title: 140820_47341c

type: RNA

city: Tsukuba

first: Chikako

last: Nishitani

state: Ibaraki

institute: National Institutes of Fruit Tree Science

hyb_protocol: The labeled samples were hybridized to custom microarray slides with a Gene Expression Hybridization Kit (Agilent).

scan_protocol: After hybridization, arrays were washed with Gene Expression Wash Buffer (Agilent) according to the manufacturer’s instructions. The dried slides were scanned on an Agilent scanner under the control of Agilent Feature Extraction software v. 9.5.1.

data_processing: In subio software (Subio), the expression of each gene was calculated as the mean of three biological replicates for each time point with the standard error of the mean (SEM).

organism: Pyrus pyrifolia

source_name: watercore susceptible line (47341) fruits harvested on 140820

label_protocol: The RNA from the samples were reverse-transcribed and the resultant cDNAs were labeled with a Low RNA Input Linear Amplification Kit PLUS, One-Color (Agilent, Palo Alto, CA), according to the manufacturer’s instructions.

extract_protocol: RNA was prepared using the Concert™ Plant RNA Reagent (Invitrogen) following the manufacturer's recommendations.

value: fruit

tag: ecotype

value: Taihaku x Akiakari

tag: line/phenotype

value: watercore susceptible line (47341)

tag: harvest date


title: S314

status: Public on Apr 24 2021

type: SRA

city: CAMBRIDGE

first: Aviv

last: Regev

state: MASSACHUSETTS

address: 415 MAIN ST

country: USA

institute: Broad Institute

description:
single-cell RNA-Seq data
C2_S314
H5TJLBGX3_S314

library_source: transcriptomic

data_processing:
Reads were aligned to a reference genome (mm10 Ensembl) using Bowtie
Expression levels were quantified using RSEM (inferred counts and tpm values), and log2(TPM+1) values generated.
cells were selected for subsequent analysis based on genecount and mapping rates to transcriptome (see manuscript)
Quality filtering parameters: (high-quality CD8 T) cells that had (1) 1,400-6,000 detected genes (defined by at least one mapped read), (2) at least 100,000 reads mapped to the transcriptome, (3) at least 20% of the reads mapped to the transcriptome, and (4) non-zero expression of either Cd3 or Cd8b1. Genes with non-zero expression in at least one cell were kept.
Genome_build: mm10
Supplementary_files_format_and_content: log2(TPM+1) values

molecule: total RNA

organism: Mus musculus

source_name: Mus musculus CD8 TILs

extract_protocol:
TILs were isolated by dissociating tumor tissue in the presence of collagenase D (2.5 mg/ml) for 20 min prior to centrifugation on a discontinuous Percoll gradient (GE Healthcare).
Smart-seq2 (Picelli et al., Nature Methods, 2014)

tag: strain background

tag: genotype/variation

value: wild type

tag: cell type

value: CD8 tumor infiltrating lymphocytes (TIL)


title: 140820_47377c

country: Japan

source_name: watercore sustainable line (47377) fruits harvested on 140820

tag: tissue

value: watercore sustainable line (47377)


title: 140820_47341b


title: T2-NSCLP

city: Miami

first: Derek

last: Van Booven

state: FL

address: 1501 NW 10th Ave

institute: University of Miami

library_source: genomic

data_processing:
Adapters trimmed with TrimGalore
Data aligned to hg19 with Bismark2
Coverage files fed into DSS for differential methylation
Genome_build: hg19

molecule: genomic DNA

organism: Homo sapiens

source_name: Saliva DNA

extract_protocol:
Buccal swab
400ng per sample. Unmethylated Lambda gDNA was spiked into each sample per the Swift Accel-NGS Methyl-Seq DNA Library procotol (v.2.0). Samples were sonicated using a 500bp protocol. Sonicated products were processed overnight using the Zymo EZ DNA Methylation Gold kit. Products were purified and stored at -20C. Library preparations were carried out using the Accel-NGS® Methyl-Seq DNA Library Kit.

value: Saliva DNA

tag: disease state

value: NSCLP-Affected Twin


title: 140820_47377a


title: 140820_47341a


title: S26

description:
single-cell RNA-Seq data
C2_S26
H5TJLBGX3_S26


title: c_t021_pio_day_5

city: Singapore

first: Nicholas

last: Ang

address: 8a Biomedical Grove, Singapore 138648

country: Singapore

institute: Singapore Immunology Network (SIgN)

description:
R10838
gene_counts.txt

data_processing:
FASTQ files were mapped using STAR to GRCh38 genome build.
Gene counts were obtained using featureCounts on GENCODE v31 annotations.
Genome_build: GRCh38
Supplementary_files_format_and_content: gene_counts.txt

source_name: Whole blood

growth_protocol: Not Applicable

extract_protocol:
Total RNA was extracted using MagMax™ for Stabilized Blood Tubes RNA Isolation Kit (Invitrogen).
Smart-Seq V2 RNA-Seq Library Preparation Protocol.

treatment_protocol: Not Applicable

value: Whole blood

tag: diagnosis

tag: severity

tag: gene deletion status

tag: pcr status

value: positive

tag: patient id


title: T1-Unaf

value: Unaffected Twin


title: 140805_47377c

source_name: watercore sustainable line (47377) fruits harvested on 140805


title: S313

description:
single-cell RNA-Seq data
C1_S313
H5TJLBGX3_S313


title: c_t016_pio_day_20

description:
R10836
gene_counts.txt


title: T1-NSCLP


title: S25

description:
single-cell RNA-Seq data
C1_S25
H5TJLBGX3_S25


title: S36

description:
single-cell RNA-Seq data
C12_S36
H5TJLBGX3_S36


title: c_t011_pio_day_11

description:
R10834
gene_counts.txt


title: c_t011_pio_day_25

description:
R10835_R
gene_counts.txt

value: negative


title: Patient3 TILs - HCMV pp65 single cell gene expression

city: Dallas

first: Ze

last: Zhang

state: TX

address: 5959 Maple Avenue, APT 1102

institute: University of Texas Southwestern Medical Center at Dallas

description: single cell RNA

data_processing:
The software Cell Ranger provided by the 10x genomics was used to analyze multiplex data for each of the sample, with the parameter setting as '-- expected-cells 5000', to generate the two processed data files.
Genome_build: hg38
Supplementary_files_format_and_content: h5 hierarchical data , csv charts

source_name: Lymphocytes

growth_protocol: TILs were expanded using anti-CD3 antibody and IL-2 and and irradiated feeder cells and maintained in AIM-V medium.

extract_protocol:
TILs were rinsed with PBS with  2% FBS and re-suspended at the concentration of 1*10^6 cell/ml.
Libraries were prepared according to 10x Genomics' instructions for V(D)J-enriched libraries and 5’ Gene Expression libraries.
5’ scRNAseq gene expression with immune profiling

treatment_protocol: TILs were co-cultured with 10ug/ml viral peptide for 24 hours.

value: TIL

tag: treatment

value: stimulated with HCMV pp65 peptide

tag: reactivity

value: Positive


title: Patient3 TILs - HCMV pp65 single cell vdj sequences

description: single cell RNA


title: 140805_47377a


title: S324

description:
single-cell RNA-Seq data
C12_S324
H5TJLBGX3_S324


title: c_t016_pio_day_23

description:
R10837
gene_counts.txt


title: S35

description:
single-cell RNA-Seq data
C11_S35
H5TJLBGX3_S35


title: 140805_47341c

source_name: watercore susceptible line (47341) fruits harvested on 140805


title: 130911_1703_3

source_name: watercore sustainable line (1703) fruits harvested on 130911

value: Hosui x Hogetsu

value: watercore sustainable line (1703)


title: Patient3 TILs - EBV BMLF1 single cell gene expression

description: single cell RNA

value: stimulated with EBV BMLF1 peptide


title: 140805_47377b

value: fruit

value: Taihaku x Akiakari


title: Patient3 TILs - Influenza A single cell vdj sequences

description: single cell RNA

value: stimulated with Influenza A peptide

value: Negative


title: 140805_47341a

value: watercore susceptible line (47341)


title: c_t005_pio_day_8

description:
R10832
gene_counts.txt


title: c_t005_d13_11_feb_2020

description:
R10833
gene_counts.txt


title: Patient3 TILs - EBV BMLF1 single cell vdj sequences

description: single cell RNA


title: c_n008_pio_day_18

description:
R10831_R
gene_counts.txt


title: S34

description:
single-cell RNA-Seq data
C10_S34
H5TJLBGX3_S34

value: wild type

value: CD8 tumor infiltrating lymphocytes (TIL)


title: 130911_1703_1


title: Patient3 TILs - Influenza A single cell gene expression

description: single cell RNA


title: S323

description:
single-cell RNA-Seq data
C11_S323
H5TJLBGX3_S323


title: c_n008_pio_day_7

description:
R10830
gene_counts.txt

value: Whole blood


title: 130911_1703_2


title: S322

description:
single-cell RNA-Seq data
C10_S322
H5TJLBGX3_S322


title: Patient3 TILs - Influenza M single cell vdj sequences

description: single cell RNA

value: stimulated with Influenza M peptide


title: S309

description:
single-cell RNA-Seq data
B9_S309
H5TJLBGX3_S309


title: 140805_47341b


title: Organ of Corti_cochlear_P33

city: Omaha

first: Jian

last: Zuo

state: Nebraska

address: 2500 California Plaza

institute: Creighton University

data_processing:
the sequence reads were aligned to mouse reference genome mm10 using the Cell Ranger pipeline (10X Genomics).
Genome_build: mm10
Supplementary_files_format_and_content: feature, barcode and matrix file

source_name: Organ of Corti

extract_protocol:
Cochleae were dissected out and incubated in solution (50% accutase (Innovative Cell Technologies, San Diego), 0.02% Trypsin(Thermofisher), 125 μg/ml Thermolysin (SIGMA-ALDRICH)) at 37°C for 3 min. 0.02 mg/ml collagenase IV (SIGMA-ALDRICH) and dispase (Worthington Biochemicals Corp) were further added to the enzyme solution and incubated for 4 min at 37°C. The enzymatically treated tissue was triturated gently using fire-polished Pasteur pipettes and aggregated cells were removed using a 40-μm strainer. The dissociated cells were centrifuged at 500 × g for 5 min and resuspended in solution (0.5% fetal bovine serum, 0.04% bovine serum albumin, 0.3 mM ethylenediaminetetraacetic acid in Hanks' balanced salt solution).
Library construction was performed using Chromium Single Cell 3’ v2 Reagent Kits (10X genomics) following the instruction manuals. Briefly, the dissociated cells were loaded into a Chromium Controller (10X Genomics) and the encapsulated cells were lysed individually. Reverse transcription within each oil droplet was performed and the cDNAs were amplified.
Single cell RNAseq  performed using an Illumina HiSeq 4000 system

value: Organ of Corti

tag: age

tag: genotype

value: Fgfr3iCre+; Atoh1-HA+; Chrna9-GFP+; Rosa26-CAG-loxP-stop-loxP-tdTomato+

value: Tamoxifen uninjected


title: 130911_1726no_3

source_name: watercore susceptible line (1726) fruits normal part harvested on 130911

value: fruit (normal part)

tag: ecotype

value: watercore susceptible line (1726)


title: Patient3 TILs - Influenza M single cell gene expression

description: single cell RNA

value: TIL

value: Positive


title: 130911_1726no_2

value: Hosui x Hogetsu

tag: line/phenotype


title: Organ of Corti_cochlear_P12_rep2


title: 130911_1726no_1

tag: harvest date


title: 140820_47377b

value: fruit

value: watercore sustainable line (47377)


title: 130911_1726wc_3

source_name: watercore susceptible line (1726) fruits watercored part harvested on 130911

value: fruit (watercored part)


title: T3-NSCLP

value: Saliva DNA

value: NSCLP-Affected Twin


title: Patient3 TILs - CEF single cell vdj sequences

description: single cell RNA

value: stimulated with CEF pool


title: Patient3 TILs - CEF single cell gene expression

description: single cell RNA


title: Patient3 Control TILs single cell vdj sequences

description: single cell RNA

value: untreated

value: Negative


title: S308

description:
single-cell RNA-Seq data
B8_S308
H5TJLBGX3_S308

tag: strain background

value: wild type

value: CD8 tumor infiltrating lymphocytes (TIL)


title: S307

description:
single-cell RNA-Seq data
B7_S307
H5TJLBGX3_S307

tag: genotype/variation


title: S20

description:
single-cell RNA-Seq data
B8_S20
H5TJLBGX3_S20

tag: cell type


title: Organ of Corti_cochlear_P12_rep1

tag: tissue

value: Organ of Corti

value: Fgfr3iCre+; Atoh1-HA+; Chrna9-GFP+; Rosa26-CAG-loxP-stop-loxP-tdTomato+


title: Organ of Corti_cHC_P33_rep2

value: Tamoxifen injected at P12-13


title: P30-P.RNAseq

city: Beijing

first: Jiaxin

last: Yang

address: Shuaifuyuan No.1

country: China

institute: PUMCH

data_processing:
We used TopHat2 to align RNA-seq reads to the reference
We quantified gene expression level through cufflinks (v2.2.1)
Gene expression levels were quantified by Fragments Per Kilobase Million (FPKM).
Genome_build: hg19
Supplementary_files_format_and_content: YST.gene_expression_hg19.txt.gz  (Tab-delimited text file containing the expression abundance (FPKM) of each gene in each sample.)

