description: Library name: iPSC_C5_mCherry

source_name: mCherry

value: mCherry


title: 711-MIF-3

value: mifepristone treatment


title: ESC E14 nucleolus 20

value: Untreated


title: iPSC mouse C4 mCherry Dom

description: Library name: iPSC_C4_mCherry


title: 711GRKO-1


title: ESC E14 nucleolus 21


title: iPSC mouse C5 BFP Supp

description: Library name: iPSC_C5_BFP


title: TP53-KO cells treated with DMSO labeled with 15 minutes of EdU plate 3

description:
Library name: JvB-128-EIF2A-NULL-DMSO-15E-F-pl3
2x100bp

value: eye

value: RPE-1 hTERT


title: C4-2, siMETTL3, direct RNA, rep 2

description: Library name: nanopore_siMETTL3_rep2


title: ESC E14 nucleolus 17


title: 711-DEX-3

value: dexamethasone treatment


title: 711-DEX-2


title: iPSC mouse C4 BFP Supp

description: Library name: iPSC_C4_BFP

value: Competition


title: 711-MIF-1


title: MZnps donor to WT host, replicate 2, 12 hpf, hCD4-selected

description:
Library name: MZnps_WT_rep2_12hpf
Single-cell RNA-seq library prepared using 10x Genomics Chromium v3 chemistry from MZnps donor cells transplanted into WT host embryos at 12 hpf, enriched using hCD4 selection.

value: embryo

value: mixed embryonic cell types

value: donor:MZnps (nanog-/-, pou5f3-/-, sox19b-/-); host:WT

value: hCD4 mRNA injection + transplantation


title: iPSC mouse C2 mCherry Dom

description: Library name: iPSC_C2_mCherry


title: iPSC mouse A2 mCherry Dom

description: Library name: iPSC_A2_mCherry


title: C4-2, siMETTL3, direct RNA, rep 1

description: Library name: nanopore_siMETTL3_rep1


title: ESC E14 nucleolus 16


title: ESC E14 nucleolus 18


title: iPSC mouse A2 BFP Supp

description: Library name: iPSC_A2_BFP


title: ESC E14 nucleolus 15


title: 711-MIF-2

value: cancer stem cells


title: C4-2, siEZH2, direct RNA, rep 2

description: Library name: nanopore_siEZH2_rep2


title: C4-2, siEZH2, direct RNA, rep 1

description: Library name: nanopore_siEZH2_rep1


title: ESC E14 nucleolus 14


title: ESC E14 nucleolus 19


title: 711-CON-2

value: no treatment


title: TP53-KO cells treated with DMSO labeled with 15 minutes of EdU plate 2

description:
Library name: JvB-128-EIF2A-NULL-DMSO-15E-F-pl2
2x100bp

value: retinal pigment epithelia

value: DMSO


title: ESC E14 nucleolus 13

value: E14 ESC


title: sub_library2 (mixture of all 7 barcoded samples)

city: Cambridge

first: Emma

last: Rawlins

address: Tennis court road

country: United Kingdom

institute: University of Cambridge

description: Library name: Parse_v3_sub_library2

data_processing:
The sequencing data was initially processed with split-pipe (Version 1.5.0, Parse Biosciences) to demultiplex with the sample loading information for each sub-library, and then combine the results of 2 sublibraries. The combined gene count matrix was used for downstream analysis in Seurat (Version 5).
Assembly: GRCh38.p14
Supplementary files format and content: An r object of scRNA-seq data in Seurat with cells clustered and annotated.

source_name: Human lung

extract_protocol: Evercode Whole Transcriptome v3 kit mini

value: Human lung

value: AT2 cells


title: TP53-KO cells treated with DMSO labeled with 15 minutes of EdU plate 1

description:
Library name: JvB-128-EIF2A-NULL-DMSO-15E-F-pl1
2x100bp


title: sub_library1 (mixture of all 7 barcoded samples)

description: Library name: Parse_v3_sub_library1


title: iPSC mouse C2 BFP Supp

description: Library name: iPSC_C2_BFP

value: BFP

value: iAML


title: 711-CON-1

value: WT


title: C4-2, siCTRL, direct RNA, rep 1

description: Library name: nanopore_siCTRL_rep1

value: siCTRL


title: 711-CON-3


title: ESC E14 nucleolus 12

value: Untreated


title: IgG control for CUT&Tag profiling, replicate 2

city: Jerusalem

first: Itamar

last: Kozlovski

address: Alexander Silberman Institute of Life Sciences

department: Ecology, Evolution and Behavior

country: Israel

institute: The Hebrew University of Jerusalem

description:
Library name: IgG_control_rep2
Nematostella vectensis total viable cells; CUT&Tag with IgG control antibody; biological replicate 2.

