title: mRNA-sperm-FE-breeder rooster2 [sperm_FOLATE_2]

city: Yangling

first: Shengru

last: Wu

address: 22 St.XiNong

department: College of Animal Science and Technology

institute: Northwest A&F University

data_processing:
Illumina Casava1.8 software used for basecalling.
Cutadapt software was used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases
After removing the low quality bases and undetermined bases, we aligned reads to the reference genome using HISAT2 package, the mapped reads of each sample were assembled using StringTie
All transcriptomes from samples were merged to reconstruct a comprehensive transcriptome using gffcompare software. After the final transcriptome was generated, StringTie and ballgown was used to estimate the expression levels of all genes by calculating fragments per kilobase of transcript per million fragments mapped (FPKM).
Differentially expressed genes (DEGs) were selected with log2 (fold change) > 1 or log2 (fold change) less than -1 with statistical significance (q value < 0.01) by R package.
Genome_build: Gallus_gallus 5.0 (GCF_000002315.4)
Supplementary_files_format_and_content: tab-delimited text file include FPKM values and function annotations of each gene

organism: Gallus gallus

source_name: sperm sample from breeder roosters which fed with  diet with extra 5mg/kg folate

growth_protocol: Based on a single factor experimental design, 80 1-day-old Arbor Acres breeder roosters with similar genetic background and similar body weight (Beijing Arbor Acres Poultry Breeding Co. Ltd. China. P. R) were randomly assigned to 2 treatments with each 5 replicates and 8 birds per replicate. The breeder roosters from each treatment were fed with a standard diet mainly consisting of corn and soybean meal, with the only difference between treatments being the concentration of folic acid (0.25, 5.00 mg folic acid /kg feed, named as folic acid-control (FCON) and folic acid-excess (FE) groups, respectively). Meanwhile, 600 1-day-old Arbor Acres breeder hens with similar genetic background and similar body weight (Beijing Arbor Acres Poultry Breeding Co. Ltd. China. P. R) were kept in the same environment with roosters and fed with corn-soybean meals containing folic acid, then a total of 160 32-week-old breeder hens with similar body weight and met with the standard body weight were selected for the mating with the roosters. The photoperiod and the supplementation of feed were available according to the recommendations established by Aviagen Broiler Breeders Company, U.S.A. The water was available ad libitum. At 32 weeks of age, one rooster closing to the average weight of each replicate was selected and mated with 16 breeder hens by artificial insemination technology. The artificial insemination sustained for 3 weeks, and at 35 weeks of age, fertile eggs of 5 consecutive days from each replication of each treatment were collected and then artificially hatched. The new-born offspring broilers were grouped according to their paternal diets (five treatment with five replicates per treatment and 8 birds per replicate were randomly selected) and were then raised to 21 days by feeding with corn-soybean meals containing folic acid. The water, diet, and photoperiod were all available ad libitum.

extract_protocol:
Total RNA from 10 sperm samples and 10 liver samples of breeder roosters, and 40 liver samples of 1 day-old offspring broilers from folic acid-control and folic acid-excess groups were extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s procedures. The RNA amount and purity of each sample was quantified using NanoDrop ND-2000 (NanoDrop, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number > 7.00, and confirmed by electrophoresis with denaturing agarose gel. 4 RNA samples of four separate 1 day-old broilers’ livers from same replication were mixed equally together as a pooled RNA sample according to the purity and quantity of total RNA.
The mRNAs with poly (A) tails from 1 µM total RNA were isolated and purified with Dynabeads oligo (dT) 25-61005 (Thermo Fisher, CA, USA) using two rounds of purification. The mRNAs were randomly segmented into small fragments using Magnesium RNA Fragmentation Module (NEB, USA) at 94 °C for 5 min. Using random hexamer primers, fragmented mRNAs were used as templates to synthesize first-stand cDNA. Following RNase I treatment, DNA polymerase I was used to synthesize second-strand cDNA. cDNA fragments which Purified by using AMPure XP beads were then ligated with sequencing adapters according to the protocol (Illumina, San Diego, USA). Following PCR amplification, target fragments were obtained by agarose gel electrophoresis to create the cDNA library. Then we performed paired-end sequencing (2 × 150 bp) on an Illumina Novaseq™ 6000 at LC-Bio (Hangzhou, China) following the vendor’s recommended protocol.

tag: breed/age

value: 35-week-old breeder roosters

value: 5 mg/kg folate group

tag: tissue

value: sperm


title: cc05_dep

status: Public on Dec 16 2020

type: SRA

city: Los Angeles

first: Gianni

last: Carraro

state: CA

address: 8700 Beverly Blvd

department: Medicine

country: USA

institute: Cedars-Sinai Medical Center

description: 10X Genomics Single Cell RNA Sequencing

library_source: transcriptomic

data_processing:
Raw reads were aligned to the transcriptome by Cellranger. Expression counts for each gene in all samples were collapsed and normalized to unique molecular identifier (UMI) counts.
Genome_build: GRCh38
Supplementary_files_format_and_content: Processed data for each sample are provided in binary format HDF5 as produced by Cellranger
Supplementary_files_format_and_content: R data set object: This object contains also datasets of proximal airway from GSE143706 (GSM4272915 - 920)

molecule: total RNA

organism: Homo sapiens

source_name: Airway Epithelium  distal

extract_protocol:
Tissue was minced, digested with Liberase, and epithelial cells were fractionated by FACS  (DAPI-,CD45-,CD31-,CD326+).
The barcoded sequencing libraries were quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems). Sequencing libraries were loaded on a NovaSeq 6000 (Illumina).