source_name: Tumor tissue sample

extract_protocol:
Total RNA from 12 freshly frozen samples of 10 YST patients was extracted using the Trizol method.
1 μg total RNA was used to generate the libraries using the NEBNext Ultra II RNA library Prep Kit (Illumina, USA).

tag: indivdual

value: YST patient

tag: patient

value: Ovary


title: Patient3 Control TILs single cell gene expression

description: single cell RNA

value: TIL


title: Organ of Corti_cHC_P33_rep1


title: S18

description:
single-cell RNA-Seq data
B6_S18
H5TJLBGX3_S18


title: S19

description:
single-cell RNA-Seq data
B7_S19
H5TJLBGX3_S19


title: P23-R.RNAseq

value: Pelvic cavity


title: Organ of Corti_cHC_P26_rep1


title: S305

description:
single-cell RNA-Seq data
B5_S305
H5TJLBGX3_S305


title: 130820_1703_3

source_name: watercore sustainable line (1703) fruits harvested on 130820

value: fruit

value: Hosui x Hogetsu

value: watercore sustainable line (1703)


title: SqY3

city: Mérida

first: Javier

last: Morales

state: Yucatán

address: Calle 43 No. 130 x 32 y 34, Chuburná de Hidalgo

department: Unidad de Bioquímica y Biología Molecular

country: Mexico

institute: Centro de Investigación Científica de Yucatán

data_processing:
Pair-end sequencing configuration (150PE x 40M raw reads) were performed on a Novaseq 6000 sequencer.We use the Corset software v1.05 (Davidson and Oshlack, 2014) for the clustering of transcripts to enhance the statistical power of DEG analysis by reducing redundancy.
The resulting raw reads were processed by FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Trimmomatic v0.36  to filter and remove low quality sequences and reads (Q≥20)
The resulted clean reads were assembled by using Trinity v.r2014-04-13p1 (Grabherr et al., 2011) with minimum kmer coverage set to 2 and all other parameters set to default.
functional annotation of all assembled transcripts was done against the following databases: NR (NCBI non-redundant protein sequences), NT (NCBI non-redundant nucleotide sequences), Pfam v33.1 (Protein family, Finn et al., 2014) KOG/COG , Swiss-Prot, KO  and GO. The evalue threshold for NT, NR, SwissProt, and KOG was 1e-5; for PFAM was 0.01; for Go 1e-6; and for KEGG was 1e-10.
Quantification of gene expression levels were estimated by mapping all the PE trimmed libraries to the de novo assembled transcriptome (filtered by Corset) by means of Bowtie2 program which is wrapped into the RNA-Seq by Expectation-Maximization (RSEM) software v1.2.26. Quantification of gene expression levels were estimated by mapping all the PE trimmed libraries to the de novo assembled transcriptome (filtered by Corset) by means of Bowtie2 program  which is wrapped into the RNA-Seq by Expectation-Maximization (RSEM) software v1.2.26. Read counts were normalized to FPKM.

organism: Stenocereus queretaroensis

source_name: suspension cells

extract_protocol:
0.1 g of SqY1 and SqR1 were used for RNA extraction with the kit PureLink™ RNA Mini Kit (Invitrogen) with some modifications: we used 1 mL of TRIzol™ Reagent buffer for lysis, homogenization was carried with glass 0.5 millimeters glass beads, 500 mL of isopropanol was added to homogenate and then was passed through kit columns according to the manufacturing procedure. RNA was eluted with 50 mL of water
1 µg of total RNA per sample was used as input material for each sequencing library by using poly-T oligo-attached magnetic beads. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations, index codes were added to attribute sequences to each sample, and library quality was assessed on the Agilent Bioanalyzer 2100 system.RNA-sequencing and de novo assembly. Pair-end sequencing configuration (150PE x 40M raw reads) were performed on a Novaseq 6000 sequencer.

tag: grow medium

value: MS. 3 % sucrose


title: 130911_1726wc_2

value: fruit (watercored part)

value: watercore susceptible line (1726)


title: 130820_1726_3

source_name: watercore susceptible line (1726) fruits harvested on 130820


title: 130820_1703_1


title: 130820_1703_2


title: Organ of Corti_cHC_P26_rep3


title: P19-R1.RNAseq

value: Instestine


title: S21

description:
single-cell RNA-Seq data
B9_S21
H5TJLBGX3_S21


title: P16-R.RNAseq


title: Organ of Corti_cHC_P26_rep2


title: S306

description:
single-cell RNA-Seq data
B6_S306
H5TJLBGX3_S306


title: S17

description:
single-cell RNA-Seq data
B5_S17
H5TJLBGX3_S17


title: P12-P.RNAseq


title: SqY2


title: P11-R.RNAseq


title: SqR3

value: MS. 8 % sucrose


title: SqR2


title: S304

description:
single-cell RNA-Seq data
B4_S304
H5TJLBGX3_S304

value: wild type


title: 130820_1726_2


title: S16

description:
single-cell RNA-Seq data
B4_S16
H5TJLBGX3_S16

value: CD8 tumor infiltrating lymphocytes (TIL)


title: P08-R1.RNAseq

value: Ascending colon


title: 130820_1726_1


title: 130730_1703_2

source_name: watercore sustainable line (1703) fruits harvested on 130730


title: P07-R3.RNAseq


title: 130730_1703_3


title: 130730_1703_1


title: S302

description:
single-cell RNA-Seq data
B2_S302
H5TJLBGX3_S302


title: E7

city: DIJON

first: Mathieu

last: Blot

address: 7 bd Jeanne d'Arc

country: France

institute: INSERM UMR1231

description:
sample name:
E7_S9_all_R1_001

data_processing:
Reads have been mapped on the rabbit genome (OryCun v2.0 Ensembl release 90) with TopHat2 v2.1.1 and counted using subreads featureCounts v1.5.2. Differential analyses were performed using DESeq2 v1.24.0.
Genome_build: OryCun v2.0 Ensembl release 90
Supplementary_files_format_and_content: DESeq output. Counts.tar contains counts of ERCC for each sample

organism: Oryctolagus cuniculus

source_name: Group E

extract_protocol:
Immediately after autopsy (H24 after bacterial inoculation, or immediately after death if occuring earlier), lung pieces were taken and conserved at -80°C in lysis buffer with 1% β-mercapto-ethanol. Total RNAs were extracted using the RNA GenElute kit (Sigma), followed by a RQ1Dnase (Promega) treatment. Total RNA quality was assessed on an Agilent Bioanalyzer 2100, using RNA 6000 pico kit (Agilent Technologies).
Directional RNA-Seq Libraries were constructed using the TruSeq mRNA Stranded library prep kit (Illumina), following the manufacturer’s instructions. Final libraries quality was assessed on an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit. Libraries were pooled in equimolar proportions and sequenced in Single Read 75 bp runs on an Illumina NextSeq500 instrument, using NextSeq 500 High Output 75 cycles kits. Demultiplexing has been done (bcl2fastq2 v2.18.12) and adapters were trimmed with Cutadapt v1.15, only reads longer than 10pb were kept.

treatment_protocol: With the condition associating pneumonia and adverse MV, rabbits were randomly allocated to receive: Nacl (control group), i.v. human umbilical cord-derived MSCs (3.106/Kg), i.m. ceftaroline-fosamil (ceftaroline) or ceftaroline+MSCs. Therapies were infused 4 hours after bacterial inoculation.

tag: strain

value: New Zealand

value: lung

tag: condition

value: pneumoccocal pneumonia + adverse mechanical ventilation

value: Ceftaroline 20mg/kg i.m. + MSCs. 3.10^6/kg i.v.


title: E6

description:
sample name:
E6_S8_all_R1_001


title: P08-R2.RNAseq

value: YST patient


title: SqY1


title: S303

description:
single-cell RNA-Seq data
B3_S303
H5TJLBGX3_S303

source_name: Mus musculus CD8 TILs


title: P07-R2.RNAseq

value: Spleen


title: P04-R.RNAseq

value: Pelvic cavity


title: E4

description:
sample name:
E4_S6_all_R1_001


title: E5

description:
sample name:
E5_S7_all_R1_001


title: 130730_1726_2

source_name: watercore susceptible line (1726) fruits harvested on 130730

value: fruit

value: Hosui x Hogetsu


title: S14

description:
single-cell RNA-Seq data
B2_S14
H5TJLBGX3_S14


title: P01-P.RNAseq

value: Ovary


title: 130730_1726_3


title: S13

description:
single-cell RNA-Seq data
B1_S13
H5TJLBGX3_S13


title: E3

description:
sample name:
E3_S5_all_R1_001


title: 130730_1726_1


title: 130711_1703_3

source_name: watercore sustainable line (1703) fruits harvested on 130711

value: watercore sustainable line (1703)


title: SqR1


title: ATII cells, siGRK6_3

city: Southampton

first: Yihua

last: Wang

address: University Road

country: United Kingdom

institute: University of Southampton

data_processing:
Quality control of RNA-seq raw paired data was performed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and MultiQC
Trim Galore (https://github.com/FelixKrueger/TrimGalore) was used to trim adapters, reads with low quality (< 30), and short length (< 50 bp).
RNA-seq reads were mapped to Human genome Ensembl GRCh38 using Hisat2 (version 2.1.0) with default code
read counts of each gene were summarized using featureCounts (version 1.6.5)
Genome_build: GRCh38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: ATII cells

growth_protocol: Alveolar epithelial type II (ATII)  cells  were cultured in DCCM-11 (Biological Industries Ltd)  supplemented with 10% new-born calf serum  (NBCS), 50 units/ml penicillin, 50μg/ml streptomycin, 2mM L-glutamine  (all from Life Technologies).

extract_protocol:
Total RNA from alveolar epithelial type II (ATII) cells transfected with indicated siRNA oligos was isolated using RNeasy mini kit (Qiagen) according to manufacturer’s instructions.
A total amount of 3 μg RNA per sample was used as input material for library construction. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, Ipswich, Massachusetts, USA) following manufacturer’s instruction
Libraries were pooled in equimolar and sequenced using the paired-end strategy (2 × 150) on the Illumina NovaSeq 6000 platform following the standard protocols

treatment_protocol: Alveolar epithelial type II (ATII)  cells were transfected with short interfering RNA (siRNA) oligos against GRK6 or control siRNA for 72 hours.