data_processing:
CUT&Tag: Raw reads were trimmed with Cutadapt (v5.0). Trimmed reads were aligned to the Nematostella vectensis genome (Nv2 assembly) using Bowtie2 (v2.5.2).Peaks were called using MACS2 (q < 0.1).
Signal tracks (bigWig) were generated from BAM files using deepTools bamCoverage with RPGC normalization.
Count matrices were generated by intersecting peaks with aligned reads using featureCounts (v2.0.1).
Assembly: Nematostella vectensis genome assembly Nv2 (Zimmermann  et al., 2023).
Supplementary files format and content: *.narrowPeak: peak files in MACS2 narrowPeak format (chrom, start, end, peak summit, –log10(p), fold enrichment, q-value). *.bw: bigWig files of normalized CUT&Tag signal across the genome.
Library strategy: CUT&Tag

organism: Nematostella vectensis

source_name: tentacle

growth_protocol: N. vectensis polyps were maintained at 18 °C in 16 ‰ sea salt water (Red Sea, Israel), used as the growth medium. Polyps were fed with Artemia salina nauplii three times a week.

extract_protocol:
Tentacle tissue was dissected and enzymatically dissociated into single cells. Ncol3::mOrange and Nep3::mOrange reporter lines were used for FACS isolation of positive and negative cnidocyte populations. For CUT&Tag, nuclei were prepared using the V3 CUT&Tag protocol with the NE1 buffer option.
CUT&Tag libraries were prepared following the V3 CUT&Tag protocol (Kaya-Okur et al., 2019) with an anti-H3K27ac antibody (Abcam, ChIP Grade cat. ab4729), using Illumina-compatible sequencing adapters.

treatment_protocol: No experimental treatment was applied. Animals were collected untreated for cell isolation.

value: tentacle

value: Total viable cells

value: Nep3::mOrange reporter line

value: untreated


title: 601GPDKO53-3

value: GPD1 knock out


title: C4-2, siCTRL, direct RNA,  rep 2

description: Library name: nanopore_siCTRL_rep2


title: ESC E14 nucleolus 11


title: NCH601-NT-4d

data_processing:
Raw reads from RNA-seq experiments of NCH601 BTSC cell cultures under different treatment conditions were aligned to hg19 (ensemble version 75) by STAR (2.5.3a) with default parameters. The quality of alignments were examined by QoRTs  and read counts were quantified by “featureCounts” in the R package Rsubread4. DESeq2 was employed to identify the diffeentially regulated genes with the option “cooksCutoff = FALSE”. Samples collected in day 3 and 4 are grouped as “long_term”, and different GPD1 shRNAs were treated as batches.
Assembly: hg19
Supplementary files format and content: tab-delimited text files include raw counts values for each Sample

growth_protocol: NCH601 patient-derived glioblastoma stem cells were established at the Heidelberg University Hospital. NCH BTSC cells were cultured in DMEM/Ham F-12 medium (1-26F09-I, Bioconcept) supplemented with 20 % BIT supplement (Pelo Biotech), 1 % PenStrep (Thermo Fisher), GlutaMAX Supplement (0.5 mM, 35050061, ThermoFisher), Epidermal growth factor (20 ng/ml, E4127-5X.1MG, Sigma) and Fibroblast growth factor 2 (20 ng/ml, F0291-25UG, Sigma).

treatment_protocol: Lentiviruses contained one non-targeting control shRNA (SHC002) and four shRNAs targeting different regions in GPD1 (sh3, TRCN0000026517; sh5, TRCN0000026521; sh7, TRCN0000157054 and sh9 TRCN0000026507) were used to knock down GPD1 in human BTSCs. Medium replacement was conducted 24 h post infection. Cells were harvest at day1, 2, 3 and 4 post lenti-virus post-transduction. RNA was extracted with RNeasy Micro kit (Qiagen, Germany) and DNAse treatment was performed before RNAseq library preparation

value: cancer stem cells

value: 4 day


title: 711-DEX-1

value: dexamethasone treatment


title: IgG control for CUT&Tag profiling, replicate 1

description:
Library name: IgG_control_rep1
Nematostella vectensis total viable cells; CUT&Tag with IgG control antibody; biological replicate 1.


