title: HIVE_MultiLin_2 RNA

description: HIVE Captures

data_processing:
Cells were multiplexed using HTOs to distinguish CITE-Seq ADT concentration or cell population. HTO barcode count matrices were obtained through the multimodal analysis workflow in Cell Ranger, prior to normalization (counts per ten thousand (CPTT)). Cell barcodes with > 30% of normalized reads assigned to multiple HTOs were annotated as doublets, with confident singlet assigned to cells with >40% of normalized reads assigned to a single HTO (HTO processing module of AltAnalyze). Cells were further filtered based on the 7 mouse/rat isotype control antibodies counts (Source code) and performed quality control filtering in Seurat V4 by nFeature_RNA>500 & nCount_RNA >1000 & percent.mt<25. This QC step filtered 393,748 cells to 315,792 high-quality single cells in the initial titration dataset and 90,889 to 72,198 cells in the final titrated CITE-Seq dataset.All Cell Ranger produced count matrices underwent ambient RNA exclusion using the software SoupX with a contamination fraction of 15% and quality control filtering by HTO. Ambient corrected transcriptome counts and associated ADTs counts were supplied as input to the software TotalVI to obtain normalized and denoised ADT counts. The Honeycomb HIVE CLX capture system (discountinued, Honeycomb Biotechnologies, Inc) was applied using the manufacturer’s instructions to the following populations: CD117-enriched bone marrow, sorted cells from basophil/mast enriched gates, and those from eosinophil enriched gates. Briefly, 30,000 cells of each population were loaded per HIVE by centrifugation and processed immediately without freezing. Libraries were quality checked and then pooled for sequencing on a Novaseq 6000 SP flow cells for PE50 using customized sequencing and indexing primers. Cells were called based UMI per cell knee plot and gene count matrix was generated by aligning to mm10-v2 for downstreatm analysis.
Assembly: mm10
Supplementary files format and content: .txt.gz count matrix

value: scRNA-Seq


title: 181121_cochlea_2C

status: Public on Oct 21 2025

type: SRA

city: Boston

first: Erin

middle: Elizabeth

last: Duffy

state: MA

address: 200 Longwood Ave, Goldenson 405

department: Neurobiology

country: USA

institute: Harvard Medical School

description: Library name: 181121_cochlea_2C

library_source: genomic

data_processing:
All experiments were sequenced on the NextSeq 500 (Illumina). Seventy-five bp paired-endreads were obtained for all datasets. ATAC-seq experiments were sequenced to a minimumdepth of 20 million reads. All samples were aligned to the mm10 genome using default parameters for the Subreadalignment software (subread-1.4.6-p3) (3) after quality trimming with Trimmomatic v0.33(4) with the following command: java -jar trimmomatic-0.33.jar SE -threads 1 -phred33 [FASTQ_FILE] ILLUMINACLIP:[ADAPTER_FILE]:2:30:10 LEADING:5TRAILING:5 SLIDINGWINDOW:4:20 MINLEN:45. Nextera adapters were specified for ATAC-seq data.
Putative cochlear GREs (regions of ATAC-seq enrichment) were identified as described previously. Briefly, peaks were determined using MACS2 (v 2.1.0) parameters -p 1e- 5–nolambda – keep-dup all – slocal 10000, as previously described 29 . Peaks from individual sample replicates were intersected to find only reproducible regions of enrichment. Blacklist regions were removed as previously described. To identify sites with enriched ATAC-seq signal (peaks), we applied the IDR pipeline using the MACS2 peak calling algorithm 60 with the following parameters: –nomodel –extsize 200 –keep-dup all. An IDR threshold of 0.01 was used for self- consistency, true replicate, and pooled-consistency analyses. The ‘optThresh’ cutoff was then used to obtain a final set of high-confidence, reproducible ATAC-seq peaks for each sample.
Assembly: mm10

molecule: genomic DNA

organism: Mus musculus

source_name: COCHLEA

extract_protocol:
Neonatal (P2, n=3; P5, n=3; P8, n=4) CD1 mice were sacrificed and cochleas were dissected bilaterally as described above. All tissue from each animal was combined and dounced 10 times in 5 ml of HB buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl 2 , 20 mM Tricine-KOH pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, DTT 1 mM [Sigma], one-half Roche proteinase inhibitor tablet) in a 7-ml Dounce homogenizer with tight pestle, then supplemented with IGEPAL CA-630 to final concentration of 0.3%, and finally dounced an additional five times with the tight pestle. Nuclei were filtered through a 40-μm strainer and gently combined 1:1 with 50% iodixanol. Nuclei were then underlaid with a gradient of 1 ml 40% iodixanol and 1 ml 30% iodixanol and centrifuged at 10,000g for 19 minutes using the no brake setting. Purified nuclei (0.8 ml) were then collected from the 30/40% interface. A 20-μl aliquot was taken for visual inspection and cell counting, with typical yield of ~100,000-200,000 cochlear nuclei per animal.
ATAC-seq (assay for transposase accessible chromatin with sequencing) was performed using the OMNI-ATAC protocol 58 immediately following the nuclear isolation steps described above. Nuclei (50,000) were pelleted at 500 g for 10 minutes at 4°C in a fixed angle centrifuge and resuspended in 50 μl transposition buffer (prepared from Illumina kit components TD buffer and 100 nM transposase, as well as 0.01% digitonin, 0.1% Tween-20) via careful pipetting. Transposition occurred at 37°C in a thermomixer at 1000 rpm for 30 minutes before resuspension in 250 μl of DNA binding buffer (Zymo DNA Clean and Concentrator Kit) and reaction cleanup per manufacture instructions, with final elution volume of 21 μl in elution buffer. Samples were amplified following NEBNext High-Fidelity 2X PCR Master Mix manufacturer protocols (NEB) in a total reaction volume of 50 μl (1.25 μM each of i5 and i7 sequencing primers) at 72°C for 5 minutes, 98°C for 30 seconds, (98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute) x10 cycles. PCR samples were re-purified following the Zymo manufacturer protocol and eluted into 21 μl of elution buffer. Size selection was performed via agarose gel (150-1000 bp) followed by Qiagen MinElute gel extraction according to the manufacturer’s instructions, with two serial 10 μl elution steps. Sequencing (75-bp paired-end) was performed on an Illumina NextSeq500.

tag: tissue

value: COCHLEA

tag: genotype

tag: post-natal age_(days)


title: p22179-s013_227438-05-Mar-2021

first: Dan

last: Barouch

address: 3 Blackfan Cir

department: CVVR

institute: Beth Israel Deaconess Medical Center

library_source: transcriptomic

data_processing:
Alignment was performed using STAR version 2.7.3a and transcripts were annotated using a composite genome reference which included GRCh38 patch 14, and SARS-CoV-2 (GCF_009858895.2,ASM985889v3,MN985325.1).
Transcript abundance estimates were calculated internal to the STAR aligner using the algorithm of htseq-count.
DESeq2 was used for normalization, producing a normalized read count.
Assembly: Genome Reference Consortium Human Build 38 patch release 14 (GRCh38.p14), SARS-CoV-2 Reference Genome (RefBank Accession: GCF_009858895.2)
Supplementary files format and content: PASC.ProcessedData.CountMatrix.Human.csv (read count matrix)

molecule: total RNA

organism: Homo sapiens

source_name: PBMC

extract_protocol:
PBMCs were lysed in 700 mL of Trizol and then extracted using the miRNeasy Mini kit (Qiagen) with on-column DNase digestion.
RNA quality was assessed using a TapeStation 4200 (Agilent) and then ten nanograms of total RNA was used as input for cDNA synthesis using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) according to the manufacturer’s instructions. Amplified cDNA was fragmented and appended with dual-indexed barcodes using the Nextera XT DNA Library Preparation kit (Illumina).
Libraries were validated by capillary electrophoresis on a TapeStation 4200 (Agilent), pooled at equimolar concentrations, and sequenced with PE100 reads on an Illumina NovaSeq 6000, yielding ~30 million reads per sample on average.