value: alveolar epithelial type II cells


title: S15

description:
single-cell RNA-Seq data
B3_S15
H5TJLBGX3_S15


title: S312

description:
single-cell RNA-Seq data
B12_S312
H5TJLBGX3_S312


title: ATII cells, siGRK6_2


title: 130711_1703_2


title: S24

description:
single-cell RNA-Seq data
B12_S24
H5TJLBGX3_S24


title: S301

description:
single-cell RNA-Seq data
B1_S301
H5TJLBGX3_S301


title: ATII cells, siGRK6_1


title: E2

description:
sample name:
E2_S4_all_R1_001


title: E1

description:
sample name:
E1_S3_all_R1_001


title: D7

description:
sample name:
D7_S2_all_R1_001

source_name: Group D

value: Ceftaroline 20mg/kg i.m.


title: S23

description:
single-cell RNA-Seq data
B11_S23
H5TJLBGX3_S23


title: ATII cells, Control_2

value: Control


title: ATII cells, Control_3


title: S311

description:
single-cell RNA-Seq data
B11_S311
H5TJLBGX3_S311

value: wild type


title: S310

description:
single-cell RNA-Seq data
B10_S310
H5TJLBGX3_S310

value: CD8 tumor infiltrating lymphocytes (TIL)


title: 130711_1703_1


title: D6

description:
sample name:
D6_S1_all_R1_001


title: ATII cells, Control_1


title: S22

description:
single-cell RNA-Seq data
B10_S22
H5TJLBGX3_S22


title: 130711_1726_3

source_name: watercore susceptible line (1726) fruits harvested on 130711

value: watercore susceptible line (1726)


title: S9

description:
single-cell RNA-Seq data
A9_S9
H5TJLBGX3_S9


title: D4

description:
sample name:
D4_S13_all_R1_001

value: New Zealand


title: D3

description:
sample name:
D3_S12_all_R1_001

value: lung


title: 130711_1726_2


title: 120913_1703_3

source_name: watercore sustainable line (1703) fruits harvested on 120913


title: S297

description:
single-cell RNA-Seq data
A9_S297
H5TJLBGX3_S297


title: 120913_1703_2


title: D2

description:
sample name:
D2_S11_all_R1_001

value: pneumoccocal pneumonia + adverse mechanical ventilation


title: S8

description:
single-cell RNA-Seq data
A8_S8
H5TJLBGX3_S8


title: AD 3 Ctx

city: Palo Alto

first: Andrew

address: 3801 Miranda Ave

institute: Stanford University

data_processing:
The Cellranger toolkit (v3.0.0) was used to de-multiplex and align reads to the mm10 genome
Genome_build: hg38
Supplementary_files_format_and_content: matrix.mtx matrices from Cellranger

source_name: Human brain vascular nuclei

extract_protocol:
Flow cytometry of brains prepared with custom vascular nuclei isolation protocol
10x Genomics v3; single-cell transcriptomics

tag: disease

value: AD

tag: brain region

value: Superior frontal cortex


title: Control 4 Ctx


title: AD 4 Ctx


title: D5

description:
sample name:
D5_S14_all_R1_001

tag: treatment


title: N6PG

description:
sample name:
N6PG_S9_all_R1_001

source_name: Group C


title: S296

description:
single-cell RNA-Seq data
A8_S296
H5TJLBGX3_S296


title: 120913_1726no_3

source_name: watercore susceptible line (1726) fruits normal part harvested on 120913

value: fruit (normal part)


title: D1

description:
sample name:
D1_S10_all_R1_001


title: S7

description:
single-cell RNA-Seq data
A7_S7
H5TJLBGX3_S7


title: 120913_1703_1


title: AD 1 Ctx


title: Control 3 Ctx


title: N5PG

description:
sample name:
N5PG_S8_all_R1_001


title: Control 2 Ctx


title: S295

description:
single-cell RNA-Seq data
A7_S295
H5TJLBGX3_S295


title: 120913_1726no_2


title: 120913_1726no_1


title: 120913_1726wc_3

source_name: watercore susceptible line (1726) fruits watercored part harvested on 120913

value: fruit (watercored part)


title: C7

description:
sample name:
C7_S7_all_R1_001


title: S6

description:
single-cell RNA-Seq data
A6_S6
H5TJLBGX3_S6


title: AD 2 Ctx


title: Control 1 Ctx


title: C4

description:
sample name:
C4_S6_all_R1_001


title: 130711_1726_1

value: fruit


title: S294

description:
single-cell RNA-Seq data
A6_S294
H5TJLBGX3_S294


title: AD 9 Hpc

value: Hippocampus


title: S5

description:
single-cell RNA-Seq data
A5_S5
H5TJLBGX3_S5


title: C3

description:
sample name:
C3_S5_all_R1_001


title: AD 8 Hpc


title: S293

description:
single-cell RNA-Seq data
A5_S293
H5TJLBGX3_S293


title: C1

description:
sample name:
C1_S3_all_R1_001


title: C2

description:
sample name:
C2_S4_all_R1_001


title: S4

description:
single-cell RNA-Seq data
A4_S4
H5TJLBGX3_S4


title: AD 7 Hpc


title: I5PG

description:
sample name:
I5PG_S2_all_R1_001

source_name: Group B

value: NaCl


title: AD 6 Hpc


title: AD 5 Hpc


title: 120823_1703_3

source_name: watercore sustainable line (1703) fruits harvested on 120823

value: Hosui x Hogetsu

value: watercore sustainable line (1703)


title: L4PG

description:
sample name:
L4PG_S12_all_R1_001


title: Control 8 Hpc


title: S291

description:
single-cell RNA-Seq data
A3_S291
H5TJLBGX3_S291


title: 120913_1726wc_1


title: 120823_1703_2


title: 120823_1726_3

source_name: watercore susceptible line (1726) fruits harvested on 120823


title: S3

description:
single-cell RNA-Seq data
A3_S3
H5TJLBGX3_S3


title: 120913_1726wc_2


title: S292

description:
single-cell RNA-Seq data
A4_S292
H5TJLBGX3_S292


title: B7

description:
sample name:
B7_S11_all_R1_001


title: AD 4 Hpc


title: Control 7 Hpc


title: B6

description:
sample name:
B6_S10_all_R1_001


title: 120823_1703_1


title: I3PG

description:
sample name:
I3PG_S1_all_R1_001


title: Control 6 Hpc


title: B2

description:
sample name:
B2_S00_all_R1_001


title: S2

description:
single-cell RNA-Seq data
A2_S2
H5TJLBGX3_S2


title: S290

description:
single-cell RNA-Seq data
A2_S290
H5TJLBGX3_S290


title: S289

description:
single-cell RNA-Seq data
A1_S289
H5TJLBGX3_S289


title: B1

description:
sample name:
B1_S8_all_R1_001


title: S1

description:
single-cell RNA-Seq data
A1_S1
H5TJLBGX3_S1


title: AD 3 Hpc


title: AD 2 Hpc


title: Control 5 Hpc


title: S300

description:
single-cell RNA-Seq data
A12_S300
H5TJLBGX3_S300


title: S12

description:
single-cell RNA-Seq data
A12_S12
H5TJLBGX3_S12


title: 120823_1726_1


title: Control 4 Hpc


title: 120802_1703_2

source_name: watercore sustainable line (1703) fruits harvested on 120802


title: Control 3 Hpc


title: 120823_1726_2


title: 120802_1726_2

source_name: watercore susceptible line (1726) fruits harvested on 120802


title: 120802_1703_3


title: A7PD

description:
sample name:
A7PD_S00_all_R1_001

source_name: Group A

value: healthy


title: 120802_1703_1


title: 120802_1726_1


title: D5PG

description:
sample name:
D5PG_S00_all_R1_001


title: 120802_1726_3


title: 120713_1703_3

source_name: watercore sustainable line (1703) fruits harvested on 120713


title: S11

description:
single-cell RNA-Seq data
A11_S11
H5TJLBGX3_S11

source_name: Mus musculus CD8 TILs


title: AD 1 Hpc


title: S299

description:
single-cell RNA-Seq data
A11_S299
H5TJLBGX3_S299


title: Control 2 Hpc

value: Control


title: A5PD

description:
sample name:
A5PD_S00_all_R1_001


title: Control 1 Hpc


title: S298

description:
single-cell RNA-Seq data
A10_S298
H5TJLBGX3_S298

value: wild type


title: A4PD

description:
sample name:
A4PD_S00_all_R1_001


title: D7PD

description:
sample name:
D7PD_S00_all_R1_001


title: 130911_1726wc_1

source_name: watercore susceptible line (1726) fruits watercored part harvested on 130911


title: A9

description:
sample name:
A9_S00_all_R1_001


title: S10

description:
single-cell RNA-Seq data
A10_S10
H5TJLBGX3_S10

value: CD8 tumor infiltrating lymphocytes (TIL)


title: 120713_1703_1


title: 120713_1703_2


title: 120713_1726_1

source_name: watercore susceptible line (1726) fruits harvested on 120713

value: watercore susceptible line (1726)


title: 120713_1726_3


title: A3

description:
sample name:
A3_S00_all_R1_001

value: New Zealand


title: Polysome selection pool 2 selected cycle 5

status: Public on Apr 23 2021

city: Stanford

first: Rhiju

last: Das

state: California

address: 279 Campus Dr, B419 Beckman Center

department: Biochemistry

institute: Stanford University School of Medicin

data_processing:
Library strategy: Barcode amplicon sequencing
Following adapter trimming, 670440 sequences with at least 10 summed read count across all libraries combined were set as the reference. Each library was aligned to this indexed reference using Bowtie2. Only uniquely mapping reads with edit distance ≤3 were retained. Alignments were further deduplicated using UMIcollapse (-p 0.05, -k 1). This results in the matrix of read count where rows are different sequence variants and columns are the samples. Normalized counts were obtained by dividing the matrix column-wise by total read counts per sample. For sequence variants with at least 15 reads in any one of the samples, a regression model was fitted on normalized read counts with the sequential selection rounds as ordinal predictors, penalizing differences between coefficients of adjacent groups (R package ordPens) . False discovery rate was estimated by Benjamini-Hochberg procedure. For choosing the final set of candidates, the criteria of ≥15 read counts in the final round polysome selection library and ≥2 fold enrichment over input in the final round was also required. For analysis of k-mers, 1 million reads (prior to alignment and analysis for highly enriched individual sequences) were sampled from each library. Position-specific k-mers were counted and statistical significance of pair-wise enrichments for each position-specific k-mers are calculated using kpLogo. The parameters were: zero-order Markov model background; 2≤k≤6; -shift 0 and -max-shift 0; binomial test with Bonferroni correction.
processed data files format and content: Table S3 contains k-mer enrichment statistics enriched after polysome selection for both input pools.