title: ESC E14 nucleolus 10


title: NCH601-NT-3d

value: 3 day


title: 601GPDKO53-1

value: no treatment


title: CUT&Tag profiling of H3K27ac in Nep3+ cells, replicate 4

description:
Library name: Nep3_pos_H3K27ac_rep4
Nematostella vectensis tentacle Nep3+ nematocytes; CUT&Tag with H3K27ac antibody; biological replicate 4.

value: Nep3-positive cnidocytes


title: 601GPDKO53-2


title: 601-MIF-3

value: mifepristone treatment


title: ESC E14 nucleolus 8

value: E14 ESC


title: ESC E14 nucleolus 9


title: NCH601-NT-2d

value: 2 day


title: CUT&Tag profiling of H3K27ac in Nep3+ cells, replicate 3

description:
Library name: Nep3_pos_H3K27ac_rep3
Nematostella vectensis tentacle Nep3+ nematocytes; CUT&Tag with H3K27ac antibody; biological replicate 3.


title: NCH601-NT-1d

value: WT

value: 1 day


title: CUT&Tag profiling of H3K27ac in Nep3+ cells, replicate 2

description:
Library name: Nep3_pos_H3K27ac_rep2
Nematostella vectensis tentacle Nep3+ nematocytes; CUT&Tag with H3K27ac antibody; biological replicate 2.


title: CUT&Tag profiling of H3K27ac in Nep3+ cells, replicate 1

description:
Library name: Nep3_pos_H3K27ac_rep1
Nematostella vectensis tentacle Nep3+ nematocytes; CUT&Tag with H3K27ac antibody; biological replicate 1.


title: ESC E14 nucleolus 7

value: Untreated


title: 601-MIF-1


title: NCH601-mock2-4d


title: NCH601-mock1-old

value: long term


title: ESC E14 nucleolus 5


title: 601-DEX-3


title: ESC E14 nucleolus 6


title: CUT&Tag profiling of H3K27ac in Nep3- cells, replicate 4

description:
Library name: Nep3_neg_H3K27ac_rep4
Nematostella vectensis tentacle Nep3⁻ cells; CUT&Tag with H3K27ac antibody; biological replicate 4.

value: tentacle

value: Nep3-negative cells


title: 601-DEX-2


title: ESC E14 nucleolus 4


title: CUT&Tag profiling of H3K27ac in Nep3- cells, replicate 2

description:
Library name: Nep3_neg_H3K27ac_rep2
Nematostella vectensis tentacle Nep3⁻ cells; CUT&Tag with H3K27ac antibody; biological replicate 2.

value: Nep3::mOrange reporter line

value: untreated


title: ESC E14 nucleolus 3


title: CUT&Tag profiling of H3K27ac in Nep3- cells, replicate 3

description:
Library name: Nep3_neg_H3K27ac_rep3
Nematostella vectensis tentacle Nep3⁻ cells; CUT&Tag with H3K27ac antibody; biological replicate 3.


title: 601-DEX-1

value: cancer stem cells


title: 601-CON-3


title: NCH601-GPD1-sh9-2d

value: GPD1 knock down


title: NCH601-GPD1-sh9-3d


title: ESC E14 nucleolus 2


title: CUT&Tag profiling of H3K27ac in Nep3- cells, replicate 1

description:
Library name: Nep3_neg_H3K27ac_rep1
Nematostella vectensis tentacle Nep3⁻ cells; CUT&Tag with H3K27ac antibody; biological replicate 1.


title: 601-MIF-2


title: 601-CON-2


title: CUT&Tag profiling of H3K27ac in Ncol3+ cells, replicate 3

description:
Library name: Ncol3_pos_H3K27ac_rep3
Nematostella vectensis tentacle cnidocytes (Ncol3⁺) isolated by FACS; CUT&Tag with H3K27ac antibody; biological replicate 3

value: Ncol3-positive cnidocytes

value: Ncol3::mOrange reporter line


title: NCH601-GPD1-sh7-4d

value: 4 day


title: 601-CON-1

value: no treatment


title: CUT&Tag profiling of H3K27ac in Ncol3+ cells, replicate 4

description:
Library name: Ncol3_pos_H3K27ac_rep4
Nematostella vectensis tentacle cnidocytes (Ncol3⁺) isolated by FACS; CUT&Tag with H3K27ac antibody; biological replicate 4