tag: cell type

value: PBMC

tag: gender

value: Female

tag: ethnicity

value: Not Hispanic or Latino

tag: age

tag: vaccine product

value: Moderna

tag: gcid

tag: subject id

tag: timepoint

tag: pasc status

value: PASC


title: cKit+CD127+ cell 77

city: Cincinnati

first: Nathan

last: Salomonis

state: OH

address: 3333 Burnet Avenue

department: Biomedical Informatics

institute: Cincinnati Children's Hospital

description:
Il7r.Cd117.Cell_77
Fluidigm C1

data_processing:
Data analyses were performed as in PMID: 32494068.
Assembly: mm10
Supplementary files format and content: RSEM transcript per million estimates text file

source_name: Bone Marrow

extract_protocol:
Fluidigm C1 captures were performed as described in PMID: 32494068.
Library preperation was performed as described in PMID: 32494068.
Library strategy was performed as described in PMID: 32494068.

value: Bone Marrow

value: cKit+CD127+ cell

value: WT

tag: treatment

tag: protocol

value: paired-end


title: p22179-s012_227438-07-Apr-2021


title: 181121_cochlea_2A

description: Library name: 181121_cochlea_2A


title: HIVE_MultiLin_1 RNA

description: HIVE Captures

extract_protocol:
CITE-seq was performed with 3 different ADT panel configurations: (1) a customized 65-plex cocktail, (2) a prototype 200-plex cocktail for antibody titration (cat number), and (3) a titrated 110-plex cocktail (cat number). Each configuration used TotalSeq-A antibodies purchased from BioLegend (Supplmentary Table X). The 65-plex cocktail was comprised of known hematopoietic stem and progenitor markers as an initial test of the technology, using CD117-enriched mouse bone marrow as input and applying antibodies at the concentrations recommended by BioLegend. The 200-plex cocktail was provided as a lyophilized mix, uniform for each TotalSeq antibody, which was rehydrated at 8x concentration and subsequently diluted to five concentrations: 4x, 2x, 1x, 0.5x, and 0.25x. 1x concentration was determined based on the performance of ADTs in the 65-plex panel or according manufacturer’s recommendation. Five sorted populations were profiled with the 200-plex panel to comprehensively represent rare cell populations in the marrow: total bone marrow cells, CD117-enriched bone marrow cells, FACS sorted HSC/MPP cells (Lin- CD117+ Sca1+ and exclusive of CD48+ CD150-), FACS sorted Multi-Lin cells (Lin- CD117+ CD34+ CD16/32+/low CD115- CD11b- Ly6C- CD150-), and FACS sorted CD127+ (Lymphoid) cells. An unique HTO antibody was added to denote each titration concentration. Same cell population stained with different titration concentrations were pooled together after washing seperately. For the titrated 110-plex panel, a customized lyophilized cocktail was created by BioLegend using selected concentrations from the 200-plex titration experiment. The titrated cocktail was applied to the same five populations except for total bone marrow cells. In addition, each sorted cell population from various gates were stained with an unique HTO, washed and pooled. In each experiment, cells were washed on Laminar Wash MINI or HT2000 System (Curiox Biosystems Inc) with the following settings: 25 cycles, flow rate of 10uL/s, initial volume of 55uL. Pooled cells were captured by 10X Chromium X 3’ version 3.1 kit (1000268, 10X Genomics). TEA-seq was performed on the same groups of cells profiled by CITE-seq following it’s published (reference eLIFE TEA-seq paper) protocol with minor modifications: (1) using a digitonin concentration of 0.025% based on optimization results, (2) the addition of protector RNAse inhibitor (Roche 3335402001) in perm, wash and tagmentation buffers. (3) 80ul instead of 160ul supernant from cDNA amplifications were used to purify pre-amplified ADT. Briefly, 100,000 to 250,000 leftover cells from CITE-seq experiment were permeablized in digitonin perm buffer, washed and then processed as nuclei according to 10X Multiome (1000283, 10X Gemonics) protocol.
Library prep was performed according to protocols with minor modifications such as reduced PCR cycles for ADT and HTO library amplifications. We found pre-PCR ADT/HTO in our libraries were abundant enough that we generally reduced 2 cycles (ADT: 10-12 cycles, HTO: 9-10 cycles). Final transcriptome, ADT and HTO libraries were quantified and analyzed by Qubit dsDNA HS (1000268, Invitrogen), High Sensitivity DNA kit (5067-4626, Agilent Technologies) on 2100 bioanalyzer (G2939BA, Agilent Technologies) and KAPA HiFi library quantification kit (KK4824, Roche).
Dual-indexed transcriptome libraries were pooled and sequenced across multiple Illumina Novaseq 6000 S4 or X plus flow cells (Illumina) with PE150+10+10 or PE100+10+10 settings. Single indexed ADT and HTO libraries were pooled and sequenced on Illumina S2 flow cells with PE50+8 setting or with transcriptome libraries. BCL files were demultiplexed into fastq files for CellRanger input. “AT” was added to the end of RPI-x ADT i7 index (6bp) to match D70X_long HTO index (8bp). HTO and ADT FASTQ files were supplied as 3P feature barcode together with transcriptome FASTQ files into Cell Ranger V6.1.2 count pipeline. Transcriptome was mapped to mm10-v2 reference genomes for downstream analysis and visualization.


title: p22179-s017_243818-18-Mar-2021

value: Pfizer


title: p22179-s011_227438-07-Jan-2021


title: HIVE_Kit+_2 RNA

description: HIVE Captures


title: cKit+CD127+ cell 75

description:
Il7r.Cd117.Cell_75
Fluidigm C1


title: HIVE_Kit+_1 RNA

description: HIVE Captures


title: mESC Tet1/2/3 TKO, RIP, PyBo, clone 26

city: Mainz

first: Alexandr

last: Gopanenko

address: Ackermannweg 4

country: Germany

institute: Institute of Molecular Biology gGmbH (IMB)

description: Library name: FIBo_seq_pilot_no_rRNA_dep_3

data_processing:
Raw read data were quality-assessed by FastQC (v.0.11.9) and aligned to reference genome using STAR (v. 2.7.10a) with the following relaxed parameters to map short overlapping reads containing C-to-T mismatches: outFilterScoreMinOverLread 0.1, --outFilterMatchNminOverLread 0.1 and --outFilterMismatchNoverReadLmax 0.05.
deepTools (v. 3.5.1) was used to generate bigWig tracks.
Assembly: GRCm39 (mm39)
Supplementary files format and content: bigWig; tracks for RIP-seq experiment

source_name: mouse embryonic stem cells

growth_protocol: mESC clones were cultured on tissue culture plates coated with 0.1% gelatin solution in 2i medium (50% Neurobasal and 50% DMEM/F-12 medium, supplemented with 1x N2, 1x B27, 2 mM L-Glutamine, 1000 U/ml Leukemia inhibitory factor, 100 U/ml Pen-Strep, 1 µM PD0325901, 3 µM CHIR99021 and 50 µg/ml BSA) at 37 °C in 5% CO2 and 20% O2.