source_name: Cell culture

extract_protocol:
Transfected RNA was isolated and fractionated by sucrose gradient as in the "polysome fraction" samples. Fractions 10-16 were pooled and RNA was purified by acidic phenol chloroform extraction followed by silica column purification (Zymo)
1.5 µg RNA in 5.5 µL was mixed with 0.5 µL 2uM RT_Nluc26_UMI12_Read1Partial (CCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNGGAACATCGTATGGGTAAACGGCCAT) and 0.5 µL 10 mM dNTPs each. The RNA samples were then denatured at 65°C for 5 min and chilled to 4°C. 3.5 µL reverse transcription mix was added to 10 µL total reaction volume: 2 µL 5x Superscript IV buffer, 0.5 µL 10 mM DTT, 0.5 µL Superase-In (ThermoFisher Scientific, AM2694), 0.5 µL Superscript IV (Thermo 18091050). The reaction was incubated at 55°C for 45 min and inactivated at 80°C for 10 min. For sequencing library preparation the RT reaction was PCR amplified under the following conditions: 1 µL RT reaction, 10 µL 2x Q5 Hot Start Master Mix (NEB M0494S), 0.2 µL 100x SYBR (Thermo S7563), 1 µL 10 µM Read1, 1 µL 10 µM Read2Partial_HBB29 in 20 µL total reaction volume. Cycling conditions were as follows: 98°C for 60 sec, and 15 cycles of 98°C for 10 sec, 68°C for 10 sec, 72°C for 10 sec. Sequencing adaptors were added using the following conditions for final round PCR: 1 µL first round PCR reaction, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10uM NEBNext Index Primer (NEB E7335, NEB E7500, NEB E7710, NEB E7730, NEB E6609), 1µL 10uM NEBNext Universal PCR Primer in 20 µL total volume. Cycling conditions are: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 10 sec. All barcoded samples were then pooled at equal volumes and purified with 1.1x SPRIselect beads Beckman Coulter B23317).

value: HEK293T cell line


title: Polysome selection pool 2 selected cycle 4


title: Polysome selection pool 2 selected cycle 2


title: Polysome selection pool 2 input cycle 1


title: Polysome selection pool 2 selected cycle 3


title: Polysome selection pool 2 input cycle 0


title: Polysome selection pool 2 selected cycle 1


title: Polysome selection pool 1 selected cycle 5


title: Polysome selection pool 2 selected cycle 0


title: Purple sea urchin muscle (Sample2581)

city: Houston

first: Olga

last: Dudchenko

address: 1 Baylor Plaza

institute: Baylor College of Medicine

data_processing:
Hi-C-guided genome assembly (3D-DNA, JBAT)
Construction of the chromosome-length Hi-C contact map (3D-DNA, Juicer)
Centromere position identification (repeat sequence mapping or structural analysis, 3D-DNA/JBAT)
Aggregate chromosome analysis (3D-DNA)
Variant calling (DRAGEN pipeline)
Chromosome-length variant phasing (3D-DNA)
Genome_build: In addition to those generate as part of the study, the following existing genome builds were used: GRCg6a (domestic chicken); Xla.v91 (African clawed frog); AaegL5.0 (Yellow fever mosquito), CpipJ3 (Southern domestic mosquito); Release_6_plus_ISO1_MT (fruit fly, with chromosome arms merged for ACA analysis resulting in Release_6_plus_ISO1_MT_merged); WBcel235 (C. elegans); sacCer3 (baker's yeast); arahy.Tifrunner.gnm2.J5K5 (ground nut); 161010_Chinese_Spring_v1.0_pseudomolecules (bread wheat).
Supplementary_files_format_and_content: fasta.gz: chromosome-length nucleotide sequences generated usign Hi-C-guided genome assembly
Supplementary_files_format_and_content: hic: contact maps generated by aligning Hi-C data to corresponding chromosome-length reference sequences. In addition to standard contact maps generated for all genomic positions across chromosomes the uploaded files include ACA (aggregate chromosome analysis) maps and phasing contact maps representing interactions between positions on the genome that contain heterogyzous SNPs. For more details see Hoencamp et al., Supplement.
Supplementary_files_format_and_content: wig: tracks showing the alignment frequency of Hi-C reads from read pairs that contain centromere repeat-specifc sequences.
Supplementary_files_format_and_content: bedpe: Files describing the tentative position of centromeres in chromosome-length genome assemblies, identified either by mapping repeat sequences associated with the centromere or via analysis of the contact map for evidence of centromere-to-telomere folding axis. For details see Hoencamp et al., Supplement.
Supplementary_files_format_and_content: vcf.gz: Variants (specifically SNPs) identivied by the DRAGEN variant calling pipeline from aligning Hi-C data to chromosome-length references generated via Hi-C-guided assembly and phased using the 3D-DNA Hi-C-guided phasing module.

organism: Strongylocentrotus purpuratus

source_name: muscle

extract_protocol:
Extraction protocol is described as part of the library construction protocol.
The Hi-C libraries were prepared following the in situ Hi-C strategy (Rao, Huntley et al., 2014). Briefly, cells or tissue were crosslinked and then lysed with nuclei permeabilized but still intact. DNA was then restricted and the overhangs filled in incorporating a biotinylated base. Free ends were then ligated together in situ. Crosslinks were reversed, the DNA was sheared to 300-500bp and then biotinylated ligation junctions were recovered with streptavidin beads. Afterward DNA was handled according to standard Illumina library construction protocol. DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo-) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3 end. After adapter ligation DNA was PCR amplified with Illumina primers and library fragments of 400-600 bp (insert plus adaptor and PCR primer sequences) were purified using SPRI beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on various Illumina instruments following the manufacturer's protocols. Fpr the tammar wallaby, a short insert-size PCR-free DNA-Seq library was prepared following the ClaSeek protocol.

value: muscle


title: Polysome selection pool 1 selected cycle 4


title: Chinese liver fluke whole animal(s) (Sample2741)

organism: Clonorchis sinensis

source_name: whole animals

value: whole animals


title: European lancelet whole animal(s) (Sample1824)

organism: Branchiostoma lanceolatum

source_name: whole animal

value: whole animal


title: Polysome selection pool 1 selected cycle 2


title: Polysome selection pool 1 selected cycle 3


title: Polysome selection pool 1 selected cycle 1


title: Polysome selection pool 1 selected cycle 0


title: Polysome selection pool 1 input cycle 1


title: California sea hare mix of CNS cells (Sample1819)

organism: Aplysia californica

source_name: Mix of CNS cells

value: Mix of CNS cells


title: 120713_1726_2


title: RNA in-solution timepoint 9

data_processing:
Library strategy: Barcode amplicon sequencing
Constant regions were trimmed using cutadapt. The trimmed reads were aligned to the indexed reference of barcode sequences using Bowtie2 with the following options: -L 11 -N 0 --nofw 89. The alignments were deduplicated based on UMIs using UMIcollapse with -p 0.05 and counted using samtools available under idxstats. This pipeline yields a matrix of barcode read counts where rows are the different constructs in the library and columns are the different samples. The count matrix was log transformed and normalized column-wise using a linear fit on the dilution series of spike-in constructs in each sample. For the calculation of RNA degradation coefficients in cells, we carried out a linear fit to log RNA abundance from the time course data, i.e. we fit an expression of Y = β0+β1 t where Y is the normalized log RNA abundance and t is the number of hours after transfection; β1 is the degradation constant. For the calculation of in solution degradation coefficients, sufficient data points were available to carry out a nonlinear fit directly to an exponential model, i.e. we fit an expression of y = A exp(-𝛕/t), where y is the fraction intact (RNA abundance normalized to initial abundance), A is the amplitude, t is the time of incubation in degradation buffer in hours, and 𝛕 is the degradation time constant. For polysome profiles, percent RNA abundances for each fraction were first calculated by scaling per-fraction values by the sum of all fractions. For the heatmap displays in the figures, column medians were also subtracted from each percent RNA value. For the calculation of ribosome load, the matrix of percent RNA abundances in fractions 4-9 (1-3 are free RNP fractions, and >9 have negligible abundance) were first multiplied by a weight vector representing the number of ribosomes in each fraction as determined by the A260 trace from the fractionator, then the weighted abundances were summed across the row. For the calculation of polysome to monosome ratio, the sum of fractions 7-9 (>3 ribosomes) abundances were divided by fraction 4 (80S) abundance. For the calculation of monosome to 40S/60S ratio, fraction 4 (80S) abundance was divided by the sum of fraction 2 (40S/60S) abundance.
processed data files format and content: Table S1 is an XLSX file containing the final polysome profile metrics and degradation statistics for each of the 233 constructs in the input library.

extract_protocol:
RNA was purified using Ampure beads + 40% polyethylene glycol 8000 (7:3)
Up to 250 ng RNA in 2.75 µL was mixed with 0.25 µL 2 µM RT_Const2_N12_Read1Partial (TCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNtcaccacggcgtgagatcagcg) and 0.25 µL 10mM dNTPs each. The RNA samples were then denatured at 65°C for 5 min and chilled to 4°C. 1.75 µL reverse transcription mix was added to 5 µL total reaction volume: 1 µL 5x Superscript IV buffer, 0.25 µL 10mM DTT, 0.25 µL Superase-In (ThermoFisher Scientific, AM2694), 0.25 µL Superscript IV (Thermo 18091050). The reaction was incubated at 55°C for 45 min and inactivated at 80°C for 10 min. First round PCR was performed under following conditions: 1 µL RT reaction, 10 µL 2x Q5 Hot Start Master Mix (NEB M0494S), 0.2 µL 100x SYBR (Thermo S7563), 1 µL 10uM Read1Partial_F (TCTTTCCCTACACGACGCTCTTCCGATCT), 1 µL 10 uM 50:50 Hbb_Fwd:Nluc_Fwd mix in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 15 cycles of 98°C for 10 sec, 68°C for 10 sec and 72°C. Second round PCR was performed under the following conditions: 1 µL first round PCR, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10 uM Read1Partial_F, 1 µL 10 uM Read2Partial_Const1_R (GGAGTTCAGACGTGTGCTCTTCCGATCTttctagagcggccgcttcgagc) in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 5 sec. Sequencing adaptors were added using the following conditions for final round PCR: 1 µL second round PCR, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10 µM NEBNext Index Primer (NEB E7335, NEB E7500, NEB E7710, NEB E7730, NEB E6609), 1 µL 10 µM NEBNext Universal PCR Primer in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 5 sec. All barcoded samples were then pooled at equal volumes and purified with 1.1x SPRIselect beads (Beckman Coulter B23317).


title: Polysome selection pool 1 input cycle 0


title: Polysome selection pool 2 input cycle 5


title: RNA in-solution timepoint 10


title: RNA in-solution timepoint 8


title: Polysome selection pool 1 input cycle 5


title: RNA in-solution timepoint 7


title: RNA in-solution timepoint 6


title: RNA in-solution timepoint 5


title: RNA in-cell timepoint 5 replicate 2

extract_protocol:
RNA was harvested with Trizol (Thermofisher scientific). RNA was extracted from the aqeous phase by silica column (Zymo)
Up to 250 ng RNA in 2.75 µL was mixed with 0.25 µL 2 µM RT_Const2_N12_Read1Partial (TCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNtcaccacggcgtgagatcagcg) and 0.25 µL 10mM dNTPs each. The RNA samples were then denatured at 65°C for 5 min and chilled to 4°C. 1.75 µL reverse transcription mix was added to 5 µL total reaction volume: 1 µL 5x Superscript IV buffer, 0.25 µL 10mM DTT, 0.25 µL Superase-In (ThermoFisher Scientific, AM2694), 0.25 µL Superscript IV (Thermo 18091050). The reaction was incubated at 55°C for 45 min and inactivated at 80°C for 10 min. First round PCR was performed under following conditions: 1 µL RT reaction, 10 µL 2x Q5 Hot Start Master Mix (NEB M0494S), 0.2 µL 100x SYBR (Thermo S7563), 1 µL 10uM Read1Partial_F (TCTTTCCCTACACGACGCTCTTCCGATCT), 1 µL 10 uM 50:50 Hbb_Fwd:Nluc_Fwd mix in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 15 cycles of 98°C for 10 sec, 68°C for 10 sec and 72°C. Second round PCR was performed under the following conditions: 1 µL first round PCR, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10 uM Read1Partial_F, 1 µL 10 uM Read2Partial_Const1_R (GGAGTTCAGACGTGTGCTCTTCCGATCTttctagagcggccgcttcgagc) in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 5 sec. Sequencing adaptors were added using the following conditions for final round PCR: 1 µL second round PCR, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10 µM NEBNext Index Primer (NEB E7335, NEB E7500, NEB E7710, NEB E7730, NEB E6609), 1 µL 10 µM NEBNext Universal PCR Primer in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 5 sec. All barcoded samples were then pooled at equal volumes and purified with 1.1x SPRIselect beads (Beckman Coulter B23317).