value: Cells isolated from dissected and FACS sorted human airway material

tag: disease

value: Normal


title: N0_control

city: Shanghai

first: Zhou

last: haiwen

address: 500 Qu-xi Road, Shanghai

department: Oral Mucosal Diseases

country: China

institute: Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine

description:
processed_data_control_samples.xlsx
GSM3765311

data_processing:
Basecalls performed using CASAVA version 1.8
circRNA-seq reads were mapped to human genome by BWA-MEM software by default parameters except -T = 19
circRNAs were detected and annotated with CIRI software
High throughput sequencing service was provided by CloudSeq Biotech (Shanghai, China). Transcriptome high throughput sequencing and subsequent bioinformatics analysis were all done by Cloud-Seq Biotech (Shanghai, China). Briefly, total RNA was used for removing the rRNAs using Ribo-Zero rRNA Removal Kits (Illumina, USA) following the manufacturer's instructions. RNA libraries were constructed by using rRNA-depleted RNAs with TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using  the BioAnalyzer 2100 system (Agilent Technologies, USA).Libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters and finally sequenced for 150 cycles on Illumina HiSeq Sequencer according to the manufacturer’s instructions.
Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30. After 3’ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3). The high quality trimmed reads were used to analyse circRNAs. The high quality reads were aligned to the reference genome/transcriptome with STAR software (v2.5.1b) and circRNAs were detected and identified with DCC software (v0.4.4). edgeR software (v3.16.5) was used to normalized the data and perform differentially expressed circRNA analysis.
Genome_build: hg19
Supplementary_files_format_and_content: excel format: Number of raw junction read

source_name: healthy oral mucosa tissue

growth_protocol: Patients with previous histories of All tissue samples were immediately frozen in liquid nitrogen after resection and stored at -80℃ until RNA retraction

extract_protocol:
RNA extraction protocol
library construction protocol

tag: diagnosis

value: healthy control

tag: time

value: fresh

value: oral mucosa


title: dd09_dep

description: 10X Genomics Single Cell RNA Sequencing


title: mRNA-sperm-FE-breeder rooster3 [sperm_FOLATE_3]

molecule: polyA RNA

value: 36-week-old breeder roosters

tag: treatment


title: H09LACT

city: Chapel Hill

first: Kenichi

last: Okuda

state: NC

address: 125 Mason Farm Road

department: Department of Medicine, Cystic Fibrosis/Pulmonary Research and Treatment Center

institute: University of North Carolina at Chapel Hill

data_processing:
Illumina bcl2fastq v2.19 used for basecalling.
Sequence reads in FASTQ files were mapped to GRCh38 reference genome with GENCODE 32 gene annotation by HISAT2, and gene expression estimates were derived from StringTie using default parameters.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample.
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each Sample.

source_name: Large airway epithelial cells

growth_protocol: Matched large airway epithelial (LAE) and small airway epithelial (SAE) cells were isolated from freshly excised normal human lungs from transplant donors with lungs unsuitable for transplant. Isolated LAE and SAE cells were separately expanded using a modified conditional reprogramming cell method, and then grown to obtain well-differentiated air-liquid interface (ALI) cultures

extract_protocol:
For RNA extraction from cultured LAE and SAE cells, TRI Reagent was used according to the manufacturer’s instructions. RNA concentrations were determined using a NanoDrop-8000 spectrophotometer. Total RNA was used for bulk RNA-seq.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: After ALI 28 days, LAE and SAE cells were utilized for RNA isolation for RNAseq.

tag: donor code

value: Primary large airway epithelial cells

tag: cell type

value: Primary cells

tag: age

value: ALI day 28

value: No treatment

tag: disease state


title: LN7

city: Shenyang

first: hua

last: Zhou

address: 155 North Najing St, Shenyang, Liaoning, China

institute: The First Hospital of China Medical University

description: circular RNA

data_processing:
CircRNA-Seq high throughput sequencing and subsequent bioinformatics analysis were all done by Cloud-Seq Biotech (Shanghai, China). Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30.
After 3’ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3). The reads were aligned to the reference genome/transcriptome with STAR software and circRNAs were detected and annotated with DCC software
circBase database and circ2Trait disease database were used to annotated the identified circRNAs. Raw junction reads for all samples were normalized by total reads number and log2 transformed.
Differentially expressed circRNAs were identified by ttest between two groups.
Genome_build: hg19
Supplementary_files_format_and_content: see headers of  CircRNA Expression Profiling.xlsx

source_name: tissue

extract_protocol:
Total RNA was extracted from the frozen kidney tissues with using TRIzol reagent
Total RNA from each sample was used to prepare the circRNA sequencing library  using standard Illumina protocols

value: lupus nephritis

tag: class

value: IV

tag: gender

value: Male


title: NGLY1Pt1i-509_EV (day 40)

city: Fort Worth

first: Yu-Chieh

last: Wang

state: Texas

address: 3500 Camp Bowie Boulevard

department: Department of Pharmaceutical Sciences

institute: University of North Texas Health Science Center

data_processing:
Cell Ranger pipeline (version 2.0.1, 10x Genomics) was performed to enable alignment, de-duplication, and filtering, feature-barcode matrices of all samples, and a single feature-barcode matrix (an aggregation) containing all the data
Output of the Cell Ranger pipeline was taken into Seurat for further QC and cell type determination and differential gene expression analysis.
Genome_build: grch38.91
Supplementary_files_format_and_content: A MatrixMarket format text file that includes UMI counts for all samples and the relevant tab-delimited files

source_name: cerebral organoids

growth_protocol: Cerebral organoids were generated using WA09 hESC subclones (with and without the edited NGLY1 gene) and NGLY1-deficient patient's hiPSCs (with and without the ectopic expression of human NGLY1.  The detailed protocol for culturing cerebral organoids in the neural induction, neural differnetiation and cortical expansion media is described in the relevant pulication.