extract_protocol:
Total RNA was isolated from cultured cells using QIAzol Lysis Reagent (Qiagen) according to the manufacturer’s protocol followed by TURBO DNase (Invitrogen) treatment and another round of QIAzol purification. RNA was chemically fragmented to 120-180 nt-long fragments followed by immunoprecipitation with an anti-f5C-antibody (1 µg anti-f5C-antibody/ 5 µg RNA; #61227, Active Motif). Samples were incubated in 900 mM pyridine borane in 600 mM sodium acetate (pH 5.2) at 37°C for 24 hours. For mock treated samples, water was added instead of pyridine borane. RNA was T4 PNK (NEB) treated before library construction.
Library construction was done with the NEXTFLEX Small RNA-Seq Kit V3 (Bioo Scientific) following Step A to Step G of Bioo Scientific`s standard protocol (V19.01) using the NEXTFLEX 3’ SR Adaptor and 5’ SR Adaptor. Step A (NEXTFLEX 3` 4N Adenylated Adapter Ligation) was performed overnight at 20°C and for step F (Bead Cleanup) the modified protocol was used to retain small RNAs. Libraries were prepared with a starting amount of 1.4 ng RNA and amplified in 21 PCR cycles. Libraries were size-selected for 18 – 362 nt insert size.

value: mouse embryonic stem cells

value: Tet1/2/3 TKO


title: mESC Tet1/2/3 CTRL, RIP, mock, clone 6

description: Library name: FIBo_seq_pilot_no_rRNA_dep_2

value: Tet1/2/3 wildtype


title: cKit+CD127+ cell 80

description:
Il7r.Cd117.Cell_80
Fluidigm C1


title: p22179-s009_217829-16-Feb-2021


title: cKit+CD127+ cell 73

description:
Il7r.Cd117.Cell_73
Fluidigm C1


title: HIVE_EoP RNA

description: HIVE Captures

value: Eosinophil


title: mESC Tet1/2/3 CTRL, RIP, PyBo, clone 6

description: Library name: FIBo_seq_pilot_no_rRNA_dep_1


title: HIVE_BMCP RNA

description: HIVE Captures

value: BMCP


title: p22179-s008_217829-17-Nov-2020


title: mESC Tet1/2/3 TKO, mock, rRNA, clone 26

description:
Library name: sample_34
total RNA, rRNA-depleted

data_processing:
Raw read data were quality-assessed by FastQC (v.0.11.9, https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Alignment to the reference genome was performed using STAR (v. 2.7.10a) allowing soft-clipping with non-default parameters: --outFilterScoreMinOverLread 0.33, --outFilterMatchNminOverLread 0.33 and --outFilterMismatchNoverReadLmax 0.02, that ensure mapping of overlapping reads and allowed for up to 2% of mismatches. The reference (GRCm39.dna_sm.primary_assembly.fa) was taken from https://ftp.ensembl.org/pub/release-107 and the genome index was generated using M.mus.GRCm39.107.gtf for annotation and --sjdbOverhang 99 (read length - 1).
C-to-T events were identified using JACUSA2 (v.2.0.4) applying 2 conditions mode (call-2, PyBo/Mal vs mock), scanning only positions with coverage >5 reads (-c 5), minimum base quality > 20 (-q 20) and mapping quality > 20 (-m 20), taking into account rev-strand specificity of the libraries (P RF-FIRSTSTRAND) and using bam files with only uniquely mapped reads as an input. Shortly, to identify PyBo/Mal-induced C-to-T events for Tet1/2/3 or Alkbh1 CTRL samples, we applied the following filtering criteria for raw JACUSA2 output files: 1) JACUSA2 score >2, 2) C-to-T conversion level > 5% in each of 3 replicate of PyBo/Mal-treated sample, 3) ≥ 5 reads (average of 3 replicates) supporting mismatched T in PyBo/Mal-treated samples, and 4) C-to-T conversion level < 2% in each of 3 replicate of mock-treated samples, and less <5 reads (average of 3 replicates) supporting mismatched T in mock-treated samples. The C-to-T events that passed filtering were annotated with hiAnnotator (v. 1.34.0) using Mus_musculus.GRCm39.107.gtf as a gene model. In cases when the C-to-T site overlapped with multiple transcripts, we employed the following strategy to clarify the transcript annotation. Firstly, the gene with the correct orientation for the C-to-T site and the highest expression level was selected. Secondly, the transcript variant was selected with the following prioritization: (1) highest expression (2) marked as canonical and (3) the longest transcript variant. The transcript expression level was determined using kallisto (v. 0.44.0).
Assembly: GRCm39 (mm39)
Supplementary files format and content: bedGraph; shared C-to-T events observed in CTRL samples detected by PyBo-assisted RNA-seq

extract_protocol:
Total RNA was isolated from cultured cells using QIAzol Lysis Reagent (Qiagen) according to the manufacturer’s protocol followed by TURBO DNase (Invitrogen) treatment and another round of QIAzol purification.
Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit v2 (human/Mouse/Rat) (NEB) following the manufacturer’s protocol and purified using the RNA Clean & Concentrator kit (Zymo Research). For PyBo-seq, 200 ng of RNA was incubated in 900 mM pyridine borane in 600 mM sodium acetate (pH 5.2) at 37°C for 24 hours. For Mal-seq, 200 ng of RNA was incubated in 150 mM malononitrile in 1x TE buffer (pH 7.4) at 37°C for 24 hours. For mock treated samples, water was added instead of chemicals.
For PyBo-seq of ribosomal RNA, total RNA was chemically fragmented to 120-180 nt-long fragments and PyBo-treated as described above.
RNA library preparation was performed using the TruSeq Stranded mRNA Library Prep kit (20020594, Illumina), entering the protocol at the RNA fragmentation step and excluding the poly(A) enrichment. 10 ng RNA was used as starting amount. Libraries were size-selected for >100 bp fragments.
Library construction of rRNA was done with the NEXTFLEX Small RNA-Seq Kit V3 (Bioo Scientific) following Step A to Step G of Bioo Scientific`s standard protocol (V19.01) using the NEXTFLEX 3’ SR Adaptor and 5’ SR Adaptor. Step A (NEXTFLEX 3` 4N Adenylated Adapter Ligation) was performed overnight at 20°C and for step F (Bead Cleanup) the modified protocol was used to retain small RNAs. Libraries were prepared with a starting amount of 1.4 ng RNA and amplified in 21 PCR cycles. Libraries were size-selected for 18 – 362 nt insert size.

value: mock


title: p22179-s007_173385-03-Mar-2021


title: BM cells, TPO, GFP-, rep6

city: Basel

first: Jonathan

last: Seguin

address: Spitalstrasse 33

country: Switzerland

institute: UKBB-DBM

data_processing:
RNA-seq reads were mapped to the mouse genome assembly, version mm10, with STAR (versions 2.7.9) with default parameters except for using the multi-map settings (outFilterMultimapNmax=10 and outSAMmultNmax=1) and filtering reads without evidence in spliced junction table (outFilterType="BySJout").
The output was sorted and indexed with samtools (version 1.15).
Coverage tracks per sample were generated by tiling the genome in 10bp windows, counting overlapping reads per window using the function bamCount from the bioconductor package bamsignals (version 1.26) and exporting these window counts in bigWig format using the export function of the bioconductor package rtracklayer (version 1.54).
The featureCounts software of Subread (version 2.0.1) was used to count the number of read (5’ends) overlapping with the exons of each gene assuming an exon union model based on the genes annotation provided by Ensembl database (version 102).
Assembly: mm10
Supplementary files format and content: raw counts matrix with genes as rows and samples as columns
Supplementary files format and content: bigWig

source_name: Bone marrow

growth_protocol: iKMT2A-MLLT3;Evi1-GFP mice (CD45.2) were injected .i.p with TPO or PBS in single-dose. 2 days later, flow cytometry-sorted progenitor cells from these mice were tail-vein injected in lethally irradiated 8-10 weeks-old B67.SJL/Crl (CD45.1) mice with 2x106 supporting normal BM cells of B6.SJL/Crl donor mice.