title: RNA in-cell timepoint 4 replicate 1


title: RNA in-cell timepoint 5 replicate 1


title: RNA in-solution timepoint 3


title: RNA in-solution timepoint 4


title: RNA in-cell timepoint 2 replicate 3


title: RNA in-cell timepoint 3 replicate 1


title: RNA in-cell timepoint 4 replicate 2


title: RNA in-cell timepoint 4 replicate 3


title: RNA in-cell timepoint 3 replicate 2


title: RNA in-solution timepoint 1


title: RNA in-solution timepoint 2


title: RNA in-cell timepoint 3 replicate 3


title: RNA in-cell timepoint 5 replicate 3


title: Polysome fraction 15

extract_protocol:
Cell culture media was replaced with cycloheximide (MilliporeSigma, C7698-1G) containing media at 100ug/mL. After 2 minutes, cells were washed, trypsinized and harvested using PBS, trypsin, and culture media containing 100 g/mL cycloheximide. ~10x106 cells were resuspended in 400 µL of following lysis buffer on ice for 30 min, vortexing every 10 min: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 15 mM MgCl2, 1 mM DTT, 8% glycerol, 1% Triton X-100, 100 µg/mL cycloheximide, 0.2 U/µL Superase-In RNase inhibitor (ThermoFisher Scientific, AM2694), 1x Halt protease inhibitor cocktail (ThermoFisher Scientific, 78430), 0.02 U/µL TURBO DNase (ThermoFisher Scientific, AM2238). After lysis, nuclei were removed by two step centrifuging, first at 1300 g for 5min and second at 10000 g for 5min, taking the supernatants from each. 25%-50% sucrose gradient was prepared in 13.2mL ultracentrifuge tubes (Beckman Coulter, 331372) using Biocomp Gradient Master with the following recipe: 25 or 50% sucrose (w/v), 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 15 mM MgCl2 , 1 mM DTT, 100 µg/mL cycloheximide. The lysate was layered onto the sucrose gradient and ultracentrifuged on Beckman Coulter SW-41Ti rotor at 40000rpm for 150min at 4°C. The gradient was density fractionated using Brandel BR-188 into 16x750 µL fractions, and in vitro transcribed spike-in RNA mix (120002B1, 120010B1, 220023B1, 310333T3; 1000, 100, 10, 1-fold dilutions respectively) were added to each fraction. 700 µL of each fraction was mixed with 100µL 10% SDS, 200µL 1.5M sodium acetate, and 900µL acid phenol-chloroform, pH 4.5 (ThermoFisher Scientific, AM9720), heated at 65°C for 5min, and centrifuged at 20000g for 15min at 4°C for phase separation. 600µL aqueous phase was mixed with 600 µL 100% ethanol and RNA was purified on silica columns (Zymo, R1013).
Up to 250 ng RNA in 2.75 µL was mixed with 0.25 µL 2 µM RT_Const2_N12_Read1Partial (TCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNtcaccacggcgtgagatcagcg) and 0.25 µL 10mM dNTPs each. The RNA samples were then denatured at 65°C for 5 min and chilled to 4°C. 1.75 µL reverse transcription mix was added to 5 µL total reaction volume: 1 µL 5x Superscript IV buffer, 0.25 µL 10mM DTT, 0.25 µL Superase-In (ThermoFisher Scientific, AM2694), 0.25 µL Superscript IV (Thermo 18091050). The reaction was incubated at 55°C for 45 min and inactivated at 80°C for 10 min. First round PCR was performed under following conditions: 1 µL RT reaction, 10 µL 2x Q5 Hot Start Master Mix (NEB M0494S), 0.2 µL 100x SYBR (Thermo S7563), 1 µL 10uM Read1Partial_F (TCTTTCCCTACACGACGCTCTTCCGATCT), 1 µL 10 uM 50:50 Hbb_Fwd:Nluc_Fwd mix in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 15 cycles of 98°C for 10 sec, 68°C for 10 sec and 72°C. Second round PCR was performed under the following conditions: 1 µL first round PCR, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10 uM Read1Partial_F, 1 µL 10 uM Read2Partial_Const1_R (GGAGTTCAGACGTGTGCTCTTCCGATCTttctagagcggccgcttcgagc) in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 5 sec. Sequencing adaptors were added using the following conditions for final round PCR: 1 µL second round PCR, 10 µL 2x Q5 Hot Start Master Mix, 0.2 µL 100x SYBR, 1 µL 10 µM NEBNext Index Primer (NEB E7335, NEB E7500, NEB E7710, NEB E7730, NEB E6609), 1 µL 10 µM NEBNext Universal PCR Primer in 20 µL total volume. Cycling conditions were: 98°C for 60 sec, and 5 cycles of 98°C for 10 sec, 72°C for 5 sec. All barcoded samples were then pooled at equal volumes and purified with 1.1x SPRIselect beads (Beckman Coulter B23317).


title: RNA in-cell timepoint 1 replicate 2


title: RNA in-cell timepoint 1 replicate 1


title: Polysome fraction 16


title: Polysome fraction 13


title: RNA in-cell timepoint 1 replicate 3


title: RNA in-cell timepoint 2 replicate 2


title: Polysome fraction 11


title: Polysome fraction 14


title: Polysome fraction 12


title: Polysome fraction 10


title: Polysome fraction 9


title: Polysome fraction 8


title: RNA in-cell timepoint 2 replicate 1


title: Polysome fraction 7


title: Polysome fraction 6


title: Polysome fraction 2


title: Polysome fraction 1


title: Polysome fraction 3


title: Polysome fraction 4


title: L1851-In_vivo_HEK239-dCas9_VEGF-rep1

first: Georgi

middle: Kolev

last: Marinov

address: 279 Campus Drive West, Beckman Center, B-257A/259

department: Genetics

institute: STANFORD UNIVERSITY

data_processing:
read alignment, ChIP-seq data: Bowtie 1.0.1
bigWig file generation: custom code
genome build: hg38
Supplementary_files_format_and_content: bedGraph of read coverage, normalized to RPM

extract_protocol:
For in vivo CasKAS experiments, HEK293T cells were seeded at 400,000 cells/well into a 6-well plate the day before RNP transfection. Media was exchanged 2 hours before transfection. For each well, 6,250 ng of Cas9 (MCLAB CAS9-200) or dCas9 (MCLAB dCAS9B-200) and 1,200 ng sgRNA was complexed with CRISPRMAX (Thermo Fisher CMAX00008) reagent in Opti-MEM (Thermo Fisher 51985091) following manufacturer's protocol. After incubation at room temperature for 15 minutes, the RNP solution was directly added to each well and gently mixed. The cells were incubated with the RNP complex for 14 hours at 37C. To harvest and perform kethoxal labeling, media was removed and room temperature 1X PBS was used to wash the cells. Cells were then dissociated with trypsin, trypsin was quenched with media, cells were pelleted at room temperature, and then resuspended in 100 uL of media supplemented with 5 мM N3-kethoxal (final concentration). Cells were incubated for 10 minutes at 37C with shaking at 500 rpm in a Thermomixer. Cells were then pelleted by centrifuging at 500 g for 5 minutes at 4C. Genomic DNA was then extracted using the Monarch gDNA Purification Kit (NEB T3010S) following the standard protocol but with elution using 85 uL 25 mM K3BO3 at pH 7.0.
The click reaction was carried out by combining 175 uL purified and sheared DNA, 5 uL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 20 uL 10x PBS in a final volume of 200 uL or 87.5 uL purified and sheared DNA, 2.5 uL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 10 uL 10x PBS in a final volume of 100 uL. The reaction was incubated at 37C for 90 minutes. DNA was purified using AMPure XP beads (50 uL for a 100 uL reaction or 100 uL for a 200 uL reaction), beads were washed on a magnetic stand twice with 80\% EtOH, and eluted in 130 uL 25mM K3BO3. Purified DNA was then sheared on a Covaris E220 instrument down to ~150-400 bp size. For streptavidin pulldown of biotin-labeled DNA, 10 uL of 10 mg/mL Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602) were separated on a magnetic stand, then washed with 300 uL of 1x TWB (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05\% Tween 20). The beads were resuspended in 300 uL of 2x Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl), the sonicated DNA was added (diluted to a final volume of 300 uL if necessary), and the beads were incubated for >15 minutes at room temperature on a rotator. After separation on a magnetic stand, the beads were washed with 300 uL of 1x TWB, and heated at 55C in a Thermomixer with shaking for 2 minutes. After removal of the supernatant on a magnetic stand, the TWB wash and 55C incubation were repeated.  Final libraries were prepared on beads using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645) as follows. End repair was carried out by resuspending beads in 50 uL 1x EB buffer, and adding 3 uL NEB Ultra End Repair Enzyme and 7 uL NEB Ultra End Repair Enzyme, followed by incubation at 20C for 30 minutes (in a Thermomixer, with shaking at 1,000 rpm) and then at 65C for 30 minutes. Adapters were ligated to DNA fragments by adding 30 uL Blunt Ligation mix, 1 uL Ligation Enhancer and 2.5 uL NEB Adapter, incubating at 20C for 20 minutes, adding 3 uL USER enzyme, and incubating at 37C for 15 minutes (in a Thermomixer, with shaking at 1,000 rpm) Beads were then separated on a magnetic stand, and washed with 300 uL TWB for 2 minutes at 55C, 1000 rpm in a Thermomixer. After separation on a magnetic stand, beads were washed in 100 uL 0.1X TE buffer, then resuspended in 15 uL 0.1X TE buffer, and heated at 98C for 10 minutes. For PCR, 5 uL of each of the i5 and i7 NEB Next sequencing adapters were added together with 25 uL 2X NEB Ultra PCR Mater Mix. PCR was carried out with a 98C incubation for 30 seconds and 12 cycles of 98C for 10 seconds, 65C for 30 seconds, and 72C for 1 minute, followed by incubation at 72C for 5 minutes. Beads were separated on a magnetic stand, and the supernatant was cleaned up using 1.8X AMPure XP beads. Libraries were sequenced in a paired-end format on a Illumina NextSeq instrument using NextSeq 500/550 high output kits (2x36 cycles).