extract_protocol: RNA libraries were prepared for sequencing using Chromium Single Cell 3' Reagent (v2) Kits, Chromium Single Cell 3' Library & Gel Bead Kits, Chromium Single Cell A Chip Kit, and Chromium i7 Multiplex Kits from 10x Genomics according to the manufacturer's protocol.

tag: cell types

value: neural cells developed in cerebral organoids

tag: developmental stages

value: 40 days

tag: ngly1 condition


title: mRNA-sperm-FE-breeder rooster1 [sperm_FOLATE_1]

value: 35-week-old breeder roosters

value: 5 mg/kg folate group


title: LN5

description: circular RNA

value: Female


title: WA09-C3 (day 40)

value: NGLY1-deficient (NGLY1 gene edited)


title: cc04_dep

description: 10X Genomics Single Cell RNA Sequencing

value: Cells isolated from dissected and FACS sorted human airway material

value: Normal


title: mRNA-sperm-FCON-breeder rooster3 [sperm_CON_3]

source_name: sperm sample from breeder roosters which fed with  diet with extra 0.25mg/kg folate

value: 0.25 mg/kg folate group

value: sperm


title: cc02_dep

description: 10X Genomics Single Cell RNA Sequencing


title: mRNA-sperm-FCON-breeder rooster2 [sperm_CON_2]


title: Input seq

city: Singapore

first: Reshma

last: Taneja

state: Singapore

address: 2 Medical Drive, MD9, National University of Singapore

department: Department of Physiology

country: Singapore

institute: National University of Singapore

description:
Input
ERMS clonal cell line derived from RD ERMS cell line derived from biopsy specimen of a 7-year-old female with pelvic RMS.

library_source: genomic

data_processing:
Sequencing reads were mapped to the human reference genome (hg19) using BWA. High quality mapped reads (MAPQ>=10) were retained and potential duplicates were removed using SAMtools. Binding sites were predicted from the libraries using MACS2 with default parameters.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: V4_ChIP2RD18_peaks.narrowPeak: Refer to the following website for the file format: https://github.com/taoliu/MACS
Supplementary_files_format_and_content: V4_ChIP2RD18_peaks.xls

molecule: genomic DNA

source_name: EHMT2 input

growth_protocol: Cells were cultured in RPMI1640 (Gibco) supplemented with 10% FBS.

extract_protocol:
Cells were lysed with Farnham lysis buffer followed by RIPA buffer, chromatin sheared and DNA was extracted as per manufacturer’s protocol ChIP kit (#17-295, Mercmillipore, Bedford, MA, USA).
30ng ChIPed DNA and corresponding input were used for ChIP-seq library construction (New England Biolabs). Library fragment size was determined using the DNA 1000 Kit on the Agilent Bioanalyzer (Agilent Technologies).
Libraries were pooled in equimolar and cluster generation was performed on the Illumina cBOT system. Sequencing (150bp single-end) was performed on the Illumina HiSeq 3000 according to manufacturer's protocol at the Duke-NUS Genome Biology Facility, Singapore. Sequencing reads were mapped against hg19 and G9a bound regions were identified by MACS2.

treatment_protocol: Cells were treated with protein crosslinker DSG (Di-Succinimidyl Glutarate) followed by chromatin crosslinking with 1% formaldehyde.

tag: cell line

value: Embryonal rhabdomyosarcoma cell line

tag: passages

tag: chip antibody


title: EHMT2_ChIPSeq

description:
G9a_Chip
ERMS clonal cell line derived from RD ERMS cell line derived from biopsy specimen of a 7-year-old female with pelvic RMS.

source_name: EHMT2 ChIP

value: Anti-EHMT2/G9A (abcam, #40542)


title: mRNA-sperm-FCON-breeder rooster1 [sperm_CON_1]

tag: breed/age

tag: tissue


title: LN3

description: circular RNA

tag: disease


title: A1-3_S3

city: Faro

first: Elvira

last: Fatsini

address: Universidade do Algarve

country: Portugal

institute: UALG

data_processing:
Adapter removal: Cutadapt 2.1
Mapping: Bowtie2 v2.4.1
Annotation: Htseq-count 0.6.0

organism: Solea senegalensis

source_name: plasma sample

extract_protocol:
SncRNAs were isolated from blood plasma samples using miRNeasy Serum/Plasma Kit (Qiagen, Germany) following manufacturer instructions, and assessment of RNA quality and quantity was performed in a 2200 TapeStation Nucleic Acid system using High Sensitivity RNA ScreenTapes (Agilent, USA). Library preparation of 30 samples were performed using NEXTflex Small RNA-Seq kit v3 (Bioo Scientific, USA) for Illumina platforms following manufacturer protocol, and libraries size, purity, and concentration were evaluated on a High Sensitivity D1000 ScreenTape (Agilent, USA). Normalized libraries were clustered in one pool and multiplexed sequencing was done in two independent runs (15 libraries per run) by the NextSeq500 sequencer using a NextSeq High Output kit v2 (75 cycles) (Illumina, San Diego, CA), following the protocol provided by the manufacturer.
NEXTflex Small RNA-Seq kit v3 (Bioo Scientific, USA)