title: NCH601-GPD1-sh7-3d

value: 3 day


title: ESC E14 nucleolus 1

value: E14 ESC


title: F123 ChIPseq H3K9me2

data_processing:
N-masked genome was created using the mm10 reference genome and the Variant Call Format (VCF) file from the mouse genome project containing the SNP information for all strains. The SNPsplit package (Krueger & Andrews, 2016) was used to read the VCF file and extract the SNP information for the S129/SvJae and CAST strains and N-mask for the mm10 genome. ChIPseq reads were aligned to the N-masked mm10 reference genome with Bowtie2 (version 2.5.0; (Langmead & Salzberg, 2012)) using default parameters. PCR duplicates were removed using samtools. SNPsplit was then used to tag the SNPs for S129/SvJae and CAST alleles in the bam file and sort the haplotype-specific bam files for both the alleles. Bigwig files were created from the bam files using bamCoverage from deeptools (Ramirez et al., 2014) using default parameters. Integrative Genome Viewer (IGV, version 2.15.4) (Robinson et al., 2011) was used to visualize and extract representative ChIPseq tracks.
Assembly: mm10
Supplementary files format and content: BED

growth_protocol: F123-ESCs (mouse male, hybrid cell line, F1 S129/Jae and Cast mouse cross) (Gribnau et al, 2003) were cultured on a layer of mitotically inactivated feeder murine embryonic fibroblasts under standard conditions (DMEM, supplemented with 15 % KSR, 1x Glutamax, 10 mM non-essential amino acids, 50 µM beta-mercaptoethanol, 1,000 U/ml LIF). Media was changed every 24h. Before harvesting, mESCs were passaged onto feeder-free ESGRO-gelatin (EMD Millipore SF008) coated plates for at least 2 passages to remove feeder cells. Cells were harvested after approximately 48 h at 70 – 80 % confluency.

extract_protocol:
ChIP was performed as previously described (Lerra et al, 2024). Briefly, 1% formaldehyde was added to cultured cells to cross-link proteins to DNA. Isolated nuclei were then lysed with lysis buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, pH 8, 1% SDS, 1X protease inhibitor complete EDTA-free cocktail, Roche). Nuclei were sonicated using a Bioruptor ultrasonic cell disruptor (Diagenode) to shear genomic DNA to an average fragment size of 200 bp. 20 μg of chromatin was diluted to a total volume of 500 μl with ChIP buffer (16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100) and incubated overnight with the ChIP-grade antibodies against H3K9me2, H3K9me3, H3K27me3, H3K4me1, H3K4me3 and H3K27ac. After washing, bound chromatin was eluted with the elution buffer (1% SDS, 100 mM NaHCO3). Upon proteinase K digestion (50 °C for 3 h) and reversion of cross- linking (65 °C, overnight), DNA was purified with phenol/chloroform, and ethanol precipitated.
The quality and quantity of the isolated DNA were determined with a Qubit® 4 Fluorometer (Life Technologies, California, USA) and the 4200 TapeStation system. Briefly, ChIP samples (10 ng) were end-repaired and polyadenylated before the ligation of Illumina compatible adapters. The adapters contain the index for multiplexing. The quality and quantity of the enriched libraries were validated using Qubit® 4 Fluorometer and the 4200 TapeStation system. The libraries were pooled to 10 nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. Sequencing was performed on the NovaSeq 6000 for 100bp single end reads (Illumina, Inc, California, USA).

value: F123 hyrbid ESC


title: F123 ChIPseq H3K9me3


title: Input DNA for 3XFlag-SyCRP1 ChIP-seq under high CO₂ condition

city: Hyderabad

first: Suraj

last: Chauhan

state: Telangana

address: Prof. C.R. Rao Road, Central University P.O., Gachibowli

department: Biotechnology and Bioinformatics

country: India

institute: University of Hyderabad

description: Library name: SyCRP1_HC_INPUT

data_processing:
Sequence reads were subjected to adaptor and quality trimming using fastp v0.23.4; a Phred quality score cutoff of 20 was applied for trimming.
Trimmed reads were aligned to the Synechocystis reference genome using BWA-MEM v0.7.17-r1188 with default parameters.
Alignments with a mapping quality score below 10 were filtered out using SAMtools.
Duplicate reads were removed using GATK v4.4.0.0. Peak calling was performed using MACS2 v2.2.9.1 with default parameters, using input DNA as the control.
Alignments were converted to BigWig format using deepTools v3.5.4, with read counts normalized to 1× genome coverage (RPGC).
Assembly: ASM972v1 (GCF_000009725.1)
Supplementary files format and content: bigWig, narrowPeak (except for INPUT sample)