extract_protocol:
Mice were sacrificed once symptomatic. Total RNA from flow sorted (GFP+, GFP- or bulk) bone marrow cells was isolated using the Quick-RNA Miniprep kit (Cat. R1054, Zymo Research).
Library preparation was performed with 15ng total RNA using the TruSeq stranded mRNA Library Kit. 10 cycles of PCR were performed.

treatment_protocol: Dox was provided in water (500mg/ml) or as impregnated food pellets corresponding to 1.2mg/day.

value: Bone marrow

value: TPO

tag: Sex

value: female


title: cKit+CD127+ cell 72

description:
Il7r.Cd117.Cell_72
Fluidigm C1


title: cKit+CD127+ cell 71

description:
Il7r.Cd117.Cell_71
Fluidigm C1


title: cKit+CD127+ cell 76

description:
Il7r.Cd117.Cell_76
Fluidigm C1


title: mESC Tet1/2/3 TKO, RIP, mock, clone 26

description: Library name: FIBo_seq_pilot_no_rRNA_dep_4


title: p22179-s006_173385-11-Feb-2021

value: PBMC

value: Female

value: Not Hispanic or Latino


title: p22179-s010_217829-01-Apr-2021

value: PASC


title: cKit+CD127+ cell 70

description:
Il7r.Cd117.Cell_70
Fluidigm C1

value: Bone Marrow

value: cKit+CD127+ cell


title: mESC Tet1/2/3 TKO, PyBo, rRNA, clone 26

description:
Library name: sample_33
total RNA, rRNA-depleted


title: cKit+CD127+ cell 74

description:
Il7r.Cd117.Cell_74
Fluidigm C1

value: WT

value: paired-end


title: BM cells, noTPO, bulk, rep2

value: PBS


title: mESC Tet1/2/3 CTRL, mock, rRNA, clone 6

description:
Library name: sample_32
total RNA, rRNA-depleted

value: mouse embryonic stem cells


title: mESC Tet1/2/3 CTRL, PyBo, rRNA, clone 6

description:
Library name: sample_31
total RNA, rRNA-depleted

value: Tet1/2/3 wildtype


title: cKit+CD127+ cell 69

description:
Il7r.Cd117.Cell_69
Fluidigm C1


title: p22179-s005_165381-18-Feb-2021

value: Male

value: Pfizer


title: p22179-s004_165381-23-Apr-2021


title: BM cells, noTPO, bulk, rep7


title: TX3

city: Lanzhou

first: Yijun

last: Chai

state: Gansu

address: No.1 Xujiaping, Gansu Lanzhou

country: China

institute: Lanzhou Veterinary Research Institute, CAS

description: Extracellular vesicles secreted from bovine serum infected with T.annulata

data_processing:
Total RNA was prepared from triplicate samples of TaXJS‐EVs, TaBC-EVs, TaDC-EVs and extracellular vesicles from cattle serum using Trizol (Thermo Fisher) according to manufacturer's guidelines. Small RNA libraries were prepared using the NEBNext® Multiplex Small RNA Library Prep Kit (NEB, USA) according to the manufacturer’s protocol. Libraries were sequenced on the Illumina NovaSeq 6000 platform, generating 50 bp single-end reads. After sequencing, the raw reads were processed to remove low-quality reads, adapters, and contaminants, following established protocols.  Clean reads were aligned to the Bos taurus and T. annulata genomes using Bowtie. Known miRNAs were identified by comparing the reads to miRBase v22, while novel miRNAs were predicted using mirDeep2. Differential expression analysis was performed using DESeq2, with differentially expressed miRNAs defined as those with a |log2 fold change| > 1 and p-value < 0.05. miRNAs flagged as questionable based on previous reports or in-house validation were omitted from further analysis. Target genes of the differentially expressed miRNAs were predicted by overlapping results from Targetfinder and miRanda databases (http://www.bioinformatics.com.cn/l) ensuring reliable candidate gene identification. For both proteomic and small RNA datasets, functional enrichment analysis was performed to explore the biological significance of differentially expressed proteins (DEPs) and miRNAs. Raw data were analyzed using the MaxQuant software package, with the NCBI T. annulata protein database (7627 proteins) used as a reference. Differentially expressed proteins were identified based on a p-value < 0.05 and |log2 fold change| > 1. Gene Ontology (GO) and InterPro (IPR) functional analyses were conducted using the InterProScan program to annotate proteins against the non-redundant protein database. 28 Additionally, the Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to analyze protein families and pathways, determining the biological and functional properties of the identified proteins and miRNAs. Differentially expressed proteins and miRNAs were subjected to volcanic map analysis, cluster heatmap visualization, and enrichment analysis of GO, IPR, and KEGG. Cytoscape and the igraph package in R software (version 3.6.1) were employed to construct and visualize the interwoven networks of proteins and non-coding RNAs involved in various signaling pathways. Protein-protein interaction (PPI) networks were constructed using the STRING database, while miRNA-protein interaction networks were inferred by integrating miRNA and proteomic data. These analyses provided insights into the molecular mechanisms by which T. annulata manipulates host cells through EV cargo.
Assembly: NCBI 353154
Supplementary files format and content: read counts and tpm

organism: Bos taurus

source_name: Bovine serum

extract_protocol:
Sample collection and preparation RNA qualitative and quantitative analysis (Novogene Experimental Department) RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA,USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the AgilentGVO Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
The small RNA libraries were prepared from a total of 2 ug total RNA isolated from each sample using NEBNext Multiplex Small RNA Library Prep Set for Illumina*(NEB, USA.) according to the manufacturer’s instruction. Briefly, purified RNA was mixed with NEB 3' SR Adapter, then the SR RT Primer hybridized to the excess of 3'SR Adapter (that remained free after the 3' ligation reaction) and transformed the single-stranded DNA adapter into a double-stranded DNA molecule. This step is important to prevent adapter-dimer formation, besides, dsDNAs are not substrates for ligation mediated by T4 RNA Ligase I and therefore do not ligate to the 5’SR Adapter in the subsequent ligation step. 5'ends adapter was ligated to 5'ends of miRNAs, siRNA and piRNA. Then first strand cDNAwas synthesized usingM-MuLV Reverse Transcriptase(RNaseH).PCR amplification was performed using LongAmp Taq2X Master Mix,SR Primer for Illumina and index (X) primer. PCR products were purified on a 8% polyacrylamide gel (100v,80 min). DNA fragments corresponding to 140-160 bp (the length of small RNA plus the 3' and 5' adapters) were recovered and dissolved in 8 μL elution buffer. At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on a Novaseq 6000 platform and50 bp single-end reads were generated.

value: Bovine serum

value: Extracellular vesicles

value: EVs were isolated from 4 ml of sera using the exoEasy Maxi Kit (QIAGEN, Germany) according to the manufacturer's instructions.