tag: chip-antibody


title: L1852-In_vivo_HEK239-dCas9_VEGF-rep2

value: Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602)


title: L1848-In_vivo_HEK239-Cas9_EMX-rep2


title: L1655-In_vitro_dCas9_EMX1_Tsai_human

extract_protocol:
In vitro CasKAS experiments were executed as follows. First, 1 uL of each synthetic sgRNA were incubated at room temperature with 1 uL of recombinant purified dCas9 (MCLab dCAS9B-200) for 20 minutes. The RNP was then incubated with 1 ug of gDNA at 37C for 10 minutes.  The KAS reaction was then carried out by adding 1 $\mu$L of 500 mM N$_3$-kethoxal (ApeXBio A8793). DNA was immediately purified using the MinElute PCR Purification Kit (Qiagen 28006), and eluted in 87.5 or 175 uL 25mM K3BO3.
The click reaction was carried out by combining 175 uL purified and sheared DNA, 5 uL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 20 uL 10x PBS in a final volume of 200 uL or 87.5 uL purified and sheared DNA, 2.5 uL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 10 uL 10x PBS in a final volume of 100 uL. The reaction was incubated at 37C for 90 minutes. DNA was purified using AMPure XP beads (50 uL for a 100 uL reaction or 100 uL for a 200 uL reaction), beads were washed on a magnetic stand twice with 80\% EtOH, and eluted in 130 uL 25mM K3BO3. Purified DNA was then sheared on a Covaris E220 instrument down to ~150-400 bp size. For streptavidin pulldown of biotin-labeled DNA, 10 uL of 10 mg/mL Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602) were separated on a magnetic stand, then washed with 300 uL of 1x TWB (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05\% Tween 20). The beads were resuspended in 300 uL of 2x Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl), the sonicated DNA was added (diluted to a final volume of 300 uL if necessary), and the beads were incubated for >15 minutes at room temperature on a rotator. After separation on a magnetic stand, the beads were washed with 300 uL of 1x TWB, and heated at 55C in a Thermomixer with shaking for 2 minutes. After removal of the supernatant on a magnetic stand, the TWB wash and 55C incubation were repeated.  Final libraries were prepared on beads using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645) as follows. End repair was carried out by resuspending beads in 50 uL 1x EB buffer, and adding 3 uL NEB Ultra End Repair Enzyme and 7 uL NEB Ultra End Repair Enzyme, followed by incubation at 20C for 30 minutes (in a Thermomixer, with shaking at 1,000 rpm) and then at 65C for 30 minutes. Adapters were ligated to DNA fragments by adding 30 uL Blunt Ligation mix, 1 uL Ligation Enhancer and 2.5 uL NEB Adapter, incubating at 20C for 20 minutes, adding 3 uL USER enzyme, and incubating at 37C for 15 minutes (in a Thermomixer, with shaking at 1,000 rpm) Beads were then separated on a magnetic stand, and washed with 300 uL TWB for 2 minutes at 55C, 1000 rpm in a Thermomixer. After separation on a magnetic stand, beads were washed in 100 uL 0.1X TE buffer, then resuspended in 15 uL 0.1X TE buffer, and heated at 98C for 10 minutes. For PCR, 5 uL of each of the i5 and i7 NEB Next sequencing adapters were added together with 25 uL 2X NEB Ultra PCR Mater Mix. PCR was carried out with a 98C incubation for 30 seconds and 12 cycles of 98C for 10 seconds, 65C for 30 seconds, and 72C for 1 minute, followed by incubation at 72C for 5 minutes. Beads were separated on a magnetic stand, and the supernatant was cleaned up using 1.8X AMPure XP beads. Libraries were sequenced in a paired-end format on a Illumina NextSeq instrument using NextSeq 500/550 high output kits (2x36 cycles).


title: Polysome fraction 5


title: L1843-In_vivo_HEK239-Cas9-no_sgRNA-rep1


title: L1846-In_vivo_HEK239-Cas9_VEGF-rep2


title: L1757-In_vitro_dCas9_EMX1_human


title: L1847-In_vivo_HEK239-Cas9_EMX-rep1


title: L1657-In_vitro_Cas9-VEGFA_site1_human


title: L1658-In_vitro_Cas9_EMX1_Tsai_human


title: L1854-In_vivo_HEK239-dCas9_EMX-rep2


title: L1844-In_vivo_HEK239-Cas9-no_sgRNA-rep2


title: L1756-In_vitro_dCas9_EMX1_human_sheared_end-repaired


title: L1758-In_vitro_Cas9_EMX1_human


title: L1755-In_vitro_dCas9_EMX1_human_sheared


title: L1853-In_vivo_HEK239-dCas9_EMX-rep1


title: L1845-In_vivo_HEK239-Cas9_VEGF-rep1


title: EC11 B

city: Ithaca

first: Amanda

middle: R

last: Loehr

state: NY

address: 618 Tower Rd

department: Biomedical Sciences

institute: Cornell University

data_processing:
Illumina pipeline software was used for base calling.
Sequenced reads were trimmed for 3' adaptor sequence and low-quality sequence and filtered to remove reads < 50nt with TrimGalore/cutadapt.
Processed reads were mapped to the reference genome/transcriptome with STAR using --quantMode GeneCounts to generate raw counts per gene.
Raw counts were analyzed in R with DEseq2.
Genome_build: mm10 (Ensembl gene annotation)
Supplementary_files_format_and_content: Matrix table with raw gene counts output from STAR (--quantMode GeneCounts)

source_name: embryonal carcinoma cells derived from a murine malignant teratocarcinoma

growth_protocol: Spontenous malignant teratocarcinomas were isolated and disaggregated with 2 mg/mL Collagenase A (Roche) in serum-free DMEM (Corning) with penicillin, streptomycin, and gentamycin at 37C for approximately 1 hour with shaking. Cells were then passed through a 40 µM filter to remove large clumps, pelleted at 1.5 x g for 5 minutes, resuspended in DMEM containing 10% FBS (Sigma) with 1x LIF (ORF Genetics) and 10 μM ROCKi (Selleckchem) and plated at various densities into 10-cm tissue culture plates. Cultures were passed at confluence until embryonal carcinoma colonies were visually observed, followed by subsequent passages to propagate each cell line. EC cell lines were maintained in EC medium (DMEM + 15% heat-inactivated Bovine Growth Serum (Hyclone) + 2% L-glutamine (Corning) + 1% non-essential amino acids (Corning) + 1% Pen/Strep (Corning) + β-mercaptoethanol + 1x LIF). Media was replaced daily, and cells were passaged by trypsinization 1:10 every other day.

extract_protocol:
Total RNA was extracted from cells using RNA Stat-60 according to the manufacturer’s protocol, with the addition of GlycoBlue Coprecipitant (Invitrogen) immediately prior to isopropanol precipitation. RNA sample quality was determined by spectrophotometry (Nanodrop) to determine concentration and chemical purity and with a Fragment Analyzer (Advanced Analytical) to determine RNA integrity.
Directional RNA-seq libraries were prepared from 1ug total RNA using the NEBNext Directional Ultra II RNA Library Prep Kit for Illumina (New England Biolabs), with initial polyA+ isolation.

treatment_protocol: To induce differentiation, cells were incubated with thioridazine hydrochloride (Sigma) (10 μM for TR14 and 20 μM for TR11) in media without LIF, for up to 2 weeks or until EC colonies were no longer observed, at which point the cultures were maintained in EC media without LIF.

tag: cell line

tag: genotype

value: Pten-/- Stra8-CreTg

value: embryonal carcinoma cells


title: TR11 A

source_name: EC11 embryonal carcinoma cells differentiated with thioridazine


title: EC11 C


title: TR11 C


title: L1654-In_vitro_dCas9-VEGFA_site1


title: EC11 A


title: TR14 B

source_name: EC14 embryonal carcinoma cells differentiated with thioridazine

value: Pten-/- Kras+/G12D Stra8-CreTg OCT4-gfpTg


title: TR14 C


title: L1849-In_vivo_HEK239-dCas9-no_sgRNA-rep1


title: TR14 A


title: L1850-In_vivo_HEK239-dCas9-no_sgRNA-rep2


title: TR11 B


title: EC14 B


title: EC14 A


title: OSM4.2_H01_S757

city: Rehovot

first: Rony

last: Chanoch

state: Israel

address: Weizmann Institute of Science, 234 Herzl Street

country: Israel

institute: Weizmann Instititue

data_processing:
Paired-end reads from mouse model were mapped to the UCSC mm10 mouse transcriptome using Bowtie. Expression values were calculated by RSEM in paired-end mode using parameters, from which TPM values for each gene were extracted. Reads from 10x were aligned using CellRanger
We examined as quality control the number of genes detected in each cell. We observed a bimodal distribution and conservatively excluded sequenced cells with fewer than 3,000 detected genes.
For the remaining cells, we calculated Eavg(i) = log2(mean(TPMi,1...n)+1) for each gene, and excluded genes with Eavg < 4
Genome_build: mm10, hg19
Supplementary_files_format_and_content: Comma-separated value containing TPM values (transcript-per-million reads).

molecule: polyA RNA

source_name: macrophage single cell

extract_protocol:
For mice samples, collected brains were mechanically and enzymatically dissociated using a papain-based brain tumor dissociation kit (Miltenyi Biotec) supplemented with 0.1% typeI collagenase (Thermo Fisher Scientific) / PBS. The dissociated cells were first stained with calcein Blue AM (Thermo Fisher Scientific) and Zombie NIR (BioLegend) for 25 min at 4 ℃, and with anti-mouse CD16/32 (BD Biosciences) for 5min. After washing cells with ice-cold 1% BSA / PBS, the cells were then stained with a combination of following antibodies; PerCP anti-mouse CD45 (clone 30-F11, BD Biosciences), phycoerythrin (PE) anti-mPdgfra (clone APA5, BioLegend), Allophycocyanin (APC) anti-mPodoplanin (clone 8.1.1, BioLegend), and APC anti-CD11b (clone M1/70, BioLegend), for 30 min at 4 ℃. Sorting was performed with Becton Dickinson Influx cytometer (Becton Dickinson). Side scatter (SSC) width versus forward scatter (FSC) area, and Trigger Pulse Width versus FSC criteria were used to discriminate doublets and gate only singleton cells. Viable single cells were identified as calcein blue AM positive and Zombine NIR negative. We sorted viable CD45 negative/ GFP positive or CD45 positive/ CD11b positive single-cells into 96-well plates containing 5uL of TCL buffer (QIAGEN) with 1% beta-mercaptoethanol. Plates were frozen immediately after sorting and stored at -80 ℃ prior to whole transcriptome amplification, library preparation and sequencing.
Libraries from isolated single cells from mouse model were generated based on the Smart-seq2 protocol (Picelli 2014) with the following modifications. RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter) before oligo-dT primed reverse transcription with Maxima reverse transcriptase and locked TSO oligonucleotide, which was followed by 20 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) with subsequent Agencourt AMPure XP bead purification as described. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina) with custom barcode adapters (sequences available upon request). Libraries from 768 cells with unique barcodes were combined and sequenced with paired-end, 38-base reads, using a NextSeq 500 sequencer (Illumina). Comma-seperated file containing TPM values (transcript-per-million reads) for 6608 genes across 679 cells. For the cell lines, scRNA-seq libraries were generated using the 10X Genomics Chromium Single Cell 3’ Kit v2 and the 10x Chromium Controller (10x Genomics) according to the 10X Single Cell 3’ v2 protocol. The cell lines were processed as a pool that consisted of 6 total cell lines, 4 of which were used in this study (MGG23, MGG75, MGH143, MGG18). Two of the used cell lines, MGG23 and MGH143 were profiled again in a different pool. Briefly, we generated a single cell suspension of the pool in 0.04% PBS-BSA (95% viability) and loaded approximately 10,500 single cells to the Chromium Controller with a targeted recovery of 6,000 cells. Single cells, reagents and single gel beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized droplets and subjected to reverse transcription. Droplets were broken and the barcoded cDNAs were purified with DynaBeads and amplified by 12 cycles of PCR (98°C for 45 s; [98°C for 20 s, 67°C for 30 s, 72°C for 1 min] x 12; 72°C for 1 min). The amplified cDNA was fragmented, end-repaired, ligated with index adaptors, and size-selected with clean-ups between each step using the SPRIselect Reagent Kit (Beckman Coulter). Quality control of the resulting barcoded libraries was performed with the Agilent TapeStation and by PCR with primers specific to the P5 and P7 sequence (NEBNext Library Quant Kit for Illumina, New England Biolabs).
single cell RNA-seq