organism: Geminocystis herdmanii PCC 6308

source_name: Synechocystis whole cells

growth_protocol: Cells were grown in BG11 media (buffered with HEPES, pH 7.8), at 34°C, under continuous illumination of 70 μmol photons m-2 s-1 and bubbled with filter sterilized air.

extract_protocol:
Cells were fixed by formaldehyde at a final concentration of 1%, the fixation was quenched with 125 mM Glycine. Cells were disrupted using glass-beads by vigorous mixing at maximum speed on a vortex mixer. The genomic DNA was fragmented by sonication using Sonics Vibracell, VC505 ultrasonicator. 3XFlag-SyCRP1 was immunoprecipitated with Anti-FLAG M2 affinity gel and eluted with 3xFLAG peptide. Protein and RNA was removed by Proteinase K and RNaseA; the DNA fragments were extracted by PCIA and precipitated by ethanol.
The input and immune-precipitated DNA was quantified by Qubit 4 flurometer from invitrogen. The NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®  from NEB was used to prepare libraries. The cleaned libraries were quantitated on Qubit 4 flurometer and appropriate dilutions loaded on a High sensitivity D1000 screen tape on Agilent 4200 tapestation to determine the average size and range of the fragments.

treatment_protocol: High CO2 (HC) treatment. 5% (v/v) CO2 in sterile air was bubbled to the culture for 1h before collection

value: Synechocystis whole cells

value: 3XFlag-SyCRP1 expressing in mutant sycrp1 background

tag: chip antibody


title: CUT&Tag profiling of H3K27ac in Ncol3+ cells, replicate 2

description:
Library name: Ncol3_pos_H3K27ac_rep2
Nematostella vectensis tentacle cnidocytes (Ncol3⁺) isolated by FACS; CUT&Tag with H3K27ac antibody; biological replicate 2


title: NCH601-GPD1-sh7-2d

value: 2 day


title: NCH601-GPD1-sh5-4d


title: NCH601-GPD1-sh7-1d

value: 1 day


title: CUT&Tag profiling of H3K27ac in Ncol3- cells, replicate 4

description:
Library name: Ncol3_neg_H3K27ac_rep4
Nematostella vectensis tentacle Ncol3⁻ cells; CUT&Tag with H3K27ac antibody; biological replicate 4.

value: tentacle

value: Ncol3-negative cells


title: F123 ChIPseq H3K4me1


title: CUT&Tag profiling of H3K27ac in Ncol3+ cells, replicate 1

description:
Library name: Ncol3_pos_H3K27ac_rep1
Nematostella vectensis tentacle cnidocytes (Ncol3⁺) isolated by FACS; CUT&Tag with H3K27ac antibody; biological replicate 1


title: NCH601-GPD1-sh5-3d


title: F123 ChIPseq H3K27me3


title: CUT&Tag profiling of H3K27ac in Ncol3- cells, replicate 2

description:
Library name: Ncol3_neg_H3K27ac_rep2
Nematostella vectensis tentacle Ncol3⁻ cells; CUT&Tag with H3K27ac antibody; biological replicate 2.


title: NCH601-GPD1-sh9-4d


title: CUT&Tag profiling of H3K27ac in Ncol3- cells, replicate 3

description:
Library name: Ncol3_neg_H3K27ac_rep3
Nematostella vectensis tentacle Ncol3⁻ cells; CUT&Tag with H3K27ac antibody; biological replicate 3.


title: F123 ChIPseq H3K27ac


title: CUT&Tag profiling of H3K27ac in Ncol3- cells, replicate 1

description:
Library name: Ncol3_neg_H3K27ac_rep1
Nematostella vectensis tentacle Ncol3⁻ cells; CUT&Tag with H3K27ac antibody; biological replicate 1.


title: NCH601-GPD1-sh5-2d


title: NCH601-GPD1-sh5-1d