title: BM cells, noTPO, bulk, rep5

value: Bone marrow

value: female


title: cKit+CD127+ cell 68

description:
Il7r.Cd117.Cell_68
Fluidigm C1


title: cKit+CD127+ cell 67

description:
Il7r.Cd117.Cell_67
Fluidigm C1


title: p22179-s003_165381-30-Dec-2020

value: PBMC


title: BM cells, TPO, GFP-, rep8

value: TPO


title: TX2


title: cKit+CD127+ cell 66

description:
Il7r.Cd117.Cell_66
Fluidigm C1


title: BM cells, TPO, bulk, rep4


title: TX1


title: p22179-s002_116521-29-Jul-2021

value: Not Hispanic or Latino

value: NOPASC


title: p22179-s001_116521-02-Apr-2021


title: cKit+CD127+ cell 65

description:
Il7r.Cd117.Cell_65
Fluidigm C1

value: Bone Marrow

value: cKit+CD127+ cell


title: sh1487,no-dox,cDNA,72hr,rep3 [R21075204LR01-Model4-doxy-3]

city: Cambridge

first: YaGuang

last: Si

address: 30 Acorn Park Dr

institute: Fogpharma

data_processing:
Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.39
The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.7.7a
Unique gene hit counts were calculated by using featureCounts from the R Subread package v.2.4.2
Read filtering, normalization, and differential expression analysis with respect to biological variation was performed with the edgeR package v.4.0.2 in R
Assembly: GRCh38
Supplementary files format and content: readcounts_CPM_allsamples_shRNA.xls
Supplementary files format and content: CPM from EdgeR for all samples in EXCEL format

molecule: polyA RNA

extract_protocol:
RNA estraction was performed by Mingma Technologies (Shanghai, China) with RNeasy Mini Kit following manufacture's instructions (QIAGEN, Hilden, Germany)
library preparation and sequencing were performed by Mingma Technologies (Shanghai, China) with TruSeq stranded mRNA library kit on Novaseq S4 Platform with PolyA enrichment.

tag: cell line

value: Colorectal adenocarcinoma

tag: guiderna

tag: dox

value: no-dox

tag: cdna

value: cDNA

tag: time

tag: replicate


title: H3

description: Healthy bovine serum extracellular vesicles

tag: cell type

tag: genotype


title: BM cells, noTPO, GFP+, rep1

tag: tissue

value: PBS


title: BM cells, TPO, GFP+, rep6


title: sh1487,no-dox,cDNA,72hr,rep2 [R21075203LR01-Model4-doxy-2]


title: BM cells, TPO, bulk, rep8


title: H2


title: cKit+CD127+ cell 64

description:
Il7r.Cd117.Cell_64
Fluidigm C1

value: WT

value: paired-end


title: cKit+CD127+ cell 63

description:
Il7r.Cd117.Cell_63
Fluidigm C1


title: cKit+CD127+ cell 62

description:
Il7r.Cd117.Cell_62
Fluidigm C1


title: BM cells, noTPO, GFP+, rep7


title: BM cells, TPO, GFP+, rep8


title: H1

value: Bovine serum


title: D3

description: Bovine Dendritic cell infected with T.annlata extracellular vesicles

source_name: Dendritic cells

value: Dendritic cells

value: Extracellular vesicles

value: EVs were isolated from the serum-free culture supernatants of TaXJS, TaBC, and TaDC cell lines using a sequential centrifugation process, followed by ultracentrifugation


title: cKit+CD127+ cell 61

description:
Il7r.Cd117.Cell_61
Fluidigm C1


title: D2


title: BM cells, TPO, GFP-, rep3

value: Bone marrow

value: female


title: cKit+CD127+ cell 60

description:
Il7r.Cd117.Cell_60
Fluidigm C1


title: sh1487,dox,cDNA,72hr,rep3 [R21075201LR01-Model4jiadoxy-3]

value: dox


title: BM cells, noTPO, GFP-, rep7


title: D1


title: shNT,no-dox,cDNA,72hr,rep3 [R21075198LR01-Model3-doxy-3]

value: shNT


title: cKit+CD127+ cell 58

description:
Il7r.Cd117.Cell_58
Fluidigm C1


title: BM cells, TPO, bulk, rep6

value: TPO


title: B3

description: Bovine B cell infected with T.annlata extracellular vesicles

source_name: B cell

value: B cell


title: cKit+CD127+ cell 59

description:
Il7r.Cd117.Cell_59
Fluidigm C1

value: Bone Marrow

value: cKit+CD127+ cell


title: sh1487,dox,cDNA,72hr,rep1 [R21075199LR01-Model4jiadoxy-1]

value: Colorectal adenocarcinoma


title: BM cells, noTPO, GFP-, rep5


title: sh1487,no-dox,cDNA,72hr,rep1 [R21075202LR01-Model4-doxy-1]

value: no-dox

value: cDNA


title: B2


title: shNT,no-dox,cDNA,72hr,rep2 [R21075197LR01-Model3-doxy-2]


title: cKit+CD127+ cell 57

description:
Il7r.Cd117.Cell_57
Fluidigm C1

value: WT


title: B1


title: BM cells, noTPO, GFP+, rep2

value: PBS


title: shNT,no-dox,cDNA,72hr,rep1 [R21075196LR01-Model3-doxy-1]


title: cKit+CD127+ cell 56

description:
Il7r.Cd117.Cell_56
Fluidigm C1

value: paired-end


title: BM cells, noTPO, GFP+, rep5


title: shNT,dox,cDNA,72hr,rep3 [R21075195LR01-Model3jiadoxy-3]


title: X3

description: Bovine lymphocytes infected with T.annlata extracellular vesicles

source_name: lymphocyte

value: lymphocyte


title: X2

value: Extracellular vesicles


title: cKit+CD127+ cell 55

description:
Il7r.Cd117.Cell_55
Fluidigm C1


title: sh1487,dox,cDNA,72hr,rep2 [R21075200LR01-Model4jiadoxy-2]


title: cKit+CD127+ cell 54

description:
Il7r.Cd117.Cell_54
Fluidigm C1


title: BM cells, noTPO, GFP-, rep1


title: X1

value: EVs were isolated from the serum-free culture supernatants of TaXJS, TaBC, and TaDC cell lines using a sequential centrifugation process, followed by ultracentrifugation


title: BM cells, TPO, GFP+, rep4

value: Bone marrow


title: cKit+CD127+ cell 53

description:
Il7r.Cd117.Cell_53
Fluidigm C1


title: shNT,dox,cDNA,72hr,rep2 [R21075194LR01-Model3jiadoxy-2]


title: BM cells, TPO, GFP-, rep4

value: female


title: shLONP1_FBXO11_3

city: Vancouver

first: Aly

last: Karsan

address: 675 West 10th Avenue

country: Canada

institute: Canada's Michael Smith Genome Sciences Centre

description:
AK_1180
CD34+ cord blood cells expressing shRNA targeting LONP1 and over-expressing FLAG-FBXO11.

data_processing:
Chastity passing reads were aligned with STAR (v2.5.2b) to hg19.
Counts generated with FeatureCounts (v2.0.0) to Ensembl v99 annotations.
TPM expression values generated with Salmon (v.0.14.2) to Ensembl v99 annotations.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: counts_with_external_IDs.tsv: Tab-delimited file of the Ensembl v99 gene ID and counts in each sample.
Supplementary_files_format_and_content: TPM_with_external_IDs.tsv: Tab-delimited file of Ensembl v99 gene ID and TPM values of expression in each sample.

source_name: Human cord-blood derived CD34+ cells

extract_protocol:
CD34+ cells were lentivirally transduced to express the corresponding vector(s) and transduced cells were selected by expression of fluorescent reporter using FACs. Sorted cells were cultured in StemSpan with CC100 cytokine cocktail with 20ng/mL hTPO for 24 hours before TRIzol RNA extraction. 40ng of total RNA was submitted per sample for ribodepletion and library construction.
Strand-specific ribodepleted RNA library construction.
150bp PE

value: CD34+ cord blood cells

tag: shRNA

value: shRNA targeting LONP1

tag: overexpression


title: cKit+CD127+ cell 52

description:
Il7r.Cd117.Cell_52
Fluidigm C1


title: sh1487,no-dox,,72hr,rep2 [R21075191LR01-Model2-doxy-2]


title: BM cells, TPO, bulk, rep3

value: TPO


title: sh1487,no-dox,,72hr,rep3 [R21075192LR01-Model2-doxy-3]


title: BM cells, TPO, GFP+, rep3


title: shLONP1_FBXO11_1

description:
AK_1178
CD34+ cord blood cells expressing shRNA targeting LONP1 and over-expressing FLAG-FBXO11.