treatment_protocol: The fish maintained in two of the four tanks (n = 31 fish) were muscularly injected with KISS2 decapeptide at a dose of 250 µg/kg body weight, based on previous reports of positive results of KISS2-10 eliciting gonadotropin release and gonadal development. The remaining fish (n = 31) were injected with phosphate buffered saline (PBS) to test the placebo effect and thus to be used as a control group. The same day, blood samples were collected 2 (2h) and 4 (4h) hours after treatment, as previously described, to determine the acute effect on hormonal levels. Further samplings were performed 2 (2d) and 4 (4d) days after the treatments, to collect blood. At this stage, also plasma was collected for detection of microRNAs. For that, samples of blood (up to 2 mL) were collected from each fish and deposited in a 2 mL Eppendorf tube previously treated with cold buffered sodium citrate (3.8 % in 0.01 M PBS), as well as the syringe. After 15 min at 4 °C, plasma was separated from other blood components through differential centrifugation (15 min at 3000 g) and collected. No visible sign of haemolysis was noted in collected plasma samples. A total of 100 μL of plasma was sampled to perform sex hormone quantification and stored at -20 °C until steroid extraction. The remaining plasma was re-centrifuged at 3000 g for 5 min (to avoid cell debris contamination) and 500 μL of supernatant plasma was collected, snap-frozen in liquid nitrogen, and stored at -80 °C until RNA isolation and analysis.

tag: Sex

value: Plasma

value: Kiss treated

tag: sampling point


title: cc01_dep

description: 10X Genomics Single Cell RNA Sequencing

source_name: Airway Epithelium distal


title: LN2

description: circular RNA

value: lupus nephritis


title: mRNA-liver-FE-offspring broiler3 [B1d_FOLATE_3]

source_name: liver sample from offspring broilers whose father fed with  diet with extra 5mg/kg folate

value: 1-day-old offspring broilers

tag: treatment

value: liver


title: LN1

description: circular RNA

value: IV


title: cc01_pep

description: 10X Genomics Single Cell RNA Sequencing

source_name: Airway Epithelium proximal


title: AIEC wt in vivo3

city: Hamilton

first: Wael

last: Elhenawy

state: ON

address: 1200 Main St West

country: Canada

institute: McMaster University

data_processing:
Raw fastq files were trimmed with Cutadapt to remove Truseq adapter sequences
Trimmed reads were aligned against the Escherichia coli O83:H1 str.NRG857c (Genome assembly: ASM18334v1, NC_017634.1, NC_017659.1) using BWA (mem algorithm)
Read counts were quantified using featureCounts
DESeq2 was used for differential gene expression analysis
Genome_build: GCF_000183345.1
Supplementary_files_format_and_content: txt files contain read counts after alignment

organism: Escherichia coli

source_name: cecum of infected animal

extract_protocol: none provided by the submitter

tag: strain

value: O83;H1 str. NRG857c


title: AIEC wt in vivo2


title: mRNA-liver-FE-offspring broiler2 [B1d_FOLATE_2]


title: NC3

description: circular RNA

value: normal control


title: NC2

description: circular RNA


title: mRNA-liver-FCON-offspring broiler3 [B1d_CON_3]

source_name: liver sample from offspring broilers whose father fed with basical diet with extra 0.25mg/kg folate


title: AIEC wt in vivo1


title: LN4

description: circular RNA


title: JS1902_09

city: West Haven

first: Sarah

last: Slavoff

state: CT

address: 300 Heffernan Dr

department: Chemistry

institute: Yale University

description: Reads filtered for number of T-to-C mutations (0-5+) and strandedness (min/pos)

data_processing:
Reads were filtered for unique sequences using FastUniq
Reads were trimmed using cutadapt (--minimum-length=20)
Reads were aligned to the GRCh38 genome using STAR aligned with default parameters and --mp 4,2 .
Reads were processed using Picard tools, including FixMateInformation, SortSam and BuildBamIndex
Reads were filtered for those that aligned uniquely (flag: 83/163, 99/147), with MAPQ ≥ 2, and without insertions.
HTSeq-count was used to identify mapped reads in UCSC transcripts (union mode)
T-to-C mutations were identified using Rsamtools and a custom R script (available upon request). Only mutations at positions with a base quality score of greater than 45 that were at least three nt from the end of the read were counted. Reads were excluded where there were greater than five T-to-C mutations and these mutations did not account for at least one third of the observed mutations (NM tag).
SNP sites were filtered from our data in two ways: 1, bcftools was used to identify T-to-C SNP sites in control samples, and these sites were excluded from analysis; 2, sites exhibiting high T-to-C mutation rates in non-s4U treated controls were excluded from analysis.
To examine the distribution of reads with each minimum number of T-to-C mutations, bam files were filtered using Picard tools.
STAR aligner was used to make genome-coverage tracks (inputAlignmentsFromBam mode, outWigType bedGraph), and tracks were normalized using DESeq2 (estimateSizeFactors).
Tracks were converted to binary format using IGVtools (toTDF).
Genome_build: GRCh38
Supplementary_files_format_and_content: bedGraph track

source_name: immortalized 293Ts

growth_protocol: 293T cells were grown in DMEM containing 1% penicillin/streptomycin, 10% FBS, at 37°C.

extract_protocol:
RNA was harvested with Trizol reagent, followed by chloroform extraction and ethanol precipitation. Genomic DNA was depleted using Turbo DNAse, followed by cleanup with RNAclean beads. RNA was treated with oxidative-nucleophilic-aromatic-substitution chemistry as described in Schofield et al. Nature Methods 2018.
Libraries were generated following standard protocols using the SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian, V2 (Takara Bio USA, cat. 634413).

treatment_protocol: s4U plus and minus cells had their media replaced with growth media supplemented with 500 uM s4U or left unsupplemented, respectively. Cells were incubated with s4U for 2h.

tag: genotype/variation

value: NBDY KO

tag: s4u feed

value: No


title: mRNA-liver-FCON-offspring broiler2 [B1d_CON_2]


title: JS1902_08

value: Yes


title: JS1902_07


title: NC1

description: circular RNA

value: Female


title: AIEC wt LB2

source_name: in vitro grown bacteria


title: cc02_pep

description: 10X Genomics Single Cell RNA Sequencing

source_name: Airway Epithelium  proximal

value: Cells isolated from dissected and FACS sorted human airway material

value: Normal


title: AIEC wt LB3


title: AIEC wt LB1


title: JS1902_12

value: Wild Type


title: 2A3, B. subtilis, in vivo (SSII)

city: Groningen

first: Danny

last: Incarnato

state: Netherlands

address: Nijenborgh 7

department: Molecular Genetics

country: Netherlands

institute: University of Groningen

data_processing:
Library strategy: SHAPE-MaP
All the relevant data analysis steps have been conducted using RNA Framework v2.6.9 (PMID: 29893890; http://www.rnaframework.com). Reads pre-processing and mapping was performed using the rf-map module (parameters: -ctn -cmn 0 -cqo -cq5 20 -b2 -mp "--very-sensitive-local"). Reads were trimmed of terminal Ns and low-quality bases (Phred < 20). Reads with internal Ns were discarded. Mapping was performed using the “very-sensitive-local” preset of Bowtie2. The mutational signal was derived using the rf-count module (parameters: -m -rd), enabling the right re-alignment of deletions. Grid search of optimal slope/intercept pairs was performed using the rf-jackknife module (parameters: -rp “-md 600 -nlp” -x) and ViennaRNA package v2.4.11, by disallowing isolated base-pairs, setting the maximum base-pairing distance to 600 nt and enabling the relaxed structure comparison mode (a basepair i/j is considered to be correctly predicted if any of the following pairs exist in the reference structure: i/j; i-1/j; i+1/j; i/j-1; i/j+1).
Supplementary_files_format_and_content: Additional data files (RC/XML files for analysis with RNA Framework [http://www.rnaframework.com]) are available at: http://www.incarnatolab.com/datasets/2A3_Marinus_2020.php

organism: Bacillus subtilis

source_name: Total RNA from B. subtilis, in vivo treated with 2A3

extract_protocol:
Cell pellets were resuspended in 200 µl TE Buffer [10 mM Tris pH 8.0; 1 mM EDTA pH 8.0], supplemented with 1.1% final SDS. 0.25 g glass beads were then added and samples were beated in a Mini-Beadbeater-24 (Glen Mills) for 1 min. Samples were then incubated on ice for 1 min, after which beating was repeated once. 1 mL ice-cold QIAzol was then added, followed by vigorous vortexing. Samples were then incubated at 70°C for 20 min, to allow complete cell lysis. 200 µl of chloroform were added, followed by vigorous vortexing for 15 sec. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12,500 x g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 mL tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase at 37°C for 20 min.
Total RNA was first fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer [65 mM Tris-HCl pH 8.0; 95 mM KCl; 4 mM MgCl2], then purified with NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 2 µl (for SSII and HIV RT) or 2.5 µl (for TGIRT-III) NF H2O. Eluted RNA was supplemented with 0.5 µl 20 µM random hexamers and either 0.25 µl (for SSII) or 0.5 µl (for HIV RT and TGIRT-III) dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 5 µl. For HIV RT, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 40 mM MgCl2], 0.5 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 5 U HIV RT (Worthington Biochemical Corporation, cat. HIVRT). For TGIRT-III, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2], 0.25 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 50 U TGIRT-III RT (InGex, cat. TGIRT-50). For SSII, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 0.5 µl DTT 0.1 M, 0.25 µl MnCl2 120 mM, 5 U SUPERase•In™ RNase Inhibitor and 50 U SuperScript II RT (ThermoFisher Scientific, cat. 18064014). Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 37°C (for HIV RT), 2 h at 57°C (for TGIRT-III), or 3 h at 42°C (for SSII). SSII and HIV RT were heat-inactivated by incubating at 75°C for 20 min. As TGIRT-III tightly binds the cDNA-RNA complex, 0.5 µg of Proteinase K were added and reaction was incubated at 37°C for 20 min, after which 0.5 µl of a 1:2 dilution of protease inhibitor cocktail (Sigma Aldrich, cat. P9599) in water was added to stop the reaction. For SSII, the buffer exchange step was omitted. Instead, 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, cat. E6111L). Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), following manufacturer instructions.

treatment_protocol: A single colony of Bacillus subtilis 168 was picked and inoculated in LB medium without antibiotics, then grown to exponential phase (OD600 ~ 0.5). 2 mL aliquots were collected by centrifugation at 1000 x g for 5 min. Cell pellets were resuspended in 450 µl 1X PBS (pH 7.4) and 50 µl of each SHAPE compound were added, followed by brief vortexing. Cells were allowed to react at 37°C for 20 min, with moderate shaking, after which 500 µl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 17,000 x g for 2 min (4°C) and supernatant discarded.

value: Bacillus subtilis 168


title: NIC, B. subtilis, in vivo (SSII)

source_name: Total RNA from B. subtilis, in vivo treated with NIC


title: mRNA-liver-FE-breeder rooster3 [R_FOLATE_3]

source_name: liver sample from breeder roosters which fed with  diet with extra 5mg/kg folate

value: 35-week-old breeder roosters

value: 5 mg/kg folate group


title: B5, B. subtilis, in vivo (SSII)

source_name: Total RNA from B. subtilis, in vivo treated with B5


title: mRNA-liver-FE-offspring broiler1 [B1d_FOLATE_1]


title: mRNA-liver-FE-breeder rooster2 [R_FOLATE_2]


title: 6A3, B. subtilis, in vivo (SSII)

source_name: Total RNA from B. subtilis, in vivo treated with 6A3


title: JS1902_11


title: 22rv1_h3k27ac_hic_rep2

city: Toronto

first: Musaddeque

last: Ahmed

address: 101 College St

institute: University health Network

data_processing:
HiC analysis performed with Juicer 1.5 pipeline
Hic Analysis with HiC pro pipeline
Genome_build: hg38 obtained from USCS and indexed with BWA
Supplementary_files_format_and_content: hic files

source_name: Prostate

growth_protocol: DMEM 10% FBS

extract_protocol:
ARIMA-HIC kit
Libraries were prepared following ARIMA-HIC protocol and the hyper prep kit illumina from Kapa Biosystems (#KK8500)
Truseq index from illumina (cat 20015960)

value: Prostate


title: mRNA-liver-FE-breeder rooster1 [R_FOLATE_1]


title: 1M4, B. subtilis, in vivo (SSII)

source_name: Total RNA from B. subtilis, in vivo treated with 1M4


title: mRNA-liver-FCON-breeder rooster3 [R_CON_3]

source_name: liver sample from breeder roosters which fed with  diet with extra 0.25mg/kg folate

value: 0.25 mg/kg folate group


title: I5, B. subtilis, in vivo (SSII)

source_name: Total RNA from B. subtilis, in vivo treated with I5


title: JS1902_10


title: mRNA-liver-FCON-offspring broiler1 [B1d_CON_1]

value: 1-day-old offspring broilers


title: N2a_YAYC_KD_rep3

type: RNA

city: Niigata

first: Tomoyuki

last: Yamanaka

address: 1-757 Asahimachidori, Chuo-ku

department: Brain Research Institute

country: Japan

institute: Niigata University

hyb_protocol: Hybridization was performed according to the Agilent standard protocol.

scan_protocol: Arrays were scanned according to the Agilent standard protocol.

data_processing: Obtained microarray data were analyzed using an Agilent GeneSpring GX software to identify differentially expressed genes (DEGs) by gene knockdown. Genes with low expressions (less than 20th percentile) were excluded to reduce false positives.

organism: Mus musculus

source_name: N2a, NFYAand NFYC KD

label_protocol: labeled cDNAs from total RNA were prepared using Agilent Low Input Quick Amp Labeling Kit One-Color.

extract_protocol: Total RNAs were prepared from transfected neuro2a cells by Qiagen RNeasy kit according to the manufacture's protocol.

tag: cell type

value: N2a mouse neuroblastoma

value: NFYA and NFYC knockdown


title: mRNA-liver-FCON-breeder rooster2 [R_CON_2]

value: liver


title: v16a_h3k27ac_hic_rep1


title: mRNA-liver-FCON-breeder rooster1 [R_CON_1]


title: v16a_h3k27ac_hic_rep2


title: N2a_YAYC_KD_rep2


title: M THY CD4SP sample 2

status: Public on Dec 15 2020

first: Gay

middle: Miriam

last: Crooks

state: California

address: 610 Charles E. Young Drive, East

department: Pathology & Laboratory Med

institute: University of California Los Angeles

description:
mature T cell population isolated from fresh murine thymus
GCAMmmThy_SP4.2

data_processing:
The STAR ultrafast universal RNA-seq aligner v2.7.0d (Dobin et al., 2013) was used to generate the genome index and perform paired-end alignments. Reads were aligned to a genome index that includes both the genome sequence (GRCm38 primary assembly) and the exon/intron structure of known gene models (Gencode M20 comprehensive genome annotation).
Alignment files were used to generate strand-specific, gene-level count summaries with STAR's built-in gene counter. Independent filtering was applied as follows: genes with less than half-count per million in all samples, count outliers or low mappability (<50bp) were filtered out for downstream analysis. This masked set included a total of 13760 mouse protein coding genes.
Counts were normalized per-sample in units of FPKMs after correcting for gene mappable length and sample total counts.
Genome_build: GRCh38 primary assembly
Supplementary_files_format_and_content: Tab delimited. Table of per-gene and per-cell type average expression estimates in units of FPKMs

source_name: mouse thymus

growth_protocol:
Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

extract_protocol:
All samples were extracted using miRNeasy Micro Kit (Qiagen).
1.5ng of total RNA was input to generate sequencing libraries with SMARTer Stranded Total RNA-Seq (Pico) Kit (Clonetech, Cat. 635005).
Paired-end 150 bp sequencing was performed on an Illumina HiSeq 3000.

treatment_protocol: The freshly sorted mouse thymic SP4 late population was pelleted for RNA extraction

value: thymus

tag: phenotype

value: Lin- CD4+ CD8- CD3+ TCRb+ CD62L+

value: mature T cell population


title: N2a_YAYC_KD_rep1


title: N2a_Vector_KD_rep3

source_name: N2a, Vector KD

value: Vector knockdown


title: NAI, B. subtilis, in vivo (SSII)

source_name: Total RNA from B. subtilis, in vivo treated with NAI


title: M THY CD8SP sample 2

description:
mature T cell population isolated from fresh murine thymus
GCAMmmThy_SP8.2

growth_protocol:
Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted mouse thymic SP8 late population was pelleted for RNA extraction

value: Lin- CD4- CD8+ CD3+ TCRb+ CD62L+


title: M THY DP late sample 1

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_DPl.1

growth_protocol:
Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). DP late (Lin- CD4+ CD8+ CD3+ TCRb+).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted mouse thymic DP late population was pelleted for RNA extraction

value: Lin- CD4+ CD8+ CD3+ TCRb+

value: immature T cell population


title: 22rv1_h3k27ac_hic_rep1


title: M THY DP early sample 1

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_DPe.1

growth_protocol:
Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). DP early (Lin- CD4+ CD8+ CD3- TCRb-).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted mouse thymic DP early population was pelleted for RNA extraction

value: Lin- CD4+ CD8+ CD3- TCRb-


title: M THY CD8SP sample 1

description:
mature T cell population isolated from fresh murine thymus
GCAMmmThy_SP8.1


title: M THY DP early sample 2

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_DPe.2


title: M THY ISP8 sample 2

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_ISP.2

growth_protocol:
Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). ISP8 (Lin- CD4- CD8+ CD3-).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted mouse thymic ISP8 population was pelleted for RNA extraction

value: Lin- CD4- CD8+ CD3-


title: M THY DN3 sample 2

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_DN3.2

growth_protocol:
Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). DN3 (Lin- CD4- CD8- CD44- CD25+).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted mouse thymic DN3 population was pelleted for RNA extraction

value: Lin- CD4- CD8- CD44- CD25+


title: M THY CD4SP sample 1

description:
mature T cell population isolated from fresh murine thymus
GCAMmmThy_SP4.1


title: M THY ISP8 sample 1

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_ISP.1


title: N2a_Vector_KD_rep1


title: M THY DN3 sample 1

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_DN3.1


title: M THY DN2 sample 1

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_DN2.1

growth_protocol:
Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted mouse thymic DN2 population was pelleted for RNA extraction

value: Lin- CD4- CD8- c-Kithi CD44hi CD25+


title: M-ATO CD4SP sample 2

description:
Mature T cell population generated in ATOs from T cell differentiation of murine bone marrow hematopoietic stem cells
GCAMmmATO_SP4.2

source_name: mouse bone marrow hematopoietic stem cells

growth_protocol:
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Flow cytometry cell sorting of ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted Mouse-ATO derived SP4 late population was pelleted for RNA extraction

value: thymic organoid


title: M THY DN2 sample 2

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_DN2.2


title: M THY ETP sample 2

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_ETP.2

growth_protocol:
Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted mouse thymic ETP population was pelleted for RNA extraction

value: Lin- CD4- CD8- c-Kithi CD44hi CD25-


title: DMSO, B. subtilis, in vivo (SSII)

source_name: Total RNA from B. subtilis, in vivo treated with DMSO

value: Bacillus subtilis 168


title: M THY ETP sample 1

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_ETP.1


title: M-ATO CD8SP sample 1

description:
Mature T cell population generated in ATOs from T cell differentiation of murine bone marrow hematopoietic stem cells
GCAMmmATO_SP8.1

growth_protocol:
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Flow cytometry cell sorting of ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted Mouse-ATO derived SP8 late population was pelleted for RNA extraction


title: M-ATO DP late sample 1

description:
immature T cell population generated in ATOs from T cell differentiation of murine bone marrow hematopoietic stem cells
GCAMmmATO_DPl.1

growth_protocol:
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Flow cytometry cell sorting of ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). DP late (Lin- CD4+ CD8+ CD3+ TCRb+).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted Mouse-ATO derived DP late population was pelleted for RNA extraction


title: M-ATO CD8SP sample 2

description:
Mature T cell population generated in ATOs from T cell differentiation of murine bone marrow hematopoietic stem cells
GCAMmmATO_SP8.2


title: M-ATO DP late sample 2

description:
immature T cell population generated in ATOs from T cell differentiation of murine bone marrow hematopoietic stem cells
GCAMmmATO_DPl.2


title: N2a_Vector_KD_rep2

value: N2a mouse neuroblastoma


title: M THY DP late sample 2

description:
immature T cell population isolated from fresh murine thymus
GCAMmmThy_DPl.2


title: M-ATO DP early sample 1

description:
immature T cell population generated in ATOs from T cell differentiation of murine bone marrow hematopoietic stem cells
GCAMmmATO_DPe.1

growth_protocol:
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Flow cytometry cell sorting of ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). DP early (Lin- CD4+ CD8+ CD3- TCRb-).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted Mouse-ATO derived DP early population was pelleted for RNA extraction


title: M-ATO ISP8 sample 2

description:
immature T cell population generated in ATOs from T cell differentiation of murine bone marrow hematopoietic stem cells
GCAMmmATO_ISP.2

growth_protocol:
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Flow cytometry cell sorting of ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). ISP8 (Lin- CD4- CD8+ CD3-).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted Mouse-ATO derived ISP8 population was pelleted for RNA extraction


title: M-ATO DP early sample 2

description:
immature T cell population generated in ATOs from T cell differentiation of murine bone marrow hematopoietic stem cells
GCAMmmATO_DPe.2


title: M-ATO ISP8 sample 1

description:
immature T cell population generated in ATOs from T cell differentiation of murine bone marrow hematopoietic stem cells
GCAMmmATO_ISP.1


title: M-ATO DN3 sample 2

description:
immature T cell population generated in ATOs from T cell differentiation of murine bone marrow hematopoietic stem cells
GCAMmmATO_DN3.2

growth_protocol:
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Flow cytometry cell sorting of ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). DN3 (Lin- CD4- CD8- CD44- CD25+).
ATOs were generated as previously described (Seet et al., 2017). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033) (in some experiments, RPMI 1640 (CellGro, Cat# 10-040-CV) was used as basal media as indicated), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044) (in some experiments, 4% B27 was used as indicated), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPC cells (1- 4000 cells / ATO) in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended in 5 µl D/F12-B27 per ATO and mixed by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12-B27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer.  Thymic fragments from the mouse thymus were finely dissected in FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were then stained for flow cytometry.  Flow cytometry cell sorting of thymic and ATO-derived T cell populations used the following surface phenotypes: (Lin- stands here for: Ter119-, TCRγδ-, B220-, CD19-, CD11c-, CD11b-, Gr1-, NK1.1-). Early Thymic Progenitor (ETP; Lin- CD4- CD8- c-Kithi CD44hi CD25-), DN2 (Lin- CD4- CD8- c-Kithi CD44hi CD25+); DN3 (Lin- CD4- CD8- CD44- CD25+); ISP8 (Lin- CD4- CD8+ CD3-); DP early (Lin- CD4+ CD8+ CD3- TCRb-); DP late (Lin- CD4+ CD8+ CD3+ TCRb+); CD8 single positive (CD8SP; Lin- CD4- CD8+ CD3+ TCRb+ CD62L+); CD4 single positive (CD4SP; Lin- CD4+ CD8- CD3+ TCRb+ CD62L+).

treatment_protocol: The freshly sorted Mouse-ATO derived DN3 population was pelleted for RNA extraction


title: 2425_400mkd_Liver

city: Indianapolis

first: Kamin

last: Johnson

state: IN

address: 9330 Zionsville Rd

institute: Corteva Agriscience

data_processing:
Basecalls performed using Illumina CASAVA software version 1.8
Raw sequencing reads were trimmed and filtered for low quality bases using the fastq-mcf tool from the ea-utils software package (v1.1.0) with the minimum remaining sequence length option set to 30bp and quality threshold set to 30
Trimmed reads were subsequently mapped to the rat reference genome (version 6.0.80) using the hisat2 mapping software (v2.0.3) run using default parameter settings and in stranded mode
Using HTSeq (v0.6.1), the reads mapped to the antisense strand of each gene were counted
Genome_build: Rattus norvegicus (version 6.0.80)
Supplementary_files_format_and_content: tab-delimited text files include raw gene count values for each Sample ...

organism: Rattus norvegicus

source_name: Liver

extract_protocol:
Total RNA was extracted from organs stored in RNAlater using a Qiagen RNeasy kit.  Samples were digested with DNAase during the RNA isolation process to avoid DNA contamination.  RNA quantity was measured using a NanoDrop ND-1000 spectrophotometer.  Total RNA quality was examined using the HT RNA Std Sens assay on a LabChip GX instrument (PerkinElmer; Waltham, MA) according to the manufacturer’s instructions.
The Illumina Truseq Stranded mRNA library preparation kit was used to prepare mRNA library templates following the manufacturer’s recommended protocol.  In brief, poly-A containing mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.  Purified mRNA was fragmented to approximately 200 bp average length using divalent cations under high temperature.  Following fragmentation, SuperScript II reverse transcriptase and random primers were used to copy the mRNA into first strand cDNA.  The cDNA was further converted into double stranded cDNA using DNA polymerase I and RNase H.  These double stranded cDNA fragments then underwent end repair, A-tailing, and ligation to indexed Illumina adapters.  Finally, adapter-ligated library products were enriched with 15 cycles of PCR and purified.

value: Liver

tag: developmental stage

value: Adult

value: Crl:CD (SD); Sprague-Dawley

tag: age at study start

value: 8-10 weeks

value: Male

tag: molecule exposure

value: Myclobutanil

tag: exposure

value: Daily via oral gavage for 14 days

tag: corn oil dose level

value: 4 ml/kg body weight

tag: dose level (mg/kg)

tag: sample deemed an outlier

value: No


title: 2426_400mkd_Liver

tag: tissue