title: OSM4.2_H05_S761


title: OSM4.2_G12_S756


title: OSM4.2_G10_S754


title: OSM4.2_H04_S760


title: OSM4.2_H12_S768


title: OSM4.2_G09_S753


title: OSM4.2_H11_S767


title: OSM4.2_H10_S766


title: OSM4.2_H03_S759


title: OSM4.2_H09_S765


title: EC14 C


title: OSM4.2_G08_S752


title: OSM4.2_H02_S758


title: OSM4.2_H07_S763


title: OSM4.2_H06_S762


title: OSM4.2_G11_S755


title: OSM4.2_G07_S751


title: OSM4.2_G06_S750


title: OSM4.2_G02_S746


title: OSM4.2_F11_S743


title: OSM4.2_F12_S744


title: OSM4.2_G01_S745


title: OSM4.2_F10_S742


title: OSM4.2_G05_S749


title: OSM4.2_G03_S747


title: OSM4.2_G04_S748


title: OSM4.2_F09_S741


title: OSM4.2_F08_S740


title: OSM4.2_H08_S764


title: OSM4.2_F03_S735


title: OSM4.2_F02_S734


title: OSM4.2_F05_S737


title: OSM4.2_F06_S738


title: OSM4.2_F04_S736


title: OSM4.2_F07_S739


title: OSM4.2_E12_S732


title: OSM4.2_E09_S729


title: OSM4.2_E07_S727


title: OSM4.2_E08_S728


title: OSM4.2_E10_S730


title: OSM4.2_E03_S723


title: OSM4.2_E04_S724


title: OSM4.2_E01_S721


title: OSM4.2_E02_S722


title: OSM4.2_E05_S725


title: OSM4.2_D09_S717


title: OSM4.2_D07_S715


title: OSM4.2_D08_S716


title: OSM4.2_D10_S718


title: OSM4.2_D05_S713


title: OSM4.2_D12_S720


title: OSM4.2_D11_S719


title: OSM4.2_D03_S711


title: OSM4.2_D02_S710


title: OSM4.2_D04_S712


title: OSM4.2_F01_S733


title: OSM4.2_E11_S731


title: OSM4.2_E06_S726


title: OSM4.2_D06_S714


title: OSM4.2_D01_S709


title: OSM4.2_C12_S708


title: OSM4.2_C10_S706


title: OSM4.2_C11_S707


title: OSM4.2_C09_S705


title: OSM4.2_C08_S704


title: OSM4.2_C07_S703


title: OSM4.2_C05_S701


title: OSM4.2_C06_S702


title: OSM4.2_B10_S694


title: OSM4.2_C02_S698


title: OSM4.2_C04_S700


title: OSM4.2_C03_S699


title: OSM4.2_C01_S697


title: OSM4.2_B05_S689


title: OSM4.2_B09_S693


title: OSM4.2_B06_S690


title: OSM4.2_B04_S688


title: OSM4.2_B12_S696


title: OSM4.2_B03_S687


title: OSM4.2_B02_S686


title: OSM4.2_B01_S685


title: Common mushroom fruiting body (Sample3828)

organism: Agaricus bisporus

source_name: fruiting body

value: fruiting body


title: African clawed frog fibroblast (XTC) (Sample4078)

organism: Xenopus laevis

source_name: Xenopus laevis tadpole-derived fibroblast line (XTC)


title: SSTP2

city: Trivandrum

first: Tessy

middle: Thomas

last: Maliekal

state: Kerala

address: Poojappura

country: India

institute: Rajiv Gandhi Centre for Biotechnology

data_processing:
mRNA-Sequencing libraries were prepared using NEBNextR Ultra™ II RNA Library PrepKit for Illumina Hiseq X Ten.   following the manufacturer’s instructions. The sequence data quality was checked using FastQC and MultiQC software. The QC passed reads were mapped onto indexed Human reference genome (GRCh38.p7) using TopHat2 and were annotated with Human GTF (GRCh38) downloaded from GENCODE. Transcripts were assembled using Cufflinks and were merged together using the Cuffmerge. Cuffcompare was used to compare the assembled transcripts with GENCODE annotation. Differential gene expression analysis was carried out using Cuffdiff.
Genome_build: GRCh38.p7
Supplementary_files_format_and_content: differential expression

growth_protocol: HSC4 cells were grown overnight in DMEM medium containing 10% FBS

extract_protocol:
RNA was isolated from the cells using RNeasy mini kit (Qiagen) as per the manufacturer’s instruction .
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: All cells were incubated overnight at 37°C in 5% CO2, were treated with peptide SSTP1 (10µm) and SSTP2 (10µm) in DMEM with 0.5% FBS for three hours.


title: SSTP1


title: OSM4.2_A11_S683


title: OSM4.2_A10_S682


title: OSM4.2_A09_S681


title: OSM4.2_B08_S692


title: OSM4.2_A07_S679


title: OSM4.2_A01_S673


title: OSM4.2_A06_S678


title: OSM4.1_H12_S576


title: OSM4.2_A05_S677


title: OSM4.2_A04_S676


title: OSM4.1_H11_S575


title: OSM4.1_H08_S572


title: OSM4.1_H07_S571


title: OSM4.1_H09_S573


title: OSM4.2_A08_S680


title: OSM4.2_A02_S674


title: OSM4.2_A03_S675


title: OSM4.1_H06_S570


title: OSM4.1_H10_S574


title: OSM4.1_H05_S569


title: OSM4.1_H03_S567


title: OSM4.1_H02_S566


title: OSM4.1_H04_S568


title: OSM4.1_H01_S565


title: OSM4.1_G12_S564


title: OSM4.1_G11_S563


title: OSM4.1_G09_S561


title: OSM4.1_G08_S560


title: OSM4.1_G06_S558


title: OSM4.1_G07_S559


title: OSM4.1_G03_S555


title: OSM4.1_F11_S551


title: OSM4.1_G02_S554


title: OSM4.1_F10_S550


title: OSM4.1_F12_S552


title: OSM4.1_G05_S557


title: OSM4.1_G01_S553


title: OSM4.2_B07_S691


title: OSM4.1_F07_S547


title: OSM4.1_F05_S545


title: OSM4.1_G04_S556


title: OSM4.1_F09_S549


title: OSM4.1_F04_S544


title: OSM4.1_F06_S546


title: OSM4.1_F08_S548


title: OSM4.1_F03_S543


title: OSM4.1_G10_S562


title: OSM4.1_F01_S541


title: OSM4.1_E11_S539


title: OSM4.1_F02_S542


title: OSM4.1_E12_S540


title: OSM4.1_E09_S537


title: OSM4.1_E08_S536


title: OSM4.1_E07_S535


title: OSM4.1_E06_S534


title: OSM4.1_E05_S533


title: OSM4.1_E04_S532


title: OSM4.1_E02_S530


title: OSM4.1_E03_S531


title: OSM4.1_D12_S528


title: OSM4.1_D11_S527


title: OSM4.1_D09_S525


title: OSM4.1_D03_S519


title: OSM4.1_E10_S538


title: OSM4.1_C11_S515


title: OSM4.1_D07_S523


title: OSM4.1_D02_S518


title: OSM4.1_C10_S514


title: OSM4.1_D08_S524


title: OSM4.1_D05_S521


title: OSM4.1_C09_S513


title: OSM4.1_D01_S517


title: OSM4.1_E01_S529


title: OSM4.1_C08_S512


title: OSM4.1_C07_S511


title: OSM4.1_C05_S509


title: OSM4.1_C06_S510


title: OSM4.1_C12_S516


title: OSM4.1_C04_S508


title: OSM4.1_C03_S507


title: OSM4.1_D06_S522


title: OSM4.1_C02_S506


title: OSM4.1_C01_S505


title: OSM4.1_D04_S520


title: OSM4.1_B12_S504


title: OSM4.1_B10_S502


title: OSM4.1_B11_S503


title: OSM4.1_B09_S501


title: OSM4.1_B08_S500


title: OSM4.1_B06_S498


title: OSM4.1_B07_S499


title: OSM4.1_B05_S497


title: OSM4.1_B03_S495


title: OSM4.1_B01_S493


title: OSM4.1_B02_S494


title: OSM4.1_A08_S488


title: OSM4.1_B04_S496


title: OSM4.1_A09_S489


title: OSM4.1_A11_S491


title: OSM4.1_A06_S486


title: OSM4.1_A07_S487


title: OSM4.1_A02_S482


title: OSM4.1_A03_S483


title: OSM4.1_A04_S484


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title: CTR2.1_A08_S8


title: CTR2.1_B09_S21


title: CTR2.1_B07_S19


title: CTR2.1_A05_S5


title: CTR2.1_B04_S16


title: CTR2.1_B10_S22


title: CTR2.1_B08_S20


title: CTR2.1_B03_S15


title: CTR2.1_A11_S11


title: CTR2.1_A09_S9


title: CTR2.1_A12_S12


title: CTR2.1_A01_S1


title: CTR2.1_A07_S7


title: TH_OSM1_0239_H10

source_name: Glioblastoma single cell


title: TH_OSM1_0239_H08


title: TH_OSM1_0239_H12


title: CTR2.1_A04_S4


title: TH_OSM1_0239_H06


title: TH_OSM1_0239_H05


title: CTR2.1_A02_S2


title: CTR2.1_A06_S6


title: TH_OSM1_0239_H03


title: CTR2.1_A03_S3


title: TH_OSM1_0239_G12


title: TH_OSM1_0239_H02


title: TH_OSM1_0239_G10


title: TH_OSM1_0239_H01


title: TH_OSM1_0239_G08


title: TH_OSM1_0239_H09


title: TH_OSM1_0239_G05


title: TH_OSM1_0239_G06


title: TH_OSM1_0239_H11


title: TH_OSM1_0239_H07


title: TH_OSM1_0239_G11


title: TH_OSM1_0239_G04


title: TH_OSM1_0239_G07


title: TH_OSM1_0239_G03


title: TH_OSM1_0239_G02


title: TH_OSM1_0239_F11


title: CTR2.1_B01_S13


title: TH_OSM1_0239_G01


title: TH_OSM1_0239_F12


title: TH_OSM1_0239_H04


title: TH_OSM1_0239_F10


title: TH_OSM1_0239_F09


title: TH_OSM1_0239_F08


title: TH_OSM1_0239_F06


title: TH_OSM1_0239_E12


title: TH_OSM1_0239_E06


title: TH_OSM1_0239_F03


title: TH_OSM1_0239_E07


title: TH_OSM1_0239_F04


title: TH_OSM1_0239_F07


title: TH_OSM1_0239_G09


title: TH_OSM1_0239_F05


title: TH_OSM1_0239_E08


title: TH_OSM1_0239_E04


title: TH_OSM1_0239_E05


title: TH_OSM1_0239_E02


title: TH_OSM1_0239_F02


title: TH_OSM1_0239_F01


title: TH_OSM1_0239_E03


title: TH_OSM1_0239_E10


title: TH_OSM1_0239_E11


title: TH_OSM1_0239_E09


title: TH_OSM1_0239_E01


title: TH_OSM1_0239_D10


title: TH_OSM1_0239_D12


title: TH_OSM1_0239_D09


title: TH_OSM1_0239_D11


title: TH_OSM1_0239_D05


title: TH_OSM1_0239_D06


title: TH_OSM1_0239_D04


title: TH_OSM1_0239_D08


title: TH_OSM1_0239_D07


title: TH_OSM1_0239_C01


title: TH_OSM1_0239_B12


title: TH_OSM1_0239_B11


title: TH_OSM1_0239_B08


title: TH_OSM1_0239_B06


title: TH_OSM1_0239_B10


title: TH_OSM1_0239_B04


title: TH_OSM1_0239_B09


title: TH_OSM1_0239_C06


title: TH_OSM1_0239_C12


title: TH_OSM1_0239_C09


title: TH_OSM1_0239_D02


title: TH_OSM1_0239_D01


title: TH_OSM1_0239_C08


title: TH_OSM1_0239_B05


title: TH_OSM1_0239_D03


title: TH_OSM1_0239_C11


title: TH_OSM1_0239_C02


title: TH_OSM1_0239_C03


title: TH_OSM1_0239_C07


title: TH_OSM1_0239_C10


title: TH_OSM1_0239_C04


title: TH_OSM1_0239_B02


title: TH_OSM1_0239_B03


title: TH_OSM1_0239_C05


title: TH_OSM1_0239_A12


title: TH_OSM1_0239_B07


title: TH_OSM1_0239_A10


title: TH_OSM1_0239_A11


title: TH_OSM1_0239_A09


title: TH_OSM1_0239_A06


title: TH_OSM1_0239_A08


title: TH_OSM1_0239_A04


title: TH_OSM1_0239_A05


title: TH_OSM1_0239_B01


title: TH_OSM1_0239_A03


title: TH_CTR_0239_H09


title: TH_CTR_0239_H12


title: TH_CTR_0239_H10


title: TH_OSM1_0239_A02


title: TH_CTR_0239_H05


title: TH_CTR_0239_H11


title: TH_CTR_0239_H02


title: TH_OSM1_0239_A01


title: TH_CTR_0239_H08


title: TH_CTR_0239_H01


title: TH_CTR_0239_H06


title: TH_CTR_0239_H03


title: TH_CTR_0239_H04


title: TH_CTR_0239_H07


title: TH_CTR_0239_G12


title: TH_CTR_0239_G08


title: TH_CTR_0239_G09


title: TH_CTR_0239_G11


title: TH_CTR_0239_G07


title: TH_OSM1_0239_A07


title: TH_CTR_0239_G06


title: TH_CTR_0239_G02


title: TH_CTR_0239_G03


title: TH_CTR_0239_G04


title: TH_CTR_0239_G01


title: TH_CTR_0239_G05


title: TH_CTR_0239_F11


title: TH_CTR_0239_F10


title: TH_CTR_0239_F04


title: TH_CTR_0239_F07


title: TH_CTR_0239_F08


title: TH_CTR_0239_F03


title: TH_CTR_0239_F05


title: TH_CTR_0239_F02


title: TH_CTR_0239_F06


title: TH_CTR_0239_F09


title: TH_CTR_0239_F01


title: TH_CTR_0239_E12


title: TH_CTR_0239_E10


title: TH_CTR_0239_E11


title: TH_CTR_0239_G10


title: TH_CTR_0239_E08


title: TH_CTR_0239_E09


title: TH_CTR_0239_E07


title: TH_CTR_0239_E06


title: TH_CTR_0239_E05


title: TH_CTR_0239_E04


title: TH_CTR_0239_E03


title: TH_CTR_0239_E02


title: TH_CTR_0239_F12


title: TH_CTR_0239_E01


title: TH_CTR_0239_D12


title: TH_CTR_0239_D11


title: TH_CTR_0239_D08


title: TH_CTR_0239_D10


title: TH_CTR_0239_D09


title: TH_CTR_0239_D06


title: TH_CTR_0239_D07


title: TH_CTR_0239_D04


title: TH_CTR_0239_D05


title: TH_CTR_0239_D01


title: TH_CTR_0239_C12


title: TH_CTR_0239_D02


title: TH_CTR_0239_C08


title: TH_CTR_0239_C11


title: TH_CTR_0239_C06


title: TH_CTR_0239_C03


title: TH_CTR_0239_C05


title: TH_CTR_0239_C02


title: TH_CTR_0239_B10


title: TH_CTR_0239_C04


title: TH_CTR_0239_B11


title: TH_CTR_0239_C01


title: TH_CTR_0239_C09


title: TH_CTR_0239_B08


title: TH_CTR_0239_D03


title: Fruit fly PnM cell line (SAMN10242997)

organism: Drosophila melanogaster

source_name: PnM cells

value: PnM cells


title: TH_CTR_0239_B09


title: TH_CTR_0239_C10


title: TH_CTR_0239_C07


title: TH_CTR_0239_B06


title: TH_CTR_0239_B05


title: TH_CTR_0239_B07


title: TH_CTR_0239_A10


title: TH_CTR_0239_B03


title: TH_CTR_0239_A12


title: TH_CTR_0239_A08


title: TH_CTR_0239_B12


title: TH_CTR_0239_B01


title: TH_CTR_0239_A05


title: TH_CTR_0239_A07


title: TH_CTR_0239_A11


title: TH_CTR_0239_B04


title: TH_CTR_0239_A04


title: Lenti_OSM_2_Day7_H09


title: Lenti_OSM_2_Day7_H10


title: Lenti_OSM_2_Day7_H12


title: Lenti_OSM_2_Day7_H11


title: TH_CTR_0239_B02


title: TH_CTR_0239_A01


title: TH_CTR_0239_A02


title: total thymic iNKT 1

first: Juliane

last: Lohmeyer

address: 269 W. Campus Drive

description: single cell RNA-seq

data_processing:
scRNAseq: Sample demultiplexing, barcode processing, alignment to the mm10/GRCm38 reference genome, and single-cell 3′ gene counting were performed using the Cell Ranger Suite version 2.1. scRNA-Seq analysis was performed using the Seurat R package, version 3.
Bulk RNAseq on iNKT sublineages: Paired-end reads were aligned to the mm10 genome (GRCm38 v90) using STAR v. 2.5.4, and genes quantified using HTSEQ24. Differential gene expression was performed using the DESeq2 version 1.22.2. To generate the genome tracks, we used samtools to make stranded BAM files for each library from the SAM file. The BAM file was used to generate a bedGraph using the genomeCoverageBed command. BedGraph from multiple replicates were aggregated using bedtoolsunionbedg, and each track was normalized to the same sequencing depth of 30 million reads. The averaged bedGraph were converted to BigWig files using the command bedGraphToBigWig. Statistics from the mapped reads were aggregated using MultiQC.
ATACseq: Sequencing reads were processed using the ENCODE ATAC-seq pipeline (https://github.com/kundajelab/atac-seq-pipeline) with default parameter to detect and removes adapters, align reads to the specified mm10 reference genome using Bowtie2, and subsequently remove unmapped, mate unmapped, multimapped, mitochondrial mapped, and PCR duplicate reads. Peaks are called on replicates using MACS2. Reads overlapping consensus peaks were quantified for each cell subset replicate using bedtools version 2.27.1. Subset enriched peaks from the consensus set were determined with DESeq2 Consensus peak region annotations with nearest gene were obtained using HOMER version 4.9.1. Gene coordinates for genes-of-interest were queried using biomaRt version 2.34.2 using mmusculus_gene_ensembl for mm10 (GRCm38.6). Transcription factor motifs enrichment were identified using HOMER version 4.9.1.
Bulk RNAseq on Tcon post-transplantation: Sequencing reads were checked using FastQC v.0.11.7. Estimated transcript counts and transcripts per million (TPM) for the mouse genome assembly GRCm38 (mm10) were obtained using the pseudo-aligner Kallisto. Transcript-level abundance was quantified and summarized into gene level using the tximport R package. Differential gene expression was performed using the DESeq2 R package version 1.22.221.
Genome_build: mm10
Supplementary_files_format_and_content: all processed files: csv, raw counts

source_name: Thymus

extract_protocol:
Bulk RNA-seq of iNKT sublineages: RNA was extracted from freshly FACS-sorted iNKT sublineages using the RNeasy mini kit (Qiagen) after pelleting 10k-50k cells, ATAC-seq: Lysis was performed in 100 μl cold buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 + 0.1% IGEPAL CA-630 + 0.1% Tween 20), and transposition was performed in 50 μl buffer containing 25 μL 2× TD buffer (Illumina #FC-121-1030), 2.5 μL Tn5 transposase (Illumina #FC-121-1030), 17.5 μL nuclease free H2O, and 5µl 1% digitonin. Bulk RNA-seq of conventional T cells after HCT: RNA was extracted using the TRIzol RNA isolation method (ThermoFisher Scientific) combined with the RNeasy MinElute Cleanup (Qiagen).
Single-cell libraries were generated using the Chromium Controller Single-Cell Instrument and Chromium Single Cell 3′ Library & Gel Bead Kit v2 (10x Genomics). Bulk cDNA libraries of sorted iNKT sublineages were prepared using the Truseq stranded mRNA kit (Illumina) and pooled for sequencing on the Illumina HiSeq 4000 platform (75 bp, paired-end). ATAC seq was performed as previously described (Buenrostro et al., Nat. Methods 2013), following the reommended library preparation protocol and sequencing on and Illumina HiSeq 4000. Bulk RNA-seq of conventional T cells after HCT: Full-length cDNA was generated using the Clontech SMARTer v4 kit (Takara Bio USA, Inc., Mountain View, CA) prior to library generation with the Nextera XT DNA Library Prep kit (Illumina, Inc., San Diego, CA). Libraries were pooled for sequencing on the Illumina HiSeq 4000 platform (75 bp, paired-end).
RNA-seq, ATAC-seq

value: invariant natural killer T cell

value: FVB/N


title: total thymic iNKT 2

description: single cell RNA-seq


title: Lenti_OSM_2_Day7_H07


title: Lenti_OSM_2_Day7_H06


title: TH_CTR_0239_A06


title: JL042 iNKT17 I CD8  d7

description:
bulkRNA-seq
BulkRNAseq_Tcon_PrePostTx_RawCounts.csv

source_name: spleen, peripheral lymph nodes

value: conventional T cells

value: GVHD, iNKT17


title: TH_CTR_0239_A09


title: Lenti_OSM_2_Day7_H01


title: Lenti_OSM_2_Day7_G11


title: Lenti_OSM_2_Day7_H05


title: Lenti_OSM_2_Day7_H02


title: TH_CTR_0239_A03


title: Lenti_OSM_2_Day7_H03


title: Lenti_OSM_2_Day7_H04


title: JL041 iNKT17 III CD4 d7


title: Lenti_OSM_2_Day7_G12


title: JL041 iNKT17 III CD8 d7


title: Lenti_OSM_2_Day7_G09


title: JL042 iNKT2 I CD8 d7

value: GVHD, iNKT2


title: JL041 iNKT17 I CD8 d7


title: JL041 iNKT17 I CD4 d7


title: JL042 iNKT17 I CD4 d7


title: JL042 iNKT2 I CD4 d7


title: JL041 iNKT2 II CD8 d7


title: Lenti_OSM_2_Day7_G08


title: Lenti_OSM_2_Day7_G10


title: JL041 iNKT2 I CD4 d7


title: JL042 iNKT1 I CD8 d7

value: GVHD, iNKT1


title: Lenti_OSM_2_Day7_G06


title: JL041 iNKT2 II CD4 d7


title: JL041 iNKT2 I CD8 d7


title: Lenti_OSM_2_Day7_G05


title: JL041 iNKT1 II CD8 d7


title: JL042 iNKT1 I CD4 d7


title: Lenti_OSM_2_Day7_G04


title: Lenti_OSM_2_Day7_G07


title: JL041 iNKT1 II CD4 d7


title: Lenti_OSM_2_Day7_G02


title: JL041 iNKT1 I CD8 d7


title: Lenti_OSM_2_Day7_G03


title: Lenti_OSM_2_Day7_H08


title: Lenti_OSM_2_Day7_F09


title: Lenti_OSM_2_Day7_F12


title: JL041 iNKT1 I CD4 d7


title: Lenti_OSM_2_Day7_F10


title: JL042 Tcon I CD4  d7

value: GVHD, untreated


title: JL042 Tcon I CD8 d7


title: Lenti_OSM_2_Day7_F07


title: JL041 Tcon I CD8 d7


title: JL041 Tcon II CD4 d7


title: JL042 CD8 Tcon d0

value: pre-transplant