title: cKit+CD127+ cell 51

description:
Il7r.Cd117.Cell_51
Fluidigm C1


title: cKit+CD127+ cell 50

description:
Il7r.Cd117.Cell_50
Fluidigm C1


title: sh1487,no-dox,,72hr,rep1 [R21075190LR01-Model2-doxy-1]


title: BM cells, noTPO, GFP-, rep2


title: cKit+CD127+ cell 49

description:
Il7r.Cd117.Cell_49
Fluidigm C1


title: sh1487,dox,,72hr,rep3 [R21075189LR01-Model2jiadoxy-3]

value: dox


title: sh1487,dox,,72hr,rep2 [R21075188LR01-Model2jiadoxy-2]


title: LONP1_3

description:
AK_1174
CD34+ cord blood cells expressing LONP1.


title: Cartilage MPRA plasmid replicate 4

first: Terence

last: Capellini

address: 11 Divinity Avenue

department: Human Evolutionary Biology

institute: Harvard University

description: Library name: 290k_Cartilage_plasmidDNA_rep4

library_source: other

data_processing:
Read merging using NGmerge v0.3
Read counts per barcode using MPRAmatch pipeline (https://github.com/tewhey-lab/MPRASuite/tree/main)
forward and reverse read merging using BBMap fuse.sh pad=100
match barcodes to oligos using MPRAcount pipeline, modified to avoid an error due to “%sequence mismatch” threshold by setting the value for all sequences below the standard threshold (https://github.com/tewhey-lab/MPRASuite/tree/main)
Differential expression between oligos was determined using MPRAmodel (https://github.com/tewhey-lab/MPRASuite/tree/main)
Assembly: hg38
Supplementary files format and content: count table of barcodes oligo pairs in each replicate as output by MPRAcount, attributes table, and condition table, for processing via MPRAmodel (https://github.com/tewhey-lab/MPRASuite/tree/main)

molecule: other

organism: synthetic construct

source_name: Plasmid DNA

growth_protocol: K562 cells were grown with RPMI 1640 Complete Medium supplemented with  + 10% FBS + 1% Penicillin-Streptomycin, TC28a2 and CHON002 cells were grown with DMEM Dulbecco's Modified Eagle Medium supplemented  + 10% FBS + 1% Penicillin-Streptomycin

extract_protocol:
Pellets were defrosted before the addition of RLT buffer containing 2M DTT (40ul for every 1ml of RLT buffer for >2x10^8 cells). Pellets were disrupted using a 10 mL syringe and 21-gauge needle before the addition of equal amounts of 70% ethanol. Samples were then processed using the Qiagen Maxi RNeasy and following the isolation procedure instructions. RNA was nano dropped and stored at -80˚C.
Half of the RNA sample was taken for MPRA clean up processing and library generation whilst saving the remaining half. Samples underwent a 1-hour DNase digest at 37˚C to remove any contaminating DNA, particularly coming from the plasmid. Following this biotagged GFP primer probes (CGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAG/3BioTEG/, CCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCC/3BioTEG/, CCTCGATGTTGTGGCGGGTCTTGAAGTTCACCTTG/3BioTEG/) were adhered to the GFP sequence of mRNA products using formamide cross linking (2.5 hours at 65˚C). This was followed by hybridization of streptavidin magnetic DynabeadsTM (15 mins, rtp, on nutator) that bind to the BioTEG portion of the probes now bound to the mRNA products of interest. Dynabeads were made RNase free prior to use following provided protocols. mRNA bound to Dynabeads was then isolated using a magnet to remove beads from the rest of the RNA containing solution in clean up stages followed by elution in 25ul of RNAse free water at both 70˚C and then 80˚C. To remove any remaining plasmid DNA, a further 1-hour 37˚C digest was performed before RNA SPRI bead cleanup (standard isolation and elution protocols) and performing first strand synthesis and reverse transcription (ThermoFisher Scientific, 18080044). Following reverse transcription, DNA SPRI was performed to remove any remaining reagents and concentrations checked using qPCR analysis. If CT value was below 18, samples were amplified at 2 cycles above their CT value before attaching unique illumina P5 and P7 adapter pairs. At this stage, four plasmid replicates were included and prepared for sequencing to assess library diversity representation for downstream processing. Samples were then assessed for concentration before pooling to form an equimolar pool. The resulting pool was sequenced five times on an Illumina NovaSeq X 10B flowcell to a depth of >125 million reads per replicate.

treatment_protocol: 300-500 million cells per technical replicate were transfected at 200ug/ml (K562, T/C-28a2) and 300ug/ml (CHON-002) of plasmid library, using a MaxCyte ATx electroporation device. Preexisting electroporation protocols established by MaxCyte were used and optimized in our hands by FACS analysis using a control plasmid on the device for each cell line. R1000 cartridges were used at ~125 million cells per cartridge. Following electroporation, cells were allowed to rest for 25 minutes in proprietary MaxCyte Buffer in a 6-well plate at 37˚C. Cells were then plated in their respective media at a 50%-70% density. Cells were harvested 24 hours post-transfection, spun down and snap frozen on dry ice before storing at -80˚C. Five technical replicates were transfected per cell-line.

value: plasmid DNA


title: BM cells, noTPO, bulk, rep1


title: shFBXO11_LONP1_1

description:
AK_1175
CD34+ cord blood cells expressing shRNA targeting FBXO11 and over-expressing LONP1.

value: shRNA targeting FBXO11


title: shNT,dox,cDNA,72hr,rep1 [R21075193LR01-Model3jiadoxy-1]

value: Colorectal adenocarcinoma

value: shNT


title: LONP1_2

description:
AK_1173
CD34+ cord blood cells expressing LONP1.


title: shNT,no-dox,,72hr,rep3 [R21075186LR01-Model1-doxy-3]

value: no-dox


title: sh1487,dox,,72hr,rep1 [R21075187LR01-Model2jiadoxy-1]


title: Cartilage MPRA plasmid replicate 2

description: Library name: 290k_Cartilage_plasmidDNA_rep2


title: shLONP1_FBXO11_2

description:
AK_1179
CD34+ cord blood cells expressing shRNA targeting LONP1 and over-expressing FLAG-FBXO11.


title: Cartilage MPRA plasmid replicate 3

description: Library name: 290k_Cartilage_plasmidDNA_rep3


title: Gene Edited Setaria viridis cv. ME034V-1 with null PGR5 Alleles - Biological Replicate 1

city: Melbourne

first: Russell

last: Woodford

state: Victoria

address: 25 Rainforest Walk, Monash University, Clayton

department: School of Biological Sciences

country: Australia

institute: Monash University

description:
Library name: pgr5_1
Column name in Processed Data File : pgr5_1

data_processing:
Raw mRNA-sequencing reads were mapped to the S. viridis v2.0 genome (GCA_005286985.1) utilising the Star Seq Toolkit (Dobin et al., 2013). Raw reads were assessed and trimmed to remove low-quality base calls (PHRED <20, -q 20) using FastQC (v0.11.9) and TrimGalore! (v0.6.7). Transcript quantification was conducted for unique matches at the gene level using StringTie (v2.2.3) against the S. viridis v2.0 genome. The resulting raw counts were imported into edgeR (v4.4.1) and counts per million (CPM) reads and log-CPM was calculated for each loci. Counts data was filtered to focus on loci detected at CPM > 1 in a minimum of three samples. Library normalisation was performed using Limma-Voom (v3.62.2), employing the trimmed mean of M-values (TMM) method to account for both sequencing depth and composition. Differential gene expression analysis was performed using edgeR on batch-corrected data. Differentially expressed genes were determined using a log-fold change cutoff of 1 and an adjusted p-value cutoff of 0.05, after correcting for false discovery rate.
Assembly: GCA_005286985.1
Supplementary files format and content: Matrix of data in .csv format. Contains geneIDs, gene descriptions, counts data, corrected Log-Counts Per Million, Log-Fold Change, p-value and adjusted p-value.

organism: Setaria viridis

source_name: Whole Leaf

growth_protocol: Plants were germinated in 0.5 L pots with seed raising mix (Debco, Tyabb, Australia) containing 5 g L-1 of Osmocote fertiliser (Scotts, Bella Vista, Australia). Seeds were covered with inverted plastic cups to maintain humidity, with the cups removed one week after germination. Plants were grown in a controlled-environment chamber with a 16 h light/8 h dark photoperiod, an irradiance of 380 µmol m−2 s−1, ambient CO2, 28°C day, 22°C night and 60% humidity.

extract_protocol:
To obtain RNA for differential gene expression analyses, leaf discs were taken from plants grown under constant daylight conditions and flash-frozen in liquid nitrogen. Frozen tissue was homogenised using a 1/8” ball bearing with TissueLyser II (Qiagen, Hilden, Germany), shaking at 25 Hz for 1 minute. Total RNA was isolated using a Qiagen RNeasy Plant Mini kit as per the manufacturer protocol. RNA concentration was quantified using a Nanodrop ND-1000 Spectrophotometer at 260 nm and confirmed to be > 280 ng μL-1 for all samples. RNA integrity score was checked using a LabChip GXII Touch (PerkinElmer, Waltham, MA, USA) and confirmed to be > 8.5 for all samples.
PolyA-enriched mRNA libraries were prepared using an Illumina TruSeq Stranded mRNA library prep kit following the manufacturer protocol. 1.8 μg of total RNA was reverse transcribed into cDNA for each sample and indexed with IDT for Illumina’s Unique Dual Index adapters. cDNA clean-up was performed using Sera-mag SpeedBeads (GE Healthcare, Chicago, IL, USA). cDNA library concentration was determined for each sample using a Qubit High Sensitivity dsDNA Quantitation kit. cDNA library quality was confirmed using a LabChip GXII Touch. Individual library molarity was determined using cDNA concentration and peak fragment size, then normalised to 2.5 nM.  Illumina Next Gen Sequencing was performed at the ACRF Biomolecular Research Facility (The Australian National University, Canberra, ACT, Australia) on the NovaSeq6000 platform (150 bp paired-end sequencing).

value: Whole Leaf

value: PGR5 Null Alleles


title: Cartilage MPRA plasmid replicate 1

description: Library name: 290k_Cartilage_plasmidDNA_rep1


title: shNT,no-dox,,72hr,rep2 [R21075185LR01-Model1-doxy-2]


title: FBXO11_3

description:
AK_1171
CD34+ cord blood cells expressing FLAG-FBXO11.


title: shNT,no-dox,,72hr,rep1 [R21075184LR01-Model1-doxy-1]


title: shFBXO11_LONP1_2

description:
AK_1176
CD34+ cord blood cells expressing shRNA targeting FBXO11 and over-expressing LONP1.


title: Gene Edited Setaria viridis cv. ME034V-1 with null NdhO Alleles - Biological Replicate 2

description:
Library name: ndhO_2
Column name in Processed Data File : ndhO_2

value: NdhO Null Alleles


title: Gene Edited Setaria viridis cv. ME034V-1 with null PGR5 Alleles - Biological Replicate 2

description:
Library name: pgr5_2
Column name in Processed Data File : pgr5_2


title: FBXO11_2

description:
AK_1170
CD34+ cord blood cells expressing FLAG-FBXO11.


title: shNT,dox,,72hr,rep3 [R21075183LR01-Model1jiadoxy-3]


title: Gene Edited Setaria viridis cv. ME034V-1 with null PGR5 Alleles - Biological Replicate 3

description:
Library name: pgr5_3
Column name in Processed Data File : pgr5_3


title: Aged_HTGTS_TCR_seq_Traj_mix_rep3

city: Shanghai

first: Haiqiang

last: Dai

address: 320 Yue Yang Road

institute: Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell  Science

description: Library name: sample42

data_processing:
For Tcra and Tcrb gene rearrangement analysis, sequencing reads were aligned to the mm10 reference genome for mouse T cells and to the hg38 reference genome for human T cells. All duplicate junctions were retained in the analyses. To quantify the utilization level of V, D and J segments in TCR α and β gene rearrangements, we defined “on-target” sequences as the 40-bp regions flanking the recombination signal sequences (RSS) (RSS ± 40 bp). which were then used to determine the usage of V(D)J segments. To quantify TCR repertoires, we employed a pipeline called "HTGTSrep" to identify the productive and non-productive V(D)J rearrangements, as well as to analyze the motifs and length distribution of CDR3 regions.
Assembly: mm10;hg38
Supplementary files format and content: tlx

source_name: Aged Thymus (16-18 month)

extract_protocol:
Cells were collected and lysed in cell lysis buffer using proteinase K. Genomic DNA was later precipitated and dissolved in TE before shearing.
3-20 μg DNA was fragmented via sonication on Qsonic biorupter and subjected to linear PCR amplification with a biotinylated primer as following PCR conditions: 95°C 2 min; 95°C 30s; 58°C 30 s; 72°C 90 s; 80 cycles; 72°C 2 min. Primers were removed by VAHTS DNA Clean Beads (Vazyme, N411). The resulting single-strand biotinylated PCR products were collected via Dynabeads MyONE C1 streptavidin beads (ThermoFisher, 65002) and ligated to bridge adapters overnight in the presence of 15% PRG8000. Ligation were conducted following 25°C 60 min; 22°C 120 min; 16°C 9-12 h. Subsequently, i5-indexed bait-specific inner primers and i7-index adapter primers were added to adapter-ligated products by nested PCR. Excess primers were removed by VAHTS DNA Clean Beads (Vazyme, N411), and the purified PCR products were further tagged with Illumina sequencing adapter sequences. Libraries were size-selected by resolving PCR products on a 2% agarose gel, isolating fragments between 300-700 bp for mouse or human samples, with recovery performed via gel extraction. The HTGTS-TCR-seq libraries were sequenced on NovaXplus (Illumina) using 150-bp paired-end reads.

value: Aged Thymus (16-18 month)

value: Thymocytes

value: WT


title: FBXO11_1

description:
AK_1169
CD34+ cord blood cells expressing FLAG-FBXO11.


title: shNT,dox,,72hr,rep2 [R21075182LR01-Model1jiadoxy-2]


title: Aged_HTGTS_TCR_seq_Trav_mix_rep4

description: Library name: sample35


title: Wild Type Setaria viridis cv. ME034V-1 - Biological Replicate 3

description:
Library name: WT_3
Column name in Processed Data File : WT_3


title: shNT,dox,,72hr,rep1 [R21075181LR01-Model1jiadoxy-1]


title: Empty_3

description:
AK_1168
CD34+ cord blood cells expressing empty vector control.

value: empty vector control


title: Aged_HTGTS_TCR_seq_Trav_mix_rep3

description: Library name: sample34


title: Gene Edited Setaria viridis cv. ME034V-1 with null NdhO Alleles - Biological Replicate 1

description:
Library name: ndhO_1
Column name in Processed Data File : ndhO_1


title: Empty_2

description:
AK_1167
CD34+ cord blood cells expressing empty vector control.


title: Young_HTGTS_TCR_seq_Trav_mix_rep4

description: Library name: sample31

source_name: Thymus

value: Thymus


title: Helicon4, 30mpk, 48hr,rep6 [SH7083_SA91288_S50]

data_processing:
Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.39
The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.7.7a
Host reads were removed with XenofilteR
Unique gene hit counts were calculated by using featureCounts from the R Subread package v.2.4.2
Read filtering, normalization, and differential expression analysis with respect to biological variation was performed with the edgeR package v.4.0.2 in R
Assembly: GRCh38
Supplementary files format and content: CPM from EdgeR for all samples in EXCEL format

extract_protocol:
RNA extraction was performed using NEBNext Ultra II Directional RNA Library Prep Kit following manufacture's instructions (New England BioLabs, Ipswich, MA)
library preparation and sequencing were performed by Fulgent Gentetics (Houston, TX) with TruSeq stranded mRNA library kit on Novaseq S4 Platform with PolyA enrichment.

tag: dose


title: LONP1_1

description:
AK_1172
CD34+ cord blood cells expressing LONP1.

value: CD34+ cord blood cells


title: Gene Edited Setaria viridis cv. ME034V-1 with null NdhO Alleles - Biological Replicate 3

description:
Library name: ndhO_3
Column name in Processed Data File : ndhO_3


title: Young_HTGTS_TCR_seq_Trav_mix_rep3

description: Library name: sample30


title: Wild Type Setaria viridis cv. ME034V-1 - Biological Replicate 2

description:
Library name: WT_2
Column name in Processed Data File : WT_2


title: Wild Type Setaria viridis cv. ME034V-1 - Biological Replicate 1

description:
Library name: WT_1
Column name in Processed Data File : WT_1


title: Empty_1

description:
AK_1166
CD34+ cord blood cells expressing empty vector control.


title: Helicon4, 30mpk, 48hr,rep5 [SH7082_SA91287_S49]


title: Aged_HTGTS_TCR_seq_Trbv_mix_rep4

description: Library name: sample27


title: 5RACE_Wapl.KO_preselectionDP_Tra_rep3

description: Library name: sample13

data_processing:
Analysis was performed using MiXCR “analyze generic-amplicon” function(v4.4.1). And then, the VDJtools (v1.2.1) command PlotFancyVJUsage was used to calculate clonal frequencies of V-J recombinations. Heatmaps were generated using the pheatmap and RColorBrewer packages.
Assembly: mm10
Supplementary files format and content: txt

extract_protocol:
Single-cell suspensions were counted, lysed with TRIzol (15596018CN, Thermofisher-Invitrogen), and total RNA was extracted.
800 ng RNA was used for template switch with Superscript II (18064014, Thermofisher-Invitrogen) with a template switch oligo (TSO). TCR amplification was performed using a universal forward primer and Trac or Trbc-specific reverse primers. PCR products (300–700 bp) were gel-purified. Libraries were prepared with the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607-01) and sequenced (2×150 bp paired-end) on the Illumina Novaseq XPlus platform.

value: Preselection DP thymocytes

value: Cd4-cre Wapl f/-


title: Helicon4, 30mpk, 48hr,rep4 [SH7082_SA91284_S46]


title: Helicon4, 30mpk, 48hr,rep3 [SH7082_SA91283_S45]


title: shLONP1_1_2

description:
AK_1164
CD34+ cord blood cells expressing shRNA targeting LONP1.

value: shRNA targeting LONP1


title: Aged_HTGTS_TCR_seq_Trbv_mix_rep3

description: Library name: sample26


title: WT-sperm-Uli-epic-GLORI-rep2

city: shanghai

first: Xu

last: Chu

address: Dong'an Road

institute: Fudan University Shanghai Cancer Center

data_processing:
Reads were trimmed using Cutadapt
HEK293T mapped to hg38  use STAR, sperm and NSC mapped to mm10  use STAR
Assembly: hg38 or mm10
Supplementary files format and content: tab-delimited text file include m6A sites and level for each Sample
Library strategy: Uli-Epic GLORI

source_name: Sperm

growth_protocol: HEK 293T cells were cultured in DMEM high glucose (GIBCO) supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin and 100 μg/mL of streptomycin. Cells were kept under standard 37 ℃ and 5% CO2 conditions. Sperm were collected from the cauda epididymis of 10-12 week old mice. NSC were isolated from embryonic 17.5 Nestin-GFP mice.

extract_protocol:
HEK293T total RNA was extracted by Trizol in accordance with the manufacturer’s protocol. mRNA were purified by VAHTS mRNA capture beads (Vazyme).    Sperm were collected from the cauda epididymis of 10-12 week old mice and released into 2 mL of phosphate-buffered saline (PBS) maintained at 37°C for a 20-minute incubation. Following incubation, the suspension was filtered through a 40 µm cell strainer to remove tissue debris. Subsequently, the sperm were treated with 10 mL somatic cell lysis buffer (0.1% SDS, 0.5% Triton X-100) on ice for 40 minutes to eliminate somatic cell contamination. The purity of the sperm sample was confirmed via microscopic examination. The sperm pellet was washed twice in 5 mL of PBS, followed by centrifugation at 1000g for 5 minutes. The sperm pellet was resuspended in TRIzol reagent (Invitrogen), homogenized, and subjected to RNA extraction. The hippocampi were isolated from embryonic 17.5 Nestin-GFP mice and dissociated using the Neural Tissue Dissociation Kit P (Miltenyi Biotec, 130-092-628). Following enzymatic digestion, the solution was diluted by adding 9 mL of Hibernate™-A medium (Invitrogen, A1247501) and centrifuged at 250 × g for 8 minutes. The cell pellets were then resuspended in 0.5 mL of 1× red blood cell lysis buffer (Biogems, 64010-00-100) and incubated for 1 minute. Subsequently, 5 mL of Hibernate™-A medium was added, and the mixture was centrifuged again at 250 × g for 6 minutes. The cells were resuspended in FACS sorting medium (0.5% BSA in Hibernate™-A), incubated with DAPI (Solarbio, C0060) at a 1:1,000 dilution for 5 minutes to label dead cells, and passed through a cell strainer. Finally, the cells were sorted using a BD FACS Aria II cell sorter. Total RNAs were isolated from sorting cells by using TRIzol reagent (Invitrogen) and MaXtractTM High Density (Qiagen).
Uli-Epic GLORI steps are combined steps of our uli-epic and reported bulk GLORI. In brief, 10ng 293T mRNA or 10ng mouse sperm ribo-depletion RNA or 10ng mouse NSC ribo-depletion RNA was added into 40 μL of reaction buffer containing 6 μL of glyoxal solution (Sigma-Aldrich, 50649), 20 μL of DMSO and nuclease-free water. The mixture was incubated at 50 °C for 30 min. Then 10 μL of saturated H3BO3 (Sigma-Aldrich, B0394) solution was added and the incubation continued at 50 °C for 30 min followed by cooling on ice for further use. The 50 μL of reaction mixture was added to premixed deamination buffer and gently mixed. The tubes were incubated for 8 h at 16 °C. RNA was purified by Oligo Clean & Concentrator (Zymo Research，D4061). Then RNA were dissolved by 50 μL deprotection buffer(文献). The mixture was incubated at 95 °C for 10 min. RNA was purified with Oligo Clean & Concentrator（Zymo Research，D4061）, and then end-repaired by PNK (NEB, M0201S) to free the 3’ OH. Then add poly (A) tailing to the 3’ end of RNA by E. coli Poly(A) Polymerase (NEB, M0276L). RNA was purified by Oligo Clean & Concentrator（Zymo Research，D4061). Reverse transcription and template switching were performed with template switching RT enzyme mix (NEB, M0466L)、 T7 primer (sequence: 5’GCCGCGAAATTAATACGACTCACTATAGGGATAATGAGCAