title: 5dpf-gut-2_S2

description: Library name: Sample 2


title: WEHI231_MS8815N_2

status: Public on Aug 02 2026

type: SRA

city: Durham

first: Arum

last: Kim

state: NC

address: Medical Sciences Research Building III  3 Genome Ct

country: USA

institute: Duke University

description: Library name: WEHI231_MS8815N_2

library_source: transcriptomic

data_processing:
The fastq files were aligned to the GRCh38 human genome (GRCh38.d1.vd1.fa) or mm10 mouse genome using STAR v2.4.2 with the following parameters: --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM.
The transcript abundance for each sample was estimated with salmon to quantify the transcriptome defined by Ensembl annotation.
Gene level counts were summed across isoforms and genes with low counts (maximum expression < 10) were filtered out for the downstream analyses.
Assembly: GRCh38/mm10
Supplementary files format and content: tab delimited matrix of gene level count estimates

molecule: total RNA

organism: Mus musculus

growth_protocol: HepG2 and Huh7cells were grown in RPMI1640 and DMEM medium, respectively, supplemented with 10% of FBS and 1% of antibiotics. WEHI-231 cells were grown in DMEM medium supplemented with 10% of FBS, 1% of antibiotics, and 55uM of 2-mercaptoethanol.

extract_protocol:
RNA was extracted by using Qiagen RNeasy Mini Kit (Qiagen, #74134).
For the construction of multiplexed RNA-sequencing libraries, 1 ug of total RNA per sample was processed by using either Illumina Truseq sample prep kit v2 or NEBNext Ultra II RNA library Prep kit (NEB, #E7770) following the instructions of the manufacture’s product manual.

treatment_protocol:
HepG2 and Huh7 cells were treated with 5uM of DMSO, MS7126, C24, MS7126N1 or MS7126N2 for 48hr. WEHI-231 cells were treated with 1uM of DMSO, MS8815, EPZ-6438, or MS8815N for 16hr.
Cells were infected with the pLKO.1-puro lentivirus containing either EZH2- or SREBP2-targeting shRNA (shEZH2) or scramble (shControl), followed by puromycin selection.

tag: cell line


title: Inflammatory_replicate_1

city: Boston

first: Ruaidhrí

last: Jackson

state: MA

address: New Research Building, 77 Avenue Louis Pasteur

department: Immunology

institute: Harvard Medical School

data_processing:
Basecalling was performed using Bream v7.0.9 and Guppy v6.0.7.
Passing individual fastq files from the same sample were merged to generate a single fastq file per sample.
FASTQ files were aligned to the reference transcriptome using minimap2 v2.24 with the parameters “-ax splice -uf -k14”.
Salmon pseudocount files were generated using the parameters “quant –ont -lU -a --numBootstraps 30" with Salmon v1.10.2.
Log2(normalized counts per million + 1) summary gene quantification data was produced using edgeR v4.2.0.
Assembly: GRCm39 (Gencode M28)
Supplementary files format and content: Excel file containing log2(normalized counts per million + 1) of gene expression with samples as columns (LCPM_expression_estimate_prior_count_1.xlsx).
Supplementary files format and content: Salmon quant.sf pseudocount files for each sample.

molecule: polyA RNA

source_name: Bone Marrow

growth_protocol: Bone marrow was extracted from male C57BL/6J mice aged 8-10 weeks. Bone marrow was flushed using PBS, filtered through a 70µm cell strainer, and plated in 150mm plastic tissue culture plates. Cells were cultured for 6 days in complete Dulbecco's Modified Eagle Medium supplemented with 4.5g/L D-Glucose, 110mg/L sodium pyruvate, 10% fetal bovine serum, 1% Penicillin-Streptomycin, and 50ng/mL macrophage colony-stimulating factor (MCSF) to generate BMDMs.

extract_protocol:
RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. PolyA RNA selection was performed using the Poly(A)Purist™ MAG Kit (Invitrogen) according to the manufacturer’s instructions.
Libraries were prepared using the Direct RNA Sequencing kit SQK-RNA002 (Oxford Nanopore Technologies) according to the manufacturer’s instructions. 500ng of polyA selected RNA was used as input for each sample.
Long read direct RNA-sequencing

treatment_protocol: BMDMs were treated with 100ng/mL lipopolysaccharide (LPS) and 20ng/mL IFNγ for 24 hours, to generate inflammatory macrophages, or 20ng/mL IL-4 and 20ng/mL IL-13 for 24 hours, to generate tissue reparative macrophages. Steady state macrophages received no additional treatment.

tag: tissue

value: Bone Marrow

tag: cell type

value: Bone Marrow Derived Macrophages

tag: genotype

tag: treatment

value: LPS + IFNγ


title: S01_CD4_naive_H3K4me1

city: Cambridge

first: Vinay

middle: S

last: Mahajan

address: 65 Landsdowne Street

institute: Brigham and Women's Hospital

description: Library name: S01

library_source: genomic

data_processing:
Sequencing reads were aligned to hg38 using Bowtie2.
CUT&RUN peaks were identified using macs2
Assembly: hg38
Supplementary files format and content: broadPeak.bed (macs2 format)
Library strategy: CUT&Run

molecule: genomic DNA

organism: Homo sapiens

source_name: tonsil

extract_protocol:
Cells were FACS-sorted, stored on ice, and prompty processed for CUT&RUN
CUT&RUN was performed with 25-50 thousand cells as described by Skene et al, 2018 (PMID: 29651053). Briefly, nuclei were immobilized and permeabilized with digitonin on ConA beads, incubated with specific antibodies against H3K4me1, H3K4me3 or H3K27ac.  pA-MNase was used to cleave and release antibody-bound chromatin. Released chromatin DNA was purified and indexed libraries were prepared for sequencing.

value: tonsil

tag: histone mark


title: WEHI231_MS8815N_1

description: Library name: WEHI231_MS8815N_1


title: WEHI231_EPZ-6438_2

description: Library name: WEHI231_EPZ-6438_2


title: Tissue_Reparative_replicate_1


title: S0020_Naive_CD45RA+CXCR5neg_PD1neg

description: Library name: S0020

data_processing:
rsem
Assembly: hg38
Supplementary files format and content: count matrix (supplementary file)

extract_protocol:
Qiagen RNAeasy kit
SmartSeq2 protocol was adapted for bulk RNAseq (PMID: 24056875)


title: WEHI231_EPZ-6438_1

description: Library name: WEHI231_EPZ-6438_1


title: MV4;11, SREBP2_cayman, CUT&RUN

description: Library name: MV4;11_SREBP2_cayman

data_processing:
All reads were mapped to the human genome (hg38) , and then the non-primary alignment and PCR duplicates were removed from aligned data respectively using the Picard, Samtools and bedtools.
Bigwig files, heatmaps and averaged plotting of CUT&RUN signals were generated using Deeptools.
Profiles of CUT&RUN read densities were displayed in the Integrative Genomics Viewer (IGV, Broad Institute).
The MACS2 software was used for peak identification with default parameters.
Assembly: hg38
Supplementary files format and content: Bigwig files
Library strategy: CUT&RUN

growth_protocol: 22Rv1 and MV4;11 cells were grown in RPMI1640 medium supplemented with 10% of FBS and 1% of antibiotics.

extract_protocol:
Cells were trypsinized with 0.05% of trypsin and pellets were collected for CUT&RUN. CUT&RUN was performed according to manufacturer’s instructions (CUTANA™ pAG-MNase for ChIC/CUT&RUN, EpiCypher, Cat# 15-1116).
1-10 ng of the purified CUT&RUN-enriched DNA was used for preparation of multiplexed sequencing libraries with the NEB Ultra II DNA Library Prep Kit per manufacturer's instructions (NEB #E7645). Multiplexed libraries were subject to pair-end sequencing using an Illumina Nextseq.

tag: cut&run antibody

value: SREBP2 (cayman)


title: WEHI231_MS8815_2

description: Library name: WEHI231_MS8815_2


title: Huh7_MS7126_2

data_processing:
The fastq files were aligned to the GRCh38 human genome (GRCh38.d1.vd1.fa) using STAR v2.4.2 with the following parameters: --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM.
The transcript abundance for each sample was estimated with salmon to quantify the transcriptome defined by Ensembl annotation.
Gene level counts were summed across isoforms and genes with low counts (maximum expression < 10) were filtered out for the downstream analyses.
Assembly: GRCh38
Supplementary files format and content: tab delimited matrix of gene level count estimates

growth_protocol: HepG2 cells were grown in RPMI1640 medium plus 10% FBS and 1% antibiotics. Huh7 cells were grown in DMEM medium plus 10% FBS and 1% antibiotics.

treatment_protocol:
HepG2 cells were treated with 5uM of DMSO, MS7126, C24, MS7126N1 or MS7126N2 for 48h.
Huh7 cells were treated with 5uM of DMSO, MS7126, C24, MS7126N1 or MS7126N2 for 24h.
Cells were infected with the pLKO.1-puro lentivirus containing either EZH2-targeting shRNA (shEZH2) or empty vector (shEV), followed by puromycin selection.
Cells were infected with the pLKO.1-puro lentivirus containing either SREBP2-targeting shRNA (shSREBP2) or empty vector (shEV), followed by puromycin selection.


title: WEHI231_MS8815_1

description: Library name: WEHI231_MS8815_1


title: S0019_Late_PreTfh_CXCR5+_PD1int

description: Library name: S0019


title: Huh7_MS7126_1


title: S0018_Early_PreTfh_CXCR5+_PD1low

description: Library name: S0018


title: MV4;11, SREBP2_abcam, CUT&RUN

description: Library name: MV4;11_SREBP2_abcam

value: SREBP2 (abcam)


title: AC-9m

city: Mayaguez

first: Maribella

last: Domenech

state: PR

address: PO Box 9000

country: Puerto Rico

institute: Universidad de Puerto Rico

description: Library name: 31470-019.R1

data_processing:
All primary analysis is carried out using CLC Genomics Server 25.0. The workflow “QIAseq miRNA Quantification” of CLC Genomics Server with standard parameters is used to map the reads to miRBase version 22. In short, the reads are processed by (1) trimming of the common sequence, UMI and adapters, and (2) filtering of reads with length < 15 nt or length > 55 nt. They are then deduplicated using their UMI. Reads are grouped into UMI groups when they (1) start at the same position based on the end of the read to which the UMI is ligated , (2) are from the same strand, and (3) have identical UMIs. Groups that contain only one read (singletons) are merged into non-singleton groups if the singleton’s UMI can be converted to a UMI of a non-singleton group by introducing an SNP (the biggest group is chosen).
All reads that don’t map to miRBase, neither with perfect matches nor as isomiRs (maximum 2 mismatches and/or alternative start/end position of 2 nt), are mapped to the human genome hg38 with ENSEMBL GRCh38 version 98 annotation. This is carried out using the “RNA-Seq Analysis” workflow of CLC Genomics Server with standard parameters.
Assembly: hg38 with ENSEMBL GRCh38 version 98 annotation.
Supplementary files format and content: microsoft excel comma separated values file containing CPM per sample

source_name: adipose

extract_protocol:
RNA was isolated from using the exoRNeasy Midi (QIAGEN) according to manufacturer’s instructions with an elution volume of 14 uL
The library preparation was done using the QIAseq miRNA Library Kit (QIAGEN). A total of 10 ng total RNA was converted into miRNA NGS libraries. After adapter ligation, UMIs were introduced in the reverse transcription step. The cDNA was amplified using PCR (19 cycles) and during the PCR indices were added. After PCR the samples were purified. Library preparation was quality controlled using capillary electrophoresis (Tape D1000). Based on quality of the inserts and the concentration measurements the libraries were pooled in equimolar ratios. The library pool(s) were quantified using qPCR. The library pool(s) were then sequenced on a AVITI (Element Biosciences, San Diego, CA, USA) sequencing instrument according to the manufacturer instructions (1x76, 2x10). Raw data was de-multiplexed and FASTQ files for each sample were generated using Element Biosciences bases2fastq version 00.000.000.4.2.4.

value: adipose

value: mesenchymal stem cells

value: WT

tag: time

value: Day 9

value: Microgravity (m)

tag: culture

value: COL


title: MV4;11, EZH2, CUT&RUN

description: Library name: MV4;11_EZH2

value: EZH2 (Cell Signaling Technology)


title: S0017_Tfh_CXCR5++_PD1hi

description: Library name: S0017


title: WEHI231_DMSO_2

description: Library name: WEHI231_DMSO_2


title: Huh7_DMSO_2


title: 22Rv1, control, CUT&RUN

description: Library name: 22Rv1_control


title: S0016_Naive_CD45RA+CXCR5neg_PD1neg

description: Library name: S0016


title: 22Rv1, SREBP2_cayman, CUT&RUN

description: Library name: 22Rv1_SREBP2_cayman


title: S0015_Late_PreTfh_CXCR5+_PD1int

description: Library name: S0015


title: RBNS, 7-deaza-guanine, FMRP, rep1

status: Public on Aug 03 2026

city: Chapel Hill

first: Daniel

last: Dominguez

address: 120 Mason Farm

department: Pharmacology

institute: UNC Chapel Hill

description:
Library name: stG4_FMRP_7dG_PrepA
Column name in stG4_raw_counts.txt: FMRP_LCD_7dG_PrepA

data_processing:
Raw reads were trimmed of the universal Illumina adapter sequences using cutadapt.
Specifically:
cutadapt -j 10 -m 18 -a TGGAATTCTCGGGTGTCAAGG -g GAGTTCTACAGTCCGACGATC -o ${line}_primer_trim.fastq ${line}.fastq
Raw counts were generated by STAR mapping against (structural_pool.fasta, structural_pool.gtf). Specifically:
#### STAR index ####
STAR   --runMode genomeGenerate      --runThreadN 8   --genomeDir STAR_idx/library  --genomeFastaFiles structural_pool.fasta     --sjdbGTFfile structural_pool.gtf --genomeSAindexNbases 8
#### STAR count ####
#### notice: no mismatch
STAR --runThreadN 16 \
--genomeDir /STAR_idx/library \
--readFilesIn ./${line}_primer_trimmed.fastq \
--outFileNamePrefix STAR/${line} \
--sjdbGTFfile structural_pool.gtf \
--outFilterMultimapNmax 1 \
--outFilterMismatchNmax 0 \
--genomeLoad NoSharedMemory \
--outSAMtype BAM Unsorted \
--outReadsUnmapped Fastx \
--quantMode GeneCounts
Assembly: structural_pool.fasta, structural_pool.gtf
Supplementary files format and content: stG4_raw_counts.txt: All raw read counts for each sequence in the pool

organism: synthetic construct

source_name: RBNS

extract_protocol:
In vitro transcribed synthetic RNA pools were made with guanine or 7dG. RT stop: RNAs were annealed in noted cations, then incubated with water or pyridostatin (where noted) for 10 minutes. RNA was reverse transcribed (RT) by SuperScript III in first strand buffer substituted with the noted cations for 40 minutes. Unless specifcially noted, RT was performed at 40ºC. After 40 minutes, samples were heated at 100ºC for 5 minutes, then proceeded to library preparation. RBNS: RNAs were annealed in KCl (or 7dG in LiCl), and allowed to incubate with noted proteins conjugated to streptavidin binding proteins. Bound RNA was eluted from streptavidin magnetic beads and concentrated by phenol-chloroform extraction. Subsequent RT was performed with SuperScript III in first strand buffer substituted with LiCl.
RT samples were PCR amplified with Phusion DNA polymerase to attach Illumina adapters and subsequently agarose gel purified. Concentrations were assessed by the Qubit dsDNA assay, and equivalent nanograms of each sample were pooled for sequencing.

value: RBNS

tag: guanine or_7dg

tag: rt temperature

tag: cation (and/or_pds)_treatment

value: LiCl

tag: biological replicate


title: WEHI231_DMSO_1

description: Library name: WEHI231_DMSO_1


title: 22Rv1, SREBP2_abcam, CUT&RUN

description: Library name: 22Rv1_SREBP2_abcam


title: S0014_Early_PreTfh_CXCR5+_PD1low

description: Library name: S0014


title: HepG2_MS7126N2_2


title: HepG2_MS7126N2_1


title: 22Rv1, EZH2, CUT&RUN

description: Library name: 22Rv1_EZH2


title: HepG2, control, CUT&RUN

description: Library name: HepG2_control

data_processing:
All reads were mapped to the human genome (hg19), and then the non-primary alignment and PCR duplicates were removed from aligned data respectively using the Picard, Samtools and bedtools.
Bigwig files, heatmaps and averaged plotting of CUT&RUN signals were generated using Deeptools.
Profiles of CUT&RUN read densities were displayed in the Integrative Genomics Viewer (IGV, Broad Institute).
The MACS2 software was used for peak identification with default parameters.
Assembly: hg19
Supplementary files format and content: Bigwig files
Library strategy: CUT&RUN

growth_protocol: Huh7 cells  were grown in DMEM medium supplemented with 10% of FBS and 1% of antibiotics. HepG2 and 22Rv1 cells were grown in RPMI1640 medium supplemented with 10% of FBS and 1% of antibiotics. HepG2 and Huh7 cells were stably expressed with HA-tagged EZH2 for HA CUT&RUN.


title: RNA AML GC7 rep 3

city: Cincinnati

first: Courtney

last: Jones

state: OH

address: 3333 Burnet Ave MLC 7013

institute: Cincinnati Children's Hospital Medical Center

description: Library name: RNA_27

data_processing:
Ribo-Seq FASTQ files were processed using the nf-core/riboseq pipeline (version 1.2.0) executed with Nextflow on the Cincinnati Children’s Hospital research high performance LSF computing cluster using Singularity containers. Quality control was performed using FastQC version 0.12.1 and validated using fq version 0.12.0. Illumina small-RNA adapters were removed using Trim Galore! version 0.6.10 and Cutadapt version 4.9, with a Phred quality threshold of 20, maximum error rate of 0.1, minimum adapter overlap of 1 nucleotide, and minimum retained read length of 18 nucleotides. Ribosomal RNA reads were depleted using SortMeRNA version 4.3.7 in paired-read mode against the eight default nf-core Rfam and SILVA default ribosomal RNA reference databases. Remaining ribosome-protected fragments were aligned using STAR version 2.7.11b to the Illumina iGenomes UCSC hg38 human reference genome and corresponding gene annotation. The STAR genome index was generated with annotated splice junctions and an sjdbOverhang of 100. Alignment was performed in basic two-pass mode with end-to-end alignment, a minimum annotated splice-junction overhang of 1 nucleotide, and a maximum of 20 reported alignments per read.  Matched paired-end bulk RNA-sequencing libraries were aligned to the human GRCh38 reference genome using STAR version 2.4.0h in a two-pass, sample-specific alignment. During the first pass, splice-junction evidence was collected using a maximum intron length of 500 kb, maximum mate gap of 1 Mb, and a maximum of 20 reported alignments per read. The resulting splice-junction coordinates were used to construct a sample-specific STAR genome index, followed by a second alignment to produce coordinate-sorted BAM files. Alignments retained standard mapping, mismatch, alignment-score, and splice-strand attributes.
Assembly: hg38 human reference genome
Supplementary files format and content: FASTQ

source_name: Hematopoietic Cells

growth_protocol: PDX cells were cultured in IMDM medium (Gibco, 12440061) supplemented with 20% heat inactivated fetal bovine serum (FBS; Corning, 35011CV), 100 U/mL penicillin-streptomycin (Gibco, 15140163), 10 ng/mL SCF (PeproTech, 300-07), 10 ng/mL Flt-3 ligand (PeproTech, 300-19), 10 ng/mL TPO (PeproTech, 300-18), 10 ng/mL IL-3 (PeproTech, 200-03), and 10 ng/mL IL-6 (PeproTech, 200-06).

extract_protocol:
Total RNA Extraction Using the Trizol Method 1) Thoroughly grind an appropriate amount of tissue in liquid nitrogen, transfer it to a 1.5 mL centrifuge tube, add 1 mL of Trizol, and mix thoroughly immediately. 2) Incubate the mixture at room temperature for 10 minutes to ensure complete lysis. 3) Add 200 μL of chloroform, shake vigorously to mix, and centrifuge at 12,000 rpm and 4C for 10 minutes. 4) Collect the upper aqueous phase, add an equal volume of phenol: chloroform (25:24), mix thoroughly, and centrifuge at 12,000 rpm and 4ÅãC for 10 minutes. 5) Collect the upper aqueous phase, add an equal volume of chloroform, mix thoroughly, and centrifuge at 12,000 rpm and 4ÅãC for 10 minutes. 6) Collect the upper aqueous phase, add an equal volume of isopropanol, incubate at -20ÅãC for 1 hour, and centrifuge at 12,000 rpm and 4ÅãC for 10 minutes. 7) Discard the supernatant, add 1 mL of 75% ethanol to wash the pellet, centrifuge at 8,000 rpm and 4ÅãC for 5 minutes, and discard the supernatant. 8) Repeat the previous step. 9) Briefly centrifuge, remove residual ethanol with a pipette, and vacuum-dry for 2–4 minutes. 10) Add 20–50 μL of RNase-free water, dissolve at room temperature for 10 minutes, mix, and briefly centrifuge. 11) Store at -80C.  For Ribosome Sequencing 1. Cell Fixation • Add harringtonine to the cell culture medium to a final concentration of 2 μg/ml and incubate at room temperature for 2 minutes. • Then, add cycloheximide to a final concentration of 100 μg/ml and incubate at room temperature for 1 minute. 2. Cell Washing • Remove culture medium and wash cells with PBS. 3. Cell Collection • Scrape the cells from the culture dish. • Collect the cells into a 1.5 mL Eppendorf tube. • Centrifuge at 500 g for 5 minutes at 4ÅãC. • Carefully discard the supernatant and collect the cell pellet. 4. Cell Lysis • Add cell lysis buffer to the cell sample. • Vortex to mix thoroughly. • Incubate on ice for 10 minutes. • Centrifuge at 17,000 g for 10 minutes at 4ÅãC. • Discard the pellet and collect the supernatant. 5. DNA and RNA Digestion • Take 400 μl of the cell supernatant. • Add 10 μl of RNase I and 6 μl of DNase I. • Incubate at room temperature for 45 minutes. 6. Ribosome Separation • Gently invert the MicroSpin S-400 Column to mix the resin and tap the walls of the tube to remove any bubbles. • Open both ends of the column to allow the liquid within the resin to naturally flow out. • Add 3 ml of 1Å~ Mammalian Polysome Buffer to the column and let it flow through by gravity. • Once the buffer has drained, place a 1.5 ml centrifuge tube under the column. • Centrifuge at 600 g for 4 minutes at room temperature. • Discard the flow-through liquid. • Place the column into a new 1.5 ml centrifuge tube. • Add the product from the previous step to the column, centrifuge again at 600 g for 2 minutes, and collect the flow-through. 7. RPF (Ribosome Protected Fragment) extraction • Add 700 μl of Lysis Reagent and incubate at room temperature for 8 minutes. • Add 140 μl of chloroform and mix thoroughly. • Incubate at room temperature for 5 minutes. • Centrifuge at 12,000 g for 15 minutes at 4ÅãC. • Transfer 350 μl of the supernatant to a new Eppendorf tube, add 525 μl of absolute ethanol, and mix well. • Load 700 μl of the sample onto a spin column and centrifuge at 10,000 rpm for 15 seconds at room temperature; discard the flow-through. • Repeat the above step. • Add 700 μl of Buffer RWT, centrifuge at 10,000 rpm for 15 seconds at room temperature, and discard the flow-through. • Add 500 μl of Buffer RPE, centrifuge at 10,000 rpm for 15 seconds at room temperature, and discard the flow-through. • Repeat the above step. • Transfer the spin column to a new 1.5 ml Eppendorf tube and add 30 μl of RNase-free water. 8. Removal of ribosomal RNA (rRNA) • Prepare a reaction for the removal of rRNA and perform PCR at 68°C for 10 minutes. Reagent Volume Ribo-Zero rRNA Reaction Buffer 4 μl Ribo-Zero Removal Solution 8 μl RNA sample（RPF） 28 μl Total 40 μl • Add the solution from the previous step to Magnetic Beads, and let it stand at room temperature for 10 minutes, followed by incubation at 50ÅãC for 5 minutes. • Purify the product from the previous step using Agencourt RNA Clean XP Beads. 9. 5’ phosphorylation reaction • Prepare the reaction system according to the table (not provided here). • Perform the reaction using PCR at 37°C for 30 minutes. Reagent Volume RNA 11 μl 10ÅBuffer 1.5 μl T4 Polynucleotide Kinase 1 μl ddH2O 1.5 μl total 15 ul
Library preparation 1) 3’ Adapter Ligation • Prepare the 3’ adapter ligation mixture. Reagent Volume Input RNA 6 μl 3ÅL SR Adaptor 1 μl 3ÅL Ligation Reaction Buffer (2X) 10 μl 3ÅL Ligation Enzyme Mix 3 μl Total 20 μl • Mix thoroughly and perform a brief centrifugation. • Incubate the reaction in a PCR machine at 25ÅãC for 1 hour. • Hold the reaction at 4ÅãC. 2) Reverse Transcription Primer Annealing • Add 1 μl of the SR RT primer specific for Illumina sequencing. • Add 4.5 μl of nuclease-free water. • Incubate in a PCR machine at 75ÅãC for 5 minutes, followed by 37ÅãC for 15 minutes, then 25ÅãC for 15 minutes. • Hold the reaction at 4ÅãC. 3) 5’ Adapter Ligation • Prepare the 5’ adapter ligation mixture. Reagent Volume Product from last step 25.5μl 5ÅL SR Adaptor 1 μl 5ÅL Ligation Reaction Buffer (10X) 1 μl 5ÅL Ligation Enzyme Mix 2.5 μl Total 30 μl • Mix thoroughly and perform a brief centrifugation. • Incubate the reaction in a PCR machine at 25C for 1 hour. • Hold the reaction at 4C. 4) Reverse Transcription Setup • Prepare the reverse transcription reaction mixture. Reagent Volume Adaptor Ligated RNA 30 μl First Strand Synthesis Reaction Buffer 8 μl Murine RNase Inhibitor 1 μl ProtoScript II Reverse Transcriptase 1 μl Total 40 μl • Incubate the reaction in a PCR machine at 50ÅãC for 1 hour to reverse transcribe the ligated RNA products into double-stranded cDNA. 5) PCR Reaction Setup: • Prepare the PCR reaction mixture as per your specific requirements or protocol. Reagent Volume LongAmp Taq 2X Master Mix 50 μl SR Primer for Illumina 2.5 μl Index (X) Primer 2.5 μl Nuclease free water 5 μl Product from last step 40 μl Total 100 μl • PCR (94C 15s，62C 30s，70C 5 min，4C hold) 6) Purification using PAGE Gel • Recover and purify DNA fragments of approximately 140 bp using a PAGE (Polyacrylamide Gel Electrophoresis) gel. • Dissolve the purified DNA fragments in 20 μl of EB (Elution Buffer) solution to complete the library construction. 7) DNA Quality and Concentration Analysis • Use the DNA 1000 Assay Kit (Agilent Technologies, Part No. 5067-1504) or the High Sensitivity DNA Assay Kit (Agilent Technologies, Part No. 5067- 4626) for analysis. • The DNA 1000 Assay Kit detects DNA fragment sizes ranging from 25 to 1000 base pairs, with concentration ranges from 0.1 to 50 ng/μL. • The High Sensitivity DNA Assay Kit allows for precise quantification of samples at picogram levels, often used as a quantification standard before sequencing, detecting fragment sizes from 50 to 7000 base pairs and concentration ranges from 5 to 500 pg/μL.

treatment_protocol: Cells were treated with 10uM DENSpm or 50uM GC7 for 24 hours.

value: Hematopoietic Cells

value: PDX Model

value: AML


title: HepG2_MS7126N1_2


title: SUDHL6_MS8815N_2

description: Library name: SUDHL6_MS8815N_2

growth_protocol: SUDHL6 cells were grown in RPMI1640 medium, supplemented with 10% of FBS and 1% of antibiotics.

treatment_protocol: SUDHL6 cells were treated with 2.5uM of DMSO, MS8815, EPZ-6438, or MS8815N for 18hr. Duplicated samples were collected for RNA preparation.


title: RNA AML GC7 rep 2

description: Library name: RNA_26


title: Huh7, control, CUT&RUN

description: Library name: Huh7_control


title: S0007_Late_PreTfh_CXCR5+_PD1int

description: Library name: S0007


title: HepG2_MS7126N1_1


title: RNA AML GC7 rep 1

description: Library name: RNA_25


title: SUDHL6_MS8815N_1

description: Library name: SUDHL6_MS8815N_1


title: HepG2_C24_2


title: S0006_Early_PreTfh_CXCR5+_PD1low

description: Library name: S0006


title: SUDHL6_EPZ-6438_2

description: Library name: SUDHL6_EPZ-6438_2


title: RNA AML DENSpm rep 3

description: Library name: RNA_24

value: DENSpm


title: SUDHL6_EPZ-6438_1

description: Library name: SUDHL6_EPZ-6438_1


title: S0005_Tfh_CXCR5++_PD1hi

description: Library name: S0005


title: HepG2_MS7126_2


title: SUDHL6_MS8815_2

description: Library name: SUDHL6_MS8815_2


title: RNA AML DENSpm rep 1

description: Library name: RNA_22


title: S0004_Naive_CD45RA+CXCR5neg_PD1neg

description: Library name: S0004


title: S0003_Late_PreTfh_CXCR5+_PD1int

description: Library name: S0003


title: RNA AML vehicle rep 3

description: Library name: RNA_21

value: Vehicle


title: SUDHL6_DMSO_2

description: Library name: SUDHL6_DMSO_2

value: DMSO


title: HepG2_MS7126_1


title: RNA AML vehicle rep 2

description: Library name: RNA_20


title: SUDHL6_DMSO_1

description: Library name: SUDHL6_DMSO_1


title: RNA AML vehicle rep 1

description: Library name: RNA_19


title: S0001_Tfh_CXCR5++_PD1hi

description: Library name: S0001

value: tonsil


title: Cisplatin plus iMSC-EVs 400 ug kidney, biological replicate 3

status: Public on Aug 01 2026

city: Pyeongchang-gun

first: Ran

state: Gangwon-do

address: 1447 Pyeongchang-daero, Daehwa-myeon

country: South Korea

institute: Seoul National University

description:
Library name: Cis_EV400_3
biological replicate 3
LD400_8_1

data_processing:
Raw sequencing reads were assessed for quality using FastQC v0.11.7.
Adapter sequences and low-quality bases were removed using Trimmomatic v0.38. Bases with quality scores below 3 were removed from both ends of the reads, sliding-window trimming was performed using a window size of 4 and a mean quality threshold of 15, and reads shorter than 36 bp were discarded.
Trimmed reads were aligned to the Mus musculus reference genome GRCm38 (mm10) using HISAT2 v2.1.0.
Reference-guided transcript assembly and expression quantification were performed using StringTie v2.1.3b with the -e option.
Gene- and transcript-level expression values were calculated as raw read counts, FPKM, and TPM.
Assembly: GRCm38 (mm10)
Supplementary files format and content: Tab-delimited text file containing gene-level raw read counts, FPKM values, and TPM values for all 12 RNA-seq samples. Rows represent genes and columns represent individual samples.
Supplementary files format and content: Tab-delimited text file containing transcript-level raw read counts, FPKM values, and TPM values for all 12 RNA-seq samples. Rows represent transcripts and columns represent individual samples.

source_name: kidney

growth_protocol: Male C57BL/6 mice were housed under controlled temperature and humidity conditions with a 12-hour light/dark cycle and provided ad libitum access to food and water.

extract_protocol:
Total RNA was isolated from mouse kidney tissues, and contaminating genomic DNA was removed by DNase treatment.
mRNA sequencing libraries were prepared from total RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina). mRNA was purified, randomly fragmented, and reverse-transcribed into cDNA. Sequencing adapters were ligated to both ends of the cDNA fragments, followed by PCR amplification and size selection to obtain insert sizes of approximately 200–400 bp. The libraries were sequenced on an Illumina NovaSeq X platform using 2 × 101-bp paired-end sequencing.

treatment_protocol: Cumulative kidney injury was induced by intraperitoneal administration of cisplatin at 9 mg/kg once weekly for five cycles. After the final cisplatin administration, mice received intravenous PBS or iMSC-EVs at doses of 200 or 400 ug every 3 days for a total of five doses. Normal control mice did not receive cisplatin or iMSC-EVs. Kidney tissues were collected 3 days after the final treatment.

value: kidney

value: wild type

value: cisplatin 9 mg/kg intraperitoneally once weekly for five cycles, followed by 400 ug iMSC-EVs intravenously every 3 days for five doses

tag: batch

value: batch 1


title: Cisplatin plus iMSC-EVs 400 ug kidney, biological replicate 2

description:
Library name: Cis_EV400_2
biological replicate 2
LD400_4_1


title: Cisplatin plus iMSC-EVs 400 ug kidney, biological replicate 1

description:
Library name: Cis_EV400_1
biological replicate 1
LD400_1_1


title: Cisplatin plus iMSC-EVs 200 ug kidney, biological replicate 3

description:
Library name: Cis_EV200_3
biological replicate 3
LD200_7_1

value: cisplatin 9 mg/kg intraperitoneally once weekly for five cycles, followed by 200 ug iMSC-EVs intravenously every 3 days for five doses


title: Cisplatin plus iMSC-EVs 200 ug kidney, biological replicate 2

description:
Library name: Cis_EV200_2
biological replicate 2
LD200_6_1


title: HepG2_C24_1


title: RNA AML DENSpm rep 2

description: Library name: RNA_23


title: Cisplatin plus iMSC-EVs 200 ug kidney, biological replicate 1

description:
Library name: Cis_EV200_1
biological replicate 1
LD200_4_1


title: Cisplatin plus PBS kidney, biological replicate 3

description:
Library name: Cis_PBS_3
biological replicate 3
LD5_1

value: cisplatin 9 mg/kg intraperitoneally once weekly for five cycles, followed by intravenous PBS every 3 days for five doses


title: SUDHL6_MS8815_1

description: Library name: SUDHL6_MS8815_1


title: Cisplatin plus PBS kidney, biological replicate 2

description:
Library name: Cis_PBS_2
biological replicate 2
LD3_1


title: Cisplatin plus PBS kidney, biological replicate 1

description:
Library name: Cis_PBS_1
biological replicate 1
LD1_1


title: S0002_Early_PreTfh_CXCR5+_PD1low

description: Library name: S0002


title: Normal control kidney, biological replicate 3

description:
Library name: Normal_3
biological replicate 3
N3_1

value: untreated normal control


title: HepG2_DMSO_2


title: Normal control kidney, biological replicate 2

description:
Library name: Normal_2
biological replicate 2
N2_1


title: Normal control kidney, biological replicate 1

description:
Library name: Normal_1
biological replicate 1
N1_1


title: 3dpf-gut-3_S6

last: Levic

address: 307 Research Drive

department: Cell Biology

description: Library name: Sample 6

data_processing:
Trimmomatic(v0.38) was used to trim sequencing adapters
Reads were aligned to the zebrafish genome (GRCz11)  using STAR 2.7.11.a
Reads were counted using featureCounts in the Subread package (v1.6.3)
Assembly: GRCz11
Supplementary files format and content: tab-delimited text files includes counts for each sample

organism: Danio rerio

source_name: Intestine

extract_protocol:
Total RNA from FACS-isolated intestinal epithelial cells was extracted using the Monarch Total RNA Miniprep Kit (NEB, T2010S) according to the manufacturer’s instructions.
RNA-sequencing libraries were made from ≥150ng of total RNA purified from zebrafish intestinal cells using the KAPA HyperPrep kit.

value: Intestine

value: cldn15 positive cells

value: Intestinal epithelial cell

value: TgKI - cldn15la-tdTomato

value: 4 dpf


title: HepG2_DMSO_1


title: Cortical NPCs, Chd1 knockout, DHT, Replicate 4

city: Reykjavik

first: Kimberley

middle: J

last: Anderson

address: Sturlugata 8

country: Iceland

institute: University of Iceland

description: Library name: CHD1_DHT_Rep4

data_processing:
RNA-seq: Reads were pseudoaligned to the GRCm39 transcriptome using Kallisto using paired-end mode and 100 bootstraps
Assembly: GRCm39
Supplementary files format and content: Kallisto abundance.tsv files

source_name: Embryonic cortex

growth_protocol: NPCs were cultured in neurobasal growth medium containing 1X B27 supplement (Thermo Fisher Scientific, 17504044), 1X Penicillin/Streptomycin (Thermo Fisher Scientific, 15140122), 1X Glutamax (Thermo Fisher Scientific, 35050038), 20 ng/mL FGF-2 (Peprotech, 100-18B), 20 ng/mL EFG (Peprotech, AF-100-15), and 2 μg/mL Heparin (MP Biomedicals, 210193125). NPCs were either sub-cultured or media was replaced every 2 days. Chd1 knockout (KO) was performed using lentivirus produced from pLV[CRISPR]-hCas9/Puro-U6>mChd1[gRNA#4675] (VB900142-6157mjg; VectorBuilder), which constitutively expresses Cas9 and a guide RNA targeting mouse Chd1. A control lentivirus expressing Cas9 alone was used as a negative control.

extract_protocol:
RNA-seq: RNA was extracted by homogenization in Tri-reagent (Thermo Fisher Scientific, 15596026), then isolated using the Direct-zol RNA isolation kit (Zymo, R2062) according to the manufacturer’s instructions. RNA quality and integrity was assessed using a Nanodrop spectrophotometer and a Bioanalyzer instrument with the RNA 6000 Nano Kit (Agilent, 5067-1511). Only samples with RNA integrity (RIN) scores above 8 were used for library preparation.
RNA-seq: PolyA-enriched RNAseq library construction and Illumina paired-end sequencing was performed by Novogene, to obtain 30 million paired-end 150 bp reads for each sample.

treatment_protocol: RNA-seq: Following selection and expansion, Chd1 KO NPCs were treated with either DMSO or 10 µM enzalutamide (MedChemExpress, HY-70002) for 30 mins, then 10nM DHT was added to appropriate wells and the cultures were incubated for an additional 24 h prior to harvesting in Tri-reagent for RNA sequencing. This was performed in 4 biological replicates, using NPCs derived from embryos from two separate litters.

value: Embryonic cortex

value: E18.5 cortical NPC

value: Neuronal progenitors

value: Chd1 KO

value: DHT


title: Cortical NPCs, Chd1 knockout, DHT + Enzalutamide, Replicate 4

description: Library name: CHD1_DHT_ENZ_Rep4

value: DHT + Enzalutamide


title: 3dpf-gut-2_S5

description: Library name: Sample 5

value: 3 dpf


title: Cortical NPCs, Chd1 knockout, DMSO, Replicate 4

description: Library name: CHD1_DMSO_Rep4

value: DMSO control


title: Cortical NPCs, Chd1 knockout, DHT + Enzalutamide, Replicate 3

description: Library name: CHD1_DHT_ENZ_Rep3


title: Cortical NPCs, Chd1 knockout, DHT, Replicate 3

description: Library name: CHD1_DHT_Rep3


title: Cortical NPCs, Wild-type, DMSO, Replicate 4

description: Library name: CTL_DMSO_Rep4

value: WT


title: Cortical NPCs, Chd1 knockout, DMSO, Replicate 3

description: Library name: CHD1_DMSO_Rep3


title: Cortical NPCs, Wild-type, DMSO, Replicate 3

description: Library name: CTL_DMSO_Rep3


title: Cortical NPCs, Chd1 knockout, DHT + Enzalutamide, Replicate 2

description: Library name: CHD1_DHT_ENZ_Rep2


title: Cortical NPCs, Chd1 knockout, DHT, Replicate 2

description: Library name: CHD1_DHT_Rep2


title: Cortical NPCs, Chd1 knockout, DMSO, Replicate 2

description: Library name: CHD1_DMSO_Rep2


title: Cortical NPCs, Wild-type, DMSO, Replicate 2

description: Library name: CTL_DMSO_Rep2


title: Inflammatory_replicate_2

value: Bone Marrow

value: Bone Marrow Derived Macrophages

value: LPS + IFNγ


title: S03_CD4_naive_H3K4me1

description: Library name: S03

value: tonsil


title: Cortical NPCs, Chd1 knockout, DHT + Enzalutamide, Replicate 1

description: Library name: CHD1_DHT_ENZ_Rep1


title: Cortical NPCs, Chd1 knockout, DHT, Replicate 1

description: Library name: CHD1_DHT_Rep1


title: Cortical NPCs, Wild-type, DMSO, Replicate 1

description: Library name: CTL_DMSO_Rep1


title: ΚΟ, dm-αKG, sample8

city: Dallas

first: Samuel

last: McBrayer

state: Texas

address: 5901 Forest Park Road

department: Children’s Research Institute

institute: University of Texas Southwestern Medical Center

description: Library name: KO-aKG-8

data_processing:
Fastq files were trimmed with TrimGalore (v.0.6.10). Pre- and post-trimming quality control was done using FastQC (v.0.12.1). RNA-seq data were aligned to the mouse reference genome (mm10) using Hisat2 (v.2.2.1). The sam files were converted to bam files by Samtools (v.1.6). Then the transcript coverage of UCSC gene annotations were performed using featureCounts (v.2.0.6). Bigwig files were created from bam files using the bamCoverage function in deepTools. Gene ontology (GO) analysis was performed via the gene ontology platform (https://geneontology.org). Volcano plots were generated using ggplot2, displaying log-transformed P values on the y-axis and log2 fold changes on the x-axis. Gene set enrichment analysis (GSEA) was conducted using GSEA software (v.4.3.3) (https://www.gsea-msigdb.org). The normalized enrichment score (NES) and the false discovery rate (FDR) Q value were calculated by permuting gene set types, with a significance threshold of FDR ≤ 0.25 used to identify significantly enriched gene sets.
Assembly: mm39
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: csv files include CPM values for each Sample

source_name: Brain

extract_protocol:
Total RNA from 10 mg brain tissue samples was extracted using the RNeasy Mini Kit according to manufacturer’s protocol (Qiagen 74004) and resuspended in 30 μl nuclease-free water.Total RNA from 10 mg brain tissue samples was extracted using the RNeasy Mini Kit according to manufacturer’s protocol (Qiagen 74004) and resuspended in 30 μl nuclease-free water.
Library preparation contains the following steps: 1. Poly-A mRNA capture and reverse transcription for cDNA synthesis: oligo-dT primer to capture just the mRNA (contains sample barcode, UMI, and Read 1 sequences). 2. Second strand synthesis and tagmentation: double stranded-cDNA is generated. Tagmentation creates fragments and incorporates Read 2 sequence. 3. Illumina library amplification: adds unique dual indices (UDI = i5, i7) and P5/P7 sequences.

treatment_protocol: Μice were treated with vehicle or 600 mg per kg dm-αKG (Sigma 349631) by intraperitoneal injection. On postnatal day 19 (P19), tissues including brain, were harvested. All tissues were immediately snap-frozen and subsequently stored in liquid nitrogen until further analysis.

value: Brain

value: dm-αKG


title: ΚΟ, dm-αKG, sample7

description: Library name: KO-aKG-7


title: Cortical NPCs, Chd1 knockout, DMSO, Replicate 1

description: Library name: CHD1_DMSO_Rep1


title: S02_CD4_naive_H3K4me1

description: Library name: S02


title: 5dpf-gut-1_S1

description: Library name: Sample 1

value: Intestine

value: cldn15 positive cells

value: Intestinal epithelial cell

value: TgKI - cldn15la-tdTomato

value: 5 dpf


title: MV4;11, control, CUT&RUN

description: Library name: MV4;11_control


title: ΚΟ, dm-αKG, sample6

description: Library name: KO-aKG-6


title: ΚΟ, dm-αKG, sample5

description: Library name: KO-aKG-5


title: ΚΟ, dm-αKG, sample4

description: Library name: KO-aKG-4


title: Steady_State_replicate_1


title: ΚΟ, dm-αKG, sample3

description: Library name: KO-aKG-3


title: ΚΟ, dm-αKG, sample2

description: Library name: KO-aKG-2


title: ΚΟ, Vehicle, sample10

description: Library name: KO-Veh-10

value: Vehicle


title: ΚΟ, Vehicle, sample8

description: Library name: KO-Veh-8


title: ΚΟ, Vehicle, sample7

description: Library name: KO-Veh-7


title: ΚΟ, Vehicle, sample4

description: Library name: KO-Veh-4


title: ΚΟ, dm-αKG, sample1

description: Library name: KO-aKG-1


title: ΚΟ, Vehicle, sample6

description: Library name: KO-Veh-6


title: ΚΟ, Vehicle, sample3

description: Library name: KO-Veh-3


title: ΚΟ, Vehicle, sample2

description: Library name: KO-Veh-2


title: ΚΟ, Vehicle, sample5

description: Library name: KO-Veh-5


title: ΚΟ, Vehicle, sample1

description: Library name: KO-Veh-1


title: WT, dm-αKG, sample8

description: Library name: WT-aKG-8


title: WT, dm-αKG, sample6

description: Library name: WT-aKG-6


title: WT, dm-αKG, sample7

description: Library name: WT-aKG-7


title: WT, dm-αKG, sample5

description: Library name: WT-aKG-5


title: WT, dm-αKG, sample3

description: Library name: WT-aKG-3


title: WT, dm-αKG, sample4

description: Library name: WT-aKG-4


title: WT, dm-αKG, sample2

description: Library name: WT-aKG-2


title: WT, dm-αKG, sample1

description: Library name: WT-aKG-1


title: WT, Vehicle, sample10

description: Library name: WT-Veh-10


title: WT, Vehicle, sample9

description: Library name: WT-Veh-9


title: WT, Vehicle, sample8

description: Library name: WT-Veh-8


title: WT, Vehicle, sample7

description: Library name: WT-Veh-7


title: WT, Vehicle, sample5

description: Library name: WT-Veh-5


title: WT, Vehicle, sample4

description: Library name: WT-Veh-4


title: WT, Vehicle, sample6

description: Library name: WT-Veh-6


title: WT, Vehicle, sample2

description: Library name: WT-Veh-2


title: WT, Vehicle, sample3

description: Library name: WT-Veh-3


title: WT, Vehicle, sample1

description: Library name: WT-Veh-1


title: B. cereus, strain 3178, non-conditioned medium, biol rep 3

city: Hannover

first: Nadja

last: Jessberger

address: Bischofsholer Damm 15

department: Institute for Food Quality and Food Safety

country: Germany

institute: University of Veterinary Medicine Hannover, Foundation

description: Library name: 3178_nk3

data_processing:
Samples were sequenced via Illumina HiSeq. 2500 Genome Sequencer with 2 x 50 bp paired-end mode. Data analysis included mapping of the samples against a reference genome (B. cereus F837/76; CP003187) and determination of gene expression.
Expression Analysis: The RNA-Seq reads are aligned to the reference genome or reference transcriptome using Bowtie gen erating genome / transcriptome alignments. TopHat identifies the potential exon-exon splice junctions of the initial alignment. Then Cufflinks identifies and quantifies the transcripts from the preprocessed RNA-Seq alignment-assembly. After this, Cuffmerge merges the identified transcript pieces to full length transcripts and annotates the transcripts based on the given annotations. Finally, merged transcripts from two or more samples / conditions are compared using Cuffdiff to determine the differential expres sion levels at transcript and gene level including a measure of significance between samples / conditions
Assembly: CP003187.fasta
Supplementary files format and content: significant gene expression k vs. nk, tsv format

organism: Bacillus cereus

source_name: CaCo-2 cells

growth_protocol: RPMI 1640 medium, no FCS, 2 % casein hydrolysate and a total glucose content of 1 %. The media were used directly (non-conditioned; nk) or after 22 h pre-incubation with CaCo-2 cells (conditioned; k) and subsequent filtering through a 0.2 µm filter. 50 ml of these media were inoculated with the bacterial pre-culture to a start OD600 of 0.05. Incubation took place statically in 80 cm2 culture flasks in a humidified incubator at 37 °C and 7 % CO2 for 3h.

extract_protocol:
Total RNA preparation and on-column DNase digestion were performed using RNeasy® Mini Kit (Qiagen) according to the instructions of the manufacturer. RNA quality was tested via spectrophotometer and agarose gel electrophoresis. RNA purity was confirmed via PCR for 16 S rRNA.
Random primed cDNA libraries were constructed according to the Illumina Genome Analyzer II protocol.

treatment_protocol: Conditioned (k; pre-incubation with CaCo-2 cells) vs. Non-conditioned (nk)

tag: strain

value: plain RPMI 1640 medium

value: CaCo-2 cells


title: B. cereus, strain 3178, conditioned medium, biol rep 3

description: Library name: 3178_k3

value: RPMI 1640 medium pre-incubated with CaCo-2 cells


title: B. cereus, strain 3178, non-conditioned medium, biol rep 2

description: Library name: 3178_nk2


title: B. cereus, strain 3178, non-conditioned medium, biol rep 1

description: Library name: 3178_nk1


title: B. cereus, strain 3178, conditioned medium, biol rep 2

description: Library name: 3178_k2


title: B. cereus, strain 3178, conditioned medium, biol rep 1

description: Library name: 3178_k1


title: B. cereus, strain 3053, non-conditioned medium, biol rep 2

description: Library name: 3053_nk2


title: Huh7_DMSO_1


title: B. cereus, strain 3053, non-conditioned medium, biol rep 3

description: Library name: 3053_nk3


title: B. cereus, strain 3053, non-conditioned medium, biol rep 1

description: Library name: 3053_nk1


title: B. cereus, strain 3053, conditioned medium, biol rep 3

description: Library name: 3053_k3


title: B. cereus, strain 3053, conditioned medium, biol rep 2

description: Library name: 3053_k2


title: B. cereus, strain 3053, conditioned medium, biol rep 1

description: Library name: 3053_k1


title: B. cereus, strain 1505, non-conditioned medium, biol rep 3

description: Library name: 1505_nk3


title: B. cereus, strain 1505, non-conditioned medium, biol rep 2

description: Library name: 1505_nk2


title: B. cereus, strain 1505, conditioned medium, biol rep 3

description: Library name: 1505_k3


title: B. cereus, strain 1505, non-conditioned medium, biol rep 1

description: Library name: 1505_nk1


title: B. cereus, strain 1505, conditioned medium, biol rep 2

description: Library name: 1505_k2


title: B. cereus, strain 1505, conditioned medium, biol rep 1

description: Library name: 1505_k1


title: Mesenteric lnn, day5, PigID_166

city: Copenhagen

first: Malene

middle: Roed

last: Spiegelhauer

address: Dyrlaegevej 68

country: Denmark

institute: University of Copenhagen

data_processing:
The libraries were sequenced on the Illumina Novaseq 6000 instrument A01426, flowcell ID H73WJDSX2. Original data was transformed to raw reads by CASAVA base recognition (base calling).
Quality was checked and adapter trimming performed.
Raw data are stored in FASTQ format files, and contain sequences of reads and corresponding quality
Paired-end clean reads were aligned to the reference genome using HISAT2 v2.0.5
Featurecounts v1.5.0-p3 was used to counts the reads numbers mapped to each gene. Differential expression was analysed with the DESeq2 R package (1.20.0)
Assembly: ncbi_sus_scrofa_gcf_000003025_6_sscrofa11_1
Supplementary files format and content: txt of read counts.
Supplementary files format and content: txt of FPKM counts.

organism: Sus scrofa

source_name: Mesenteric lymph node

growth_protocol: Preterm pigs delivered by C-section were kept in NICU for five days. They were nourished with parenteral nutrition and fed enteral nutrition.

extract_protocol:
Mesenteric lymph nodes were collected immediately after euthanasia and snap-frozen in liquid nitrogen. 20-25 mg of the tissue was homogenized, and total RNA was extracted using RNeasy Mini Kit (Qiagen, Copenhagen, Denmark).
Library was constructed by Novogene NGS RNA Library prep set. Messenger RNA was purified from extracted RNA with poly-T magnetic beads, and cDNA was synthesized using random hexamer primers.

treatment_protocol: On day 1 and 2, piglets received an oral dose of either native fecal virome (nFV), chemostat-propagated virome (cFV), chemostat-propagated virome with oligosaccharides (cFV-OS), or a specific phage cocktail (sPC). The control group received a sterile buffer (SM)

value: Mesenteric lymph node

tag: animal id

value: sPC


title: Mesenteric lnn, day5, PigID_165

value: cFV-OS


title: Mesenteric lnn, day5, PigID_164

value: nFV


title: Mesenteric lnn, day5, PigID_163

value: cFV


title: Mesenteric lnn, day5, PigID_161

value: SM


title: Mesenteric lnn, day5, PigID_160


title: Mesenteric lnn, day5, PigID_159


title: Mesenteric lnn, day5, PigID_157


title: Mesenteric lnn, day5, PigID_162


title: Mesenteric lnn, day5, PigID_156


title: Mesenteric lnn, day5, PigID_158


title: Mesenteric lnn, day5, PigID_155


title: Mesenteric lnn, day5, PigID_154


title: S0013_Tfh_CXCR5++_PD1hi

description: Library name: S0013

value: tonsil


title: Mesenteric lnn, day5, PigID_153


title: Mesenteric lnn, day5, PigID_150


title: Mesenteric lnn, day5, PigID_151


title: Mesenteric lnn, day5, PigID_149


title: Mesenteric lnn, day5, PigID_148


title: Mesenteric lnn, day5, PigID_152


title: Mesenteric lnn, day5, PigID_147


title: Mesenteric lnn, day5, PigID_146


title: Mesenteric lnn, day5, PigID_145


title: Mesenteric lnn, day5, PigID_144


title: Mesenteric lnn, day5, PigID_143


title: Mesenteric lnn, day5, PigID_141


title: S0008_Naive_CD45RA+CXCR5neg_PD1neg

description: Library name: S0008


title: Mesenteric lnn, day5, PigID_142


title: Mesenteric lnn, day5, PigID_140


title: Mesenteric lnn, day5, PigID_139


title: Mesenteric lnn, day5, PigID_138


title: Mesenteric lnn, day5, PigID_137


title: Mesenteric lnn, day5, PigID_136


title: Mesenteric lnn, day5, PigID_135


title: Mesenteric lnn, day5, PigID_133


title: Mesenteric lnn, day5, PigID_131


title: Mesenteric lnn, day5, PigID_130


title: Mesenteric lnn, day5, PigID_134


title: Mesenteric lnn, day5, PigID_132


title: Mesenteric lnn, day5, PigID_128


title: Mesenteric lnn, day5, PigID_127


title: Mesenteric lnn, day5, PigID_126


title: ΚΟ, Vehicle, sample9

description: Library name: KO-Veh-9

value: Brain


title: Mesenteric lnn, day5, PigID_129


title: Mesenteric lnn, day5, PigID_125


title: Mesenteric lnn, day5, PigID_123


title: Mesenteric lnn, day5, PigID_122


title: Mesenteric lnn, day5, PigID_120


title: Mesenteric lnn, day5, PigID_118


title: Mesenteric lnn, day5, PigID_119


title: Mesenteric lnn, day5, PigID_116


title: Mesenteric lnn, day5, PigID_117


title: Mesenteric lnn, day5, PigID_115


title: Mesenteric lnn, day5, PigID_112


title: Mesenteric lnn, day5, PigID_111


title: Mesenteric lnn, day5, PigID_110


title: Mesenteric lnn, day5, PigID_113


title: Mesenteric lnn, day5, PigID_109


title: Mesenteric lnn, day5, PigID_106


title: Mesenteric lnn, day5, PigID_105


title: Mesenteric lnn, day5, PigID_107


title: Mesenteric lnn, day5, PigID_102


title: HCT116, LagTag, M.EcoGII-mCherry, rep2

city: Pasadena

first: Yodai

last: Takei

state: California

address: 1200 E California Blvd

institute: California Institute of Technology

description: Library name: HCT116_LagTag_mCherry_rep2

data_processing:
The demultiplexing was performed with ‘bases2fastq’ tool (Element Biosciences). For the barcoded nb-Tn5 samples, the demultiplexing of the adapter barcodes was further performed with custom bash scripts making use of basic shell commands and the ‘seqtk’ package (seqtk-v1.5 https://github.com/lh3/seqtk).
Sequencing data was processed using the cutnrun pipeline v3.2.2 from nf-core (https://nf-co.re/cutandrun/3.2.2) with Nextflow v23.10.1.
Reads were aligned to the UCSC mm10 or hg38 genome using bowtie2 v.2.4.4, keeping only reads with a minimum alignment q score of 20. For the adapter barcoded samples, 42 bp from the 5’ end were removed with parameters --clip_r1 42 --clip_r2 42.
Aligned reads were masked using the CUT&RUN suspect list regions.
Bigwigs were generated from the alignments using the bamCoverage function of deeptools v3.5.1.
Assembly: mm10 or hg38
Supplementary files format and content: bigWig

growth_protocol: E14 mouse embryonic stem cells (E14Tg2a.4) were maintained on 0.1% gelatin coated plates under the serum/LIF condition in DMEM supplemented with 15% ES-grade fetal bovine serum (FBS), 1,000 units/mL leukemia inhibitory factor (LIF), 1× non-essential amino acids, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, and 1× Penicillin-Streptomycin-Glutamine. mESC-Gata4 cells were maintained on 0.1% gelatin coated plates under the serum/LIF/2i condition by supplementing the serum/LIF medium with 1 μM PD0325901 and 3 μM CHIR99021. The human HCT116 cells (ATCC CCL‐247) were maintained in McCoy's 5A (modified) medium supplemented with 10% FBS and 1× Penicillin-Streptomycin. All cell lines were cultured at 37°C with 5% CO2.

extract_protocol:
Cells were fixed with 50 μL of 1% formaldehyde in 1× PBS at room temperature for 5 minutes on 96-well plates.
Cells were permeabilized with 0.5% Triton-X in 1× PBS. For LagTag, additional treatment with 5% RNaseA/T1 Mix as well as NaOH denaturation were performed. These treatments were omitted in CUT&Tag experiments unless noted. Samples were incubated with primary antibodies in blocking buffer (1× PBS, 1% BSA, and 0.05% Triton-X), then with nb-Tn5 transposomes (in-house) in wash 300 buffer (20 mM HEPES pH 7.4, 300 mM NaCl, and 0.05% Triton-X) supplemented with 0.1% BSA. Tagmentation was then performed in wash 300 buffer supplemented with 10 mM MgCl2 and 0.1% BSA. The DNA fragments were extracted using 20 μL SDS release buffer (10 mM TAPS buffer pH 8.5, 0.2% SDS, and 5% thermolabile proteinase K), then neutralized with 12 μL of 10% Triton-X for PCR input. Libraries were prepared by mixing 21 μL input, 2 μL each of i5 and i7 primers (10 μM stock), and 25 μL of NEBNext High-Fidelity 2X PCR Master Mix. PCR was performed as follows: 5 min at 58°C, 5 min at 72°C, and 45 sec at 98°C followed by 13-15 cycles of 15 sec at 98°C and 10 sec at 60°C, and a final extension at 72°C for 1 min. The amplified libraries were purified using 1.3× magnetic beads.

treatment_protocol: The mESC-Gata4 differentiation to endodermal cells was induced by 1 μg/mL Dox induction and LIF withdrawal for 24 hours. For LagTag, the adenine methyltransferase fusion proteins were stabilized with 1 μM Shield-1 for 16 hours in the engineered cells.

value: colonocyte

value: DD-linker-M.EcoGII-mCherry, polyclonal

value: 1 μM Shield-1 for 16 hours

tag: antibody

value: m6A (Invitrogen MA5-35350)


title: Mesenteric lnn, day5, PigID_101


title: Mesenteric lnn, day5, PigID_100


title: HCT116, LagTag, M.EcoGII-mCherry, rep1

description: Library name: HCT116_LagTag_mCherry_rep1

tag: cell line


title: PBMC; SeqWell; Animal T627; Timepoint ART3; Array 4D

first: Alex

last: Shalek

address: 77 Massachusetts Ave

institute: Massachusetts Institute of Technology

description:
Library name: T627_ART3_4D
T627_ART3_4D

library_source: transcriptomic single cell

data_processing:
BCL files were converted to fastQs via Terra cumulus/bcl2fastq/5
For processed data: A custom genome combining  mmul10 and SIVmac251 was created using jgould/dropseq_bundle/10 (Terra). Alignment was performed using cumulus/dropseq_workflow/11 (Terra).
Assembly: custom mmul10 + SIVmac251
Supplementary files format and content: RDS, a seurat object of all cells combined from all samples directly following alignment (untrimmed, filtered)

organism: Macaca mulatta

source_name: PBMC

extract_protocol:
PBMC samples from rhesus macaque were processed for single-cell analysis using SeqWellS3 as described in Hughes, et al; Immunity 2020.
scRNAseq libraries were prepared via NexteraXT as described in Hughes, et al; Immunity 2020

tag: tissue

value: PBMC

tag: animalid

tag: timepoint

tag: array

tag: assay

value: SeqWell

tag: hassiv

value: yes

tag: onart

tag: weekssinceinfection

tag: weeksonart


title: PBMC; SeqWell; Animal T627; Timepoint ART3; Array 6F

description:
Library name: T627_ART3_6F
T627_ART3_6F


title: PBMC; SeqWell; Animal T625; Timepoint ART3; Array 8H

description:
Library name: T625_ART3_8H
T625_ART3_8H


title: PBMC; SeqWell; Animal T625; Timepoint ART3; Array 7G

description:
Library name: T625_ART3_7G
T625_ART3_7G


title: PBMC; SeqWell; Animal T623; Timepoint ART3; Array 5E; techrep1; NextSeq2000

description:
Library name: T623_ART3_5E_techrep1
T623_ART3_5E


title: PBMC; SeqWell; Animal T625; Timepoint ART3; Array 2B

description:
Library name: T625_ART3_2B
T625_ART3_2B


title: PBMC; SeqWell; Animal T623; Timepoint ART3; Array 3C; techrep1; NextSeq2000

description:
Library name: T623_ART3_3C_techrep1
T623_ART3_3C


title: PBMC; SeqWell; Animal T537; Timepoint ART3; Array 8H

description:
Library name: T537_ART3_B1_8H
T537_ART3_B1_8H


title: PBMC; SeqWell; Animal T537; Timepoint ART3; Array 2B; techrep1; NextSeq2000

description:
Library name: T537_ART3_B1_2B_techrep1
T537_ART3_B1_2B


title: PBMC; SeqWell; Animal T530; Timepoint ART3; Array 4D

description:
Library name: T530_ART3_4D
T530_ART3_4D


title: PBMC; SeqWell; Animal T537; Timepoint ART3; Array 1A; techrep 1; NextSeq2000

description:
Library name: T537_ART3_B1_1A_techrep1
T537_ART3_B1_1A


title: PBMC; SeqWell; Animal T530; Timepoint ART3; Array 3C

description:
Library name: T530_ART3_3C
T530_ART3_3C


title: R1_ESCs_n2_PCS_R10

city: Quebec City

first: Samer

last: Hussein

state: Quebec

address: 9 rue Mcmahon

department: Departement of medecine

country: Canada

institute: CHU de Québec - Université Laval

description: Library name: Sample 45

data_processing:
Raw fast5 files from R9 sequencing were converted to the more recent pod5 format using the pod5-file-format software from nanoporetech's github. pod5 files from R9 and R10 libraries files were then basecalled using Guppy v.6.5.7 using the super accurate basecalling settings.
Samples were demultiplexed according to barcodes with the guppy_barcoder software.
The resulting FASTQ reads were aligned to the mm10 mouse reference genome with Minimap2 version 2.24-r1122. Reads with MAPQ<13 were filtered out.
Aligned reads were quality-controlled with the NanoPlot software.
Strand for spliced transcripts were detemined using Minimap2's ts tag ("transcript strand"). As this tag is empty for mono-exonic transcripts, these were oriented with the pychopper software from nanoporetech's github. Stranded BAM files are provided as raw files (..._sup.XS_tag.bam files)
Transcriptome assembly was performed using the Tama software, with the tama_collapse.py script.
Isoform classification was performed on the transcriptome assembly using the SQANTI3 software. SQANTI3 QC was used to classify isoforms. Artifactual isoforms were then filtered using SQANTI3 filter with the "Rules filter" using default rules. Filtered-out isoforms were then rescued using SQANTI3 rescue. A final filter was performed to remove isoforms without a detected polyA as well as isoforms derived from RT-switching events.
Read counts were performed using the Isoquant software, using the previously generated and filtered transcriptome assembly.
Assembly: mm10
Supplementary files format and content: GSE282319_Isoquant.gene_name_id.cpm.txt.gz: Normalized gene-level read counts (in Counts per million, CPM) obtained with the Isoquant software. Annotation file used for this analysis was the "Repro_filter_tst.gtf" file included as a processed data file.
Supplementary files format and content: GSE282319_Isoquant.transcript_name_id.tpm.txt.gz: Normalized transcript-level read counts (in Transcripts per million, TPM) obtained with the Isoquant software. Annotation file used for this analysis was the "Repro_filter_tst.gtf" file included as a processed data file.
Supplementary files format and content: GSE282319_Repro_filter_tst.gtf.gz: Filtered transcriptome annotation file generated with the Tama software from the long-read RNA sequencing data presented here and filtered with the SQANTI3 pipeline.
Supplementary files format and content: GSE282319_Repro_unfiltered_tst.gtf.gz: Transcriptome annotation file generated with the Tama software from the long-read RNA sequencing data presented here (before isoform QC and filtering).
Supplementary files format and content: GSE282319_Repro_unfiltered_tst_classification.txt.gz: SQANTI3 classification file for GSE282319_Repro_unfiltered_tst.gtf.gz transcriptome annotation.

growth_protocol: Stably transfected Oct4-GFP ESC-derived NPC cell lines were grown in NPC medium on Geltrex™-coated plates (hESC-qualified, growth factor-reduced, ThermoFisher Scientific, #A1413302). NPC medium consists of N2B27 medium with added supplements. N2B27 medium consists of a 1:1 mixture of DMEM/F-12 media mix (ThermoFisher Scientific #11330057) supplemented with 1X N-2 supplement (ThermoFisher Scientific #17502048) and 20μg/mL human recombinant Insulin (ThermoFisher Scientific #12585014), and Neurobasal™ media (ThermoFisher Scientific #21103049) supplemented with 1X B-27™ supplement (ThermoFisher Scientific #17504044), 1mM sodium pyruvate (ThermoFisher Scientific #11360070), 2mM L-glutamine (ThermoFisher Scientific #35050061), 1X MEM Non-Essential Amino Acid solution (ThermoFisher Scientific #11140050) and 10-4M 2-Mercaptoethanol (ThermoFisher Scientific #21985023). Supplements to add for NPC media : 20ng/mL human recombinant EGF, 20ng/mL human recombinant FGF-2 and 500ng/mL heparin sodium salt. Medium was refreshed every other day and NPCs were passaged every 2-3 days. For passaging, cells were washed with DPBS (ThermoFisher Scientific #14190250) and treated with StemPro™ Accutase™ Cell Dissociation Reagent (ThermoFisher Scientific #A1110501) for 5 minutes. Then, StemPro™ Accutase™ Cell Dissociation Reagent was neutralized using DMEM (high glucose, ThermoFisher Scientific #11965118) with 5% KSR (ThermoFisher Scientific #10828-028) and cells were passaged as single-cells (usually 1/6-1/8 ratio). All cell lines were maintained in a humidified incubator at 37°C under 5% CO2 and were negative for mycoplasma.

extract_protocol:
Cell lysates were homogenized with QIAshredder columns (Qiagen #79656) and total RNA was extracted using RNeasy Kit (Qiagen #74106) according to the manufacturer’s instructions. RNA was quantified using a NanoDropTM 2000 Spectrophotometer (ThermoFisher #ND-2000C) and then DNase-treated using TURBOTM DNase (ThermoFisher Scientific #AM2238) according to the manufacturer’s instructions. 300ng of DNase-treated and non-treated RNA were loaded onto a 1% Agarose A (Bio Basic #D0012) gel to verify the RNA integrity and purity. RNA was also quantified using the Qubit™ RNA Broad Range Kit (ThermoFisher Scientific #Q10210) on Qubit™ 4 Fluorometer (ThermoFisher Scientific #Q33238) according to the manufacturer’s instructions. RNA quality was further verified using an RNA 6000 Nano Kit (Agilent Technologies #5067-1511) on a 2100 Bioanalyzer instrument (Agilent Technologies #G2939B) according to the manufacturer’s instructions. Samples all had RNA integrity numbers of 7.8 or more. For samples sequenced on R9 Flow Cells, polyadenylated RNA was enriched from 7.5-20μg of total RNA using NEB®Next Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and a DynaMag™-2 Magnet (ThermoFisher Scientific #12-321-D). PolyA+-enriched RNA was quantified using the Qubit™ RNA High Sensitivity Kit (ThermoFisher Scientific #Q32852) on Qubit™ 4 Fluorometer according to the manufacturer’s instructions and RNA quality was verified using an RNA 6000 Pico Kit (Agilent Technologies #5067-1513) on a 2100 Bioanalyzer instrument according to the manufacturer’s instructions. For samples sequenced on R10 Flow Cells, total RNA was used for RT (Poly(A) enrichment step was omitted).
R9 samples were processed with Oxford Nanopore Technologies' (ONT) SQK-DCS109 kit according to the manufacturer's protocol: "Direct cDNA Native Barcoding (SQK-DCS109 with EXP-NBD104 and EXP-NBD114)" version DCB_9091_v109_revF_04Feb2019. 55-100ng of poly(A)+ RNA were used for each sample's RT reaction. Samples were barcoded with the ONT EXP-NBD104 native barcoding expansion. Barcoded samples were pooled, loaded on a MinION sequencer (ONT) using eight R9.4.1 Flow Cells (ONT #FLO-MIN106D), and sequenced using the MinKNOW (v19) software. Purification of DNA between each step was performed with KAPA Hyperpure Beads (Roche). Flow cells were washed and reloaded with the Flow Cell Wash Kit (ONT #EXP-WSH003) to increase sequencing depth obtained per flow cell. R10 samples were processed with ONT's SQK-NBD114.24 kit using 50ng of total RNA, with each sample assigned to a specific barcode. Briefly, an oligo-dT primer (VNP, sequence: 5'-5phos/ACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTVN-3' , where V=A, C, or G, and N=A, C, G, or T) was used to reverse-transcribe poly(A) RNA into cDNA with the Maxima H Minus RT (ThermoFisher Scientific). A Strand Switching Primer (SSP 5'-TTTCTGTTGGTGCTGATATTGCTmGmGmG-3' , where mG = 2' O-Methyl RNA bases) was used for strand switching during the RT reaction. Libraries were PCR amplified for 14 cycles with LongAmp Taq polymerase (NEB #M0287) or NEBNext Ultra II Q5 Master Mix (NEB #M0544L). cDNA was quantified using the Qubit™ 1X dsDNA High Sensitivity Kit (ThermoFisher Scientific #Q33230) on Qubit™ 4 Fluorometer according to the manufacturer’s instructions and cDNA smear quality was verified using a DNA 12000 Kit (Agilent Technologies #5067-1508) on a 2100 Bioanalyzer instrument (Agilent Technologies #G2939B) according to the manufacturer’s instructions. The blunt DNA ends were prepared using the NEBNext Ultra II End Repair/dA-Tailing module (NEB), adding a 3' dA tail and phosphorylating the 5' end. Barcodes were ligated to the DNA fragments with a Blunt/TA ligase (NEB), barcoded samples were pooled and motor protein adapters were finally ligated with the NEBNext Quick Ligation Module (NEB). Purification of DNA between each step was performed with KAPA Hyperpure Beads (Roche). Library was loaded on a PromethION 2 Solo (ONT) sequencer using five R10.4.1 Flow Cells (ONT #FLO-PRO114M), and sequenced using the MinKNOW (v23.07.15) software.

treatment_protocol: Stably transfected Oct4-GFP ESC-derived NPC cell lines were seeded at 3 x 103 cells/cm2 on a 0.1% porcine gelatin (Sigma-Aldrich #G1890) coating in NPC medium supplemented with 2%  Fetal Bovine Serum (FBS) to promote adhesion. The following day, medium was replaced with FBS-LIF supplemented with 1µg/mL doxycycline hyclate (Sigma-Aldrich #D9891) to induce overexpression of the reprogramming factors. FBS-LIF medium consists of DMEM (high glucose, ThermoFisher Scientific #11965118) supplemented with 15% FBS, ThermoFisher Scientific #12483020), 1mM sodium pyruvate (ThermoFisher Scientific #11360070), 2mM L-glutamine (ThermoFisher Scientific #35050061), 1X MEM Non-Essential Amino Acid solution (ThermoFisher Scientific #11140050) and 10-4M 2-mercaptoethanol (ThermoFisher Scientific #21985023). Medium was refreshed every other day until D6. From D6, samples noted as "2i-LIF at D6" (D14 and D20) were maintained in 2i-LIF medium and passaged every 2-4 days depending on colonies density. 2i-LIF medium consists of Leukemia Inhibitory Factor (LIF, home-made from pGEX-2T-MLIF, Mereau et al., J. Cell. Biol. 1993) at 20ng/mL, 3μM CHIR-99021 (Selleck Chemicals #S2924) and 1μM PD0325901 (Selleck Chemicals #S1036) in N2B27 media. D10, D14 and D20 samples noted as "FBS-LIF + dox" were instead maintained in FBS-LIF medium supplemented with 1µg/mL doxycycline hyclate. For passaging or collection of time points, cells were washed with DPBS (ThermoFisher Scientific #14190250) and treated with TrypLE Express Enzyme (ThermoFisher Scientific #12604021) for 5 minutes. Then, TrypLE Express Enzyme was neutralized using DMEM (high glucose, ThermoFisher Scientific #11965118) with 5% KSR (ThermoFisher Scientific #10828-028) and cells were passaged as single-cells (1/3-1/30 ratio depending on density). Long-read RNAseq time points were pelletted and flash frozen in liquid nitrogen, then kept in a -80°C Bio-freezer until RNA extraction. Evolution of the proportion of mCherry+ and GFP+ cells was monitored using fluorescence microscopy and flow cytometry with a BD Accuri™ C6 Plus flow cytometer (BD Biosciences). iPSCs could readily be maintained in 2i-LIF medium after 20 days. All cell lines were negative for mycoplasma. Mouse ESC control lines are maintained in 2i-LIF medium. A clonally amplified line from the R1 cell line was also used as an mESC control (R1-Clone3). NPC control lines were maintained in NPC medium.

tag: cell type

value: Embryonic stem cells

tag: genotype

value: WT

tag: treatment


title: R1_ESCs_n1_PCS_R10

description: Library name: Sample 44


title: PBMC; SeqWell; Animal T530; Timepoint ART3; Array 1A

description:
Library name: T530_ART3_1A
T530_ART3_1A


title: R1_ESCs_c3_n1_PCS_R10

description: Library name: Sample 42


title: O4G_ESCs_n2_PCS_R10

description: Library name: Sample 41


title: d18_2iL_OKS_ctrl_PCS_R10

description: Library name: Sample 39

value: Reprogramming timeline to induced pluripotent stem cells

value: OKS-Luciferase-AS


title: O4G_ESCs_n1_PCS_R10

description: Library name: Sample 40


title: d18_OKMS_ctrl_PCS_R10

description: Library name: Sample 36

value: OKMS-Luciferase-AS

value: FBS-LIF + dox


title: d18_OKS_ctrl_PCS_R10

description: Library name: Sample 37


title: NPCs_WT_Rep3_PCS_R10

extract_protocol:
Cell lysates were homogenized with QIAshredder columns (Qiagen #79656) and total RNA was extracted using RNeasy Kit (Qiagen #74106) according to the manufacturer’s instructions. RNA was quantified using a NanoDropTM 2000 Spectrophotometer (ThermoFisher #ND-2000C) and then DNase-treated using TURBOTM DNase (ThermoFisher Scientific #AM2238) according to the manufacturer’s instructions. 300ng of DNase-treated and non-treated RNA were loaded onto a 1% Agarose A (Bio Basic #D0012) gel to verify the RNA integrity and purity. RNA was also quantified using the Qubit™ RNA Broad Range Kit (ThermoFisher Scientific #Q10210) on Qubit™ 4 Fluorometer (ThermoFisher Scientific #Q33238) according to the manufacturer’s instructions. RNA quality was further verified using an RNA 6000 Nano Kit (Agilent Technologies #5067-1511) on a 2100 Bioanalyzer instrument (Agilent Technologies #G2939B) according to the manufacturer’s instructions. Samples all had RNA integrity numbers of 7.8 or more. For samples sequenced on R9 Flow Cells, polyadenylated RNA was enriched from 7.5-20μg of total RNA using NEB®Next Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and a DynaMag™-2 Magnet (ThermoFisher Scientific #12-321-D). PolyA+-enriched RNA was quantified using the Qubit™ RNA High Sensitivity Kit (ThermoFisher Scientific #Q32852) on Qubit™ 4 Fluorometer according to the manufacturer’s instructions and RNA quality was verified using an RNA 6000 Pico Kit (Agilent Technologies #5067-1513) on a 2100 Bioanalyzer instrument according to the manufacturer’s instructions. For samples sequenced on R10 Flow Cells, total RNA was used for RT (Poly(A) enrichment step was omitted).
R9 samples were processed with Oxford Nanopore Technologies' (ONT) SQK-DCS109 kit according to the manufacturer's protocol: "Direct cDNA Native Barcoding (SQK-DCS109 with EXP-NBD104 and EXP-NBD114)" version DCB_9091_v109_revF_04Feb2019. 55-100ng of poly(A)+ RNA were used for each sample's RT reaction. Samples were barcoded with the ONT EXP-NBD104 native barcoding expansion. Barcoded samples were pooled, loaded on a MinION sequencer (ONT) using eight R9.4.1 Flow Cells (ONT #FLO-MIN106D), and sequenced using the MinKNOW (v19) software. Purification of DNA between each step was performed with KAPA Hyperpure Beads (Roche). Flow cells were washed and reloaded with the Flow Cell Wash Kit (ONT #EXP-WSH003) to increase sequencing depth obtained per flow cell. R10 samples were processed with ONT's SQK-NBD114.24 kit using 50ng of total RNA, with each sample assigned to a specific barcode. Briefly, an oligo-dT primer (VNP, sequence: 5'-5phos/ACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTVN-3' , where V=A, C, or G, and N=A, C, G, or T) was used to reverse-transcribe poly(A) RNA into cDNA with the Maxima H Minus RT (ThermoFisher Scientific). A Strand Switching Primer (SSP 5'-TTTCTGTTGGTGCTGATATTGCTmGmGmG-3' , where mG = 2' O-Methyl RNA bases) was used for strand switching during the RT reaction. Libraries were PCR amplified for 14 cycles with LongAmp Taq polymerase (NEB #M0287). cDNA was quantified using the Qubit™ 1X dsDNA High Sensitivity Kit (ThermoFisher Scientific #Q33230) on Qubit™ 4 Fluorometer according to the manufacturer’s instructions and cDNA smear quality was verified using a DNA 12000 Kit (Agilent Technologies #5067-1508) on a 2100 Bioanalyzer instrument (Agilent Technologies #G2939B) according to the manufacturer’s instructions. The blunt DNA ends were prepared using the NEBNext Ultra II End Repair/dA-Tailing module (NEB), adding a 3' dA tail and phosphorylating the 5' end. Barcodes were ligated to the DNA fragments with a Blunt/TA ligase (NEB), barcoded samples were pooled and motor protein adapters were finally ligated with the NEBNext Quick Ligation Module (NEB). Purification of DNA between each step was performed with KAPA Hyperpure Beads (Roche). Library was loaded on a PromethION 2 Solo (ONT) sequencer using two R10.4.1 Flow Cells (ONT #FLO-PRO114M), and sequenced using the MinKNOW (v23.07.15) software.

treatment_protocol: Stably transfected Oct4-GFP ESC-derived NPC cell lines were seeded at 3 x 103 cells/cm2 on a 0.1% porcine gelatin (Sigma-Aldrich #G1890) coating in NPC medium supplemented with 2%  Fetal Bovine Serum (FBS) to promote adhesion. The following day, medium was replaced with FBS-LIF supplemented with 1µg/mL doxycycline hyclate (Sigma-Aldrich #D9891) to induce overexpression of the reprogramming factors. FBS-LIF medium consists of DMEM (high glucose, ThermoFisher Scientific #11965118) supplemented with 15% FBS, ThermoFisher Scientific #12483020), 1mM sodium pyruvate (ThermoFisher Scientific #11360070), 2mM L-glutamine (ThermoFisher Scientific #35050061), 1X MEM Non-Essential Amino Acid solution (ThermoFisher Scientific #11140050) and 10-4M 2-mercaptoethanol (ThermoFisher Scientific #21985023). Medium was refreshed every other day until D6. From D6, samples noted as "2i-LIF at D6" (D14 and D20) were maintained in 2i-LIF medium and passaged every 2-4 days depending on colonies density. 2i-LIF medium consists of Leukemia Inhibitory Factor (LIF, home-made from pGEX-2T-MLIF, Mereau et al., J. Cell. Biol. 1993) at 20ng/mL, 3μM CHIR-99021 (Selleck Chemicals #S2924) and 1μM PD0325901 (Selleck Chemicals #S1036) in N2B27 media. D10, D14 and D20 samples noted as "FBS-LIF + dox" were instead maintained in FBS-LIF medium supplemented with 1µg/mL doxycycline hyclate. For passaging or collection of time points, cells were washed with DPBS (ThermoFisher Scientific #14190250) and treated with TrypLE Express Enzyme (ThermoFisher Scientific #12604021) for 5 minutes. Then, TrypLE Express Enzyme was neutralized using DMEM (high glucose, ThermoFisher Scientific #11965118) with 5% KSR (ThermoFisher Scientific #10828-028) and cells were passaged as single-cells (1/3-1/30 ratio depending on density). Long-read RNAseq time points were pelletted and flash frozen in liquid nitrogen, then kept in a -80°C Bio-freezer until RNA extraction. Evolution of the proportion of mCherry+ and GFP+ cells was monitored using fluorescence microscopy and flow cytometry with a BD Accuri™ C6 Plus flow cytometer (BD Biosciences). iPSCs could readily be maintained in 2i-LIF medium after 20 days. All cell lines were negative for mycoplasma. Mouse ESC control lines are maintained in 2i-LIF medium. NPC control lines were maintained in NPC medium.

value: Neural progenitor cells

value: NPC medium


title: NPCs_OKS_ctrl_PCS_R10


title: R1_ESCs_2iL_PCS_R10


title: NPCs_OKMS_ctrl_PCS_R10


title: d6_OKS_ctrl_PCS_R10


title: d4_OKMS_ctrl_PCS_R10


title: d2_OKS_ctrl_PCS_R10


title: d2_OKMS_ctrl_PCS_R10


title: D2_FBSL_ctrl_Rep3_PCS_R10

value: Control non-reprogramming cells


title: d10_OKMS_ctrl_PCS_R10


title: D2_FBSL_ctrl_Rep2_PCS_R10


title: zebrafish_FACS_purified_cranial_motor_neurons_72hpf_biorep_03_lane2

city: Seattle

first: Austin

last: Seroka

state: WA

address: 1100 Fairview Ave. North

department: Basic Sciences

institute: Fred Hutchinson Cancer Center

description:
Library name: 72_8_Lane2
10X Genomics Chromium 3' mRNA v3

data_processing:
The demultiplexing, barcoded processing and gene counting were done using the Cell Ranger software v7.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: Danio_rerio.GRCz11.dna.primary_assembly
Supplementary files format and content: Tab-separated values files and matrix files

source_name: FACS purified hindbrain

extract_protocol:
Tg:(isl1:kaede) embryos were raised to 24, 48, 72 or 144hpf respectively prior to anesthetization in 400mg/L MESAB. Hindbrains were dissected in Calcium-Free Ringers / EDTA / 10% MESAB, prior to dissociation to single-cell suspension by pipetting in 0.25% trypsin-EDTA for 5 minutes. Cells were briefly spun down, transferred to dPBS + BSA and passed through a 40uM filter. Prior to FACS, cell health was evaluated using 2pg/mL DAPI. Kaede+ cells were isolated on a BD FACS ARIA II flow cytometer using a 100uM nozzle and collected into dPBS + 2% BSA. Collected cells were concentrated by centrifugation at 300 x g for 5 minutes at 4°C and resuspended to an optimal concentration of 1,200 cells/uL. Cell viability was assessed using a TC20 Automated Cell Counter. Libraries were prepared using the 10X Genomics Chromium 3’ mRNA v3 kit, prior to sequencing on a Novaseq S1 Flowcell
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction.

value: FACS purified hindbrain


title: zebrafish_FACS_purified_cranial_motor_neurons_72hpf_biorep_03_lane1

description:
Library name: 72_8_Lane1
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_48hpf_biorep_02_lane2

description:
Library name: 48_7_Lane2
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_72hpf_biorep_02_lane2

description:
Library name: 72_6_Lane2
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_48hpf_biorep_02_lane1

description:
Library name: 48_7_Lane1
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_72hpf_biorep_02_lane1

description:
Library name: 72_6_Lane1
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_72hpf_biorep_01_lane2

description:
Library name: 72_5_Lane2
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_72hpf_biorep_01_lane1

description:
Library name: 72_5_Lane1
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_48hpf_biorep_01_lane1

description:
Library name: 48_3_Lane1
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_28hpf_biorep_02_lane1

description:
Library name: 28_2_Lane1
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_28hpf_biorep_01_lane1

description:
Library name: 28_1_Lane1
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_28hpf_biorep_01_lane2

description:
Library name: 28_1_Lane2
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_28hpf_biorep_02_lane2

description:
Library name: 28_2_Lane2
10X Genomics Chromium 3' mRNA v3


title: zebrafish_FACS_purified_cranial_motor_neurons_6dpf_biorep_02

description:
Library name: 6_02
10X Genomics Chromium 3' mRNA v3


title: scRNAseq direct XEN TET-TKO rep2

city: Jerusalem

first: Shlomtzion

last: Lahav

address: Edmond J. Safra Campus, Givat Ram

country: Israel

institute: The Hebrew University of Jerusalem

description: cells lacking all three TET proteins (TET-TKO)

data_processing:
Basecalls were performed using illumina nextseq and bcl2fastq
ChIP-seq reads were aligned to the mm9 genome assembly using Bismark and bowtie
Data were aligned paired ended (PE)
Single CpGs were extracted using Bismark_methylation_extractor program.
We applied HOMER to find peaks and used bedtools to intersect peaks with reads
We counted the number of methylated vs. non methylated CpGs within 200-bp bins.
Single cell RNA-Seq aligned to mm10 followed by in-house perl pipeline to produce single cell matrix
Assembly: mm9 (ChIP) and mm10 (RNA)
Supplementary files format and content: peaks
Supplementary files format and content: segements of 200bp
Supplementary files format and content: segements of 200bp relative to TSS
Supplementary files format and content: segements of 200bp with methylated vs. non methylated CpGs
Supplementary files format and content: matirx files

growth_protocol: ESCs were grown on 0.1% gelatin-coated dishes and maintained in DMEM with 15% ESC-grade fetal calf serum, 50 μg/mL penicillin, 50 μg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, and 1000 U/ml LIF, supplemented with 1 μM PD0325901, an inhibitor of the MEK/ERK pathway, and 3 μMCHIR99021, an inhibitor of GSK3. For retinoic acid-induced differentiation, cells were grown on 0.1% gelatin-coated dishes for 2/5 days in DMEM with 10% ESC-grade fetal calf serum, 50 μg/mL penicillin, 50 μg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, and 1 μM retinoic acid. For XEN differentiation, cells were grown on 0.1% gelatin-coated dishes for 9 days in RPMI 1640 with 15% ESC-grade fetal calf serum, 0.1 mM β-mercaptoethanol, Penicillin/Streptomycin 100 U/mL, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10 ng/mL Activin A and 0.1 μM retinoic acid.

extract_protocol:
For ChIP and ChIP-BS: cells were lysed, treated with MNAse, and histone-DNA complexes were isolated with antibody. For bulk RNA:mRNA extraction was performed using Invitrogen Dynabeads mRNA DIRECT Purification Kit (61011) according to the manufacturer’s protocol. For scRNA: cells were collected into  reverse transcription/lysis buffer mix.
For ChIP-BS libraries: DNA samples were converted to blunt-ended, phosphorylated DNA using End-It™ DNA End-Repair Kit. DNA was purified using ratio of 1:1.8 sample: AMPure XP beads. An adenosine was added to the 3’ end of the DNA fragments using Klenow (3’-5’ exo-) (New England Biolabs, M0212L). DNA was purified using ratio of 1:1.8 sample:AMPure XP beads. Methylated adapters were added by ligation using DNA ligase (New England Biolabs, M2200S). DNA was purified using ratio of 1:1.2 sample:AMPure XP beads. Next the DNA was subjected to bisulfite conversion using the EZ DNA Methylation Gold kit (Zymo Research) according to the manufacturer’s instructions. The bisulfite-converted DNA samples were amplified using KAPA HiFi HotStart Uracil (KK2801) with 25 μM full-length primer mix containing Illumina library indices for multiplexing. Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads and eluted in 10 mM Tris-HCl [pH 8]. Library fragments were selected and isolated using the PureLink DNA gel extraction kit (Invitrogen). The resulting libraries had an average size of 350-550 bp. For ChIP libraries: 10ng DNA samples were converted to blunt ended, phosphorylated DNA using the End-It DNA End-Repair Kit (Lucigen). DNA was purified using ratio of 1:1.8 sample to AMPure XP beads. Adenosine was added to the 3’ end of the DNA fragments using Klenow (3’-5’ exo-) (New England Biolabs). DNA was purified using ratio of 1:1.6 sample to AMPure XP beads. Adapters containing Illumina library indices for multiplexing were 20 Article Title added (P7-index-PE2 and P5-PE1 long primers) by ligation using DNA ligase (New England Biolabs). DNA was purified using ratio of 1:1.2 sample to AMPure XP beads. DNA samples were amplified using KAPA HiFi HotStart (Roche) with 25μM primers (P7 and P5-PE1 short primers). Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads and eluted in 22μℓ 10mM Tris-HCl [pH 8]. Resulting libraries were run in a 2% agarose gel, and the desired 250–600 bp DNA library fragments were selected and isolated using the PureLink DNA gel extraction kit (Invitrogen). For bulk RNA libraries:  500ng mRNA samples were fragmented using RNA Fragmentation Reagents (Thermo, AM8740) by combining 9μl of sample with 1μl of RNA fragmentation reagent and incubating at 70 °C for 2 min. The sample was transferred to ice, and 40μl fragmentation stop mix containing 5μl fragmentation stop solution and 35μl 10mM Tris- HCl [pH 8] was added. Fragmented RNA was purified with 1.5x reaction volume of AMPure XP beads and eluted in 10μl 10mM Tris- HCl [pH 8]. The fragmented RNA was reverse transcribed using an oligo dT Primer as follows: First, 10μl RNA was mixed with 2μl of 50μM oligo dT primer (IDT) and 1μl of 25mM dNTP mix (NEB). The sample was incubated for 5 min at 65 °C and transferred to ice. The following components were added to the reaction to a total volume of 20μl: 4μl of 5x First-Strand SuperScript III buffer (Thermo), 2μl of 0.1M DTT (Thermo), 1μl murine RNase inhibitor (NEB), and 1μl of 200 U/μl Super-Script III reverse transcriptase (Thermo) and the sample was incubated for 1 hour at 50 °C 15 minutes at 70 °C. cDNA was purified with 1.5x reaction volume of AMPure XP beads and eluted in 17μl 10mM Tris- HCl [pH 8]. For second-strand synthesis, 17μl were combined with 2μl second-strand synthesis (SSS) buffer and 1μl of SSS enzyme mix from the NEBNext mRNA Second Strand Synthesis Module (NEB) and incubated at 16 °C for 2.5 h, followed by 20 min at 65 °C. cDNA was purified with 1.2x reaction volume of AMPure XP beads and eluted in 14μl 10mM Tris- HCl [pH 8]. DNA samples were converted to blunt-ended, phosphorylated DNA using the End-It DNA End-Repair Kit (Lucigen). DNA was purified using 1.8x reaction volume of AMPure XP beads.  Adenosine was added to the 3’ end of the DNA fragments using Klenow (3’-5’ exo-) (New England Biolabs). DNA was purified using 1.6x reaction volume of AMPure XP beads. Adapters containing Illumina library indices for multiplexing (P7-index-PE2 and P5-PE1 long primers) were added by ligation using DNA ligase (New England Biolabs). DNA was purified using 0.7x reaction volume of AMPure XP beads. The DNA samples were amplified using KAPA HiFi HotStart (Roche) with 25μM primers (P7 and P5-PE1 short primers). Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads and eluted in 22μl 10mM Tris-HCl [pH 8]. Resulting libraries were run in a 2% agarose gel, and the desired 250–600 bp DNA library fragments were selected and isolated using a PureLink DNA gel extraction kit (Invitrogen). For scRNA libraries: The reverse transcription/lysis mix consisted of 180 μL of 5X First-Strand buffer (Invitrogen), 27 μL 10% (v/v) IGEPAL CA-630 (Sigma), 18 μL 25 mM dNTPs (NEB), 30 μL 0.1 M DTT (Invitrogen), 45 μL 1 M Tris-HCl [pH 8.0] (Biological Industries), 30 μL murine RNase inhibitor (NEB), 45 μL SuperScript III reverse transcriptase (200 U/μL, Invitrogen), and 75 μL nuclease-free water (Biological Industries) in a total volume 450 μL. The carrier oil was HFE-7500 with 3% (w/w) fluorosurfactant (RAN Biotechnologies. The collection tubes were exposed to 6.5 J/cm2of 365-nm UV light for 10 min to release photocleavable barcoding primers from the barcoding beads. Next, the collection tubes containing the UV-exposed emulsions were transferred to a reverse transcription reaction at 50°C for 2 h followed by 15 min at 70°C. Each sample was demulsified by addition of 50 μL perfluoro-1-octanol (Sigma) to release the barcoded cDNA from the droplets. To remove unused primers and primer dimers, the upper, aqueous phase containing the barcoded cDNA (~50 μL) was combined with 50 μL digestion mix containing 5 μL ExoI enzyme (NEB), 5 μL HinFI enzyme (NEB), 5 μL ExoI buffer (NEB), 5 μL CutSmart buffer (NEB), and 30 μL of nuclease-free water and incubated for 1 h at 37°C and 10 min at 80°C. The reaction product was purified with 1.5x reaction volume of AMPure XP beads and eluted in 13.5 μL TE buffer (10 mM Tris–HCl [pH 8.0], 0.1 mM EDTA). For second-strand synthesis, 13.5 μL digestion reaction product was combined with 1.5 μL second-strand synthesis (SSS) buffer and 1 μL of SSS enzyme mix from the NEBNext mRNA Second Strand Synthesis Module (NEB) and incubated at 16°C for 2.5 h, followed by 20 min at 65°C. For in vitro transcription, SSS reaction products (16 μL) were combined with 24 μL HiScribe T7 High Yield RNA Synthesis Kit (NEB) reagent mix containing 4 μL T7 Buffer, 4 μL ATP, 4 μL CTP, 4 μL GTP, 4 μL UTP, and 4 μL T7 enzyme mix. The reaction was incubated at 37°C for 13 h, and the resulting RNA was purified with 1.3x reaction volume of AMPure XP beads and eluted with 20 μL TE buffer. RNA was fragmented using RNA fragmentation kit (Ambion), and 9 μL of this sample was combined with 1 μL of RNA fragmentation reagent and incubated at 70°C for 2 min. The sample was transferred to ice, and 40 μL fragmentation stop mix containing 5 μL fragmentation stop solution and 35 μL TE buffer was added. Fragmented RNA was purified with 1.3x reaction volume of AMPure XP beads and eluted in 10 μL TE buffer. The resulting amplified and fragmented RNA was reverse transcribed using a random hexamer primer as follows: First, 10 μL RNA was mixed with 2 μL of 100 μM PvG748-SBS12-RT random hexamer primer (IDT) and 1 μL of 10 mM dNTP mix (NEB). The sample was incubated for 3 min at 65°C and transferred to ice. The following components were added to the reaction to a total volume of 20 μL: 4 μL of 5x First-Strand SuperScript III buffer (Invitrogen), 1 μL of 0.1 M DTT (Invitrogen), 1 μL murine RNase inhibitor (NEB), and 1 μL of 200 U/μL SuperScript III reverse transcriptase (Invitrogen). The resulting cDNA was purified with 1.2x reaction volume of AMPure XP beads and eluted in 11.5 μL TE buffer. The resulting libraries were PCR amplified using standard PE1/PE2 full-length primer mix containing Illumina library indices for multiplexing. Each PCR reaction contained 11.5 μL post-RT cDNA library, 12.5 μL 2x KAPA HiFi HotStart PCR mix (Kapa Biosystems), and 1 μL of 25 μM PE1/PE2 index primer mix. Amplification was performed in 14 cycles. Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads and eluted in 30 μL nuclease-free water. Aliquots of 15 μL of each resulting library were run in 2% agarose gels, and 200–700 bp DNA library fragments were isolated using PureLink DNA gel extraction kit (Invitrogen).  For all libraries quality was confirmed using an Agilent 2200 TapeStation nucleic acids system and an Agilent High Sensitivity D1000 DS DNA kit. Size-selected libraries were diluted to 4nM concentration and combined for paired-end, single index sequencing on the Illumina NextSeq 550 and 2000 instrument using an Illumina 550 High Output v2 (75 cycles) kit or NextSeq 1000/2000 P2 Reagents (100 Cycles) v3 kit. Cycle distribution was 45 cycles for Read 1, 35 cycles for Read 2, and 8 cycles for the library index read or 50 cycles for Read 1, 70 cycles for Read 2, and 8 cycles for the library indexes reads for Illumina 2000.

value: TET-TKO

value: extraembryonic endoderm-induced differentiated cells

tag: agent

value: Di


title: scRNAseq direct XEN TET-TKO rep1


title: scRNAseq direct XEN WT rep2


title: scRNAseq direct XEN WT rep1


title: RPMI8226_Water extract of Astragalus mongholicus Bunge (High dose)_rep1

city: Daejeon

first: Musun

last: Park

address: 1672 Yuseong-daero, Yuseong-gu

institute: Korean Institute of Oriental Medicine

description: sample 2-2-4

data_processing:
Quality of RNA-seq raw data was assessed using FastQC (v0.11.9), and the common regions of MGISEQ adapter sequences were removed using TrimGalore (v0.6.5). The trimmed reads were then aligned to the human reference genome hg38 using the STAR (v2.7.3a) with default settings. Transcript abundance per gene including expected read counts and transcripts per million (TPM) were quantified using RSEM (v1.3.3) with gene annotation GRCh38.84.
Assembly: GRCh38.84
Supplementary files format and content: tab-delimited text files include Expected counts for each Sample

growth_protocol: RPMI8226 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in the medium recommended by the ATCC with 100 IU penicillin, and 100 µg/mL streptomycin in a humidified incubator. When cells reached 80–90% confluency, the growth medium was replaced every 3~4 days. Mycoplasma contamination was checked using the MycoAlert PLUS mycoplasma detection kit (Lonza, Rockland, ME, USA) and passed all.

extract_protocol:
Total RNA was isolated according to the manufacturer’s protocol. The concentration of the isolated RNA was determined using an Agilent RNA 6000 Nano Kit (Agilent Technologies, Waldbronn, Germany), and RNA quality was evaluated by assessing the RNA integrity number (RIN > 7).
A total RNA (500 ng) was processed to prepare a mRNA sequencing library using the MGIEasy RNA Directional Library Prep kit (#1000006386, MGI Tech Co., Ltd., China) according to the manufacturer’s instruction. The library was quantified using the QauntiFluor® ssDNA System (E3190, Promega Corporation, WI, USA). The prepared DNA nanoball was sequenced e MGIseq system (MGI Tech Co., Ltd., China) with 100 bp paired-end reads.

treatment_protocol: To determine the maximum concentration, we performed cell cytotoxicity tests and all drugs did not influence cell viability. The highest treatment concentrations (500ug/mL) were selected considering the solubility and clinical application of the drugs. To confirm the influence of concentration, we treated the cells with two concentrations (500 and 125 ug/mL). The cells treated with PBS were used as the vehicle control. Cells were plated at a density of 5x105 cells / well in a 12-well microplate and treated with two concentrations of drugs. After 24 hours, cells were washed two times with ice-cold PBS, and total RNA was isolated using QIAzol RNA Isolation Reagents (Thermo Fisher Scientific) according to the manufacturer's instructions.

value: B lymphocyte

value: Water extract of Astragalus mongholicus Bunge 500 μg/mL

tag: time


title: RPMI8226_Water extract of Panax ginseng C. A. Meyer (Low dose)_rep1

description: sample 2-2-2

value: Water extract of Panax ginseng C. A. Meyer 125 μg/mL


title: RPMI8226_Water extract of Panax ginseng C. A. Meyer (Low dose)_rep2

description: sample 2-2-3


title: RPMI8226_Water extract of Panax ginseng C. A. Meyer (High dose)_rep2

description: sample 2-1-11

value: Water extract of Panax ginseng C. A. Meyer 500 μg/mL


title: RPMI8226_Water extract of Panax ginseng C. A. Meyer (High dose)_rep3

description: sample 2-1-12


title: RPMI8226_Water extract of Panax ginseng C. A. Meyer (High dose)_rep1

description: sample 2-1-10


title: RPMI8226_Water extract of BJIKT (Low dose)_rep3

description: sample 2-1-9

value: Water extract of BJIKT 125 μg/mL


title: RPMI8226_Water extract of BJIKT (Low dose)_rep2

description: sample 2-1-8


title: RPMI8226_Water extract of BJIKT (High dose)_rep2

description: sample 2-1-5

value: Water extract of BJIKT 500 μg/mL


title: RPMI8226_Water extract of BJIKT (High dose)_rep3

description: sample 2-1-6


title: RPMI8226_Water extract of BJIKT (High dose)_rep1

description: sample 2-1-4


title: RPMI8226_Vehicle(PBS)_rep3

description: sample 2-1-3

value: PBS


title: RPMI8226_Vehicle(PBS)_rep2

description: sample 2-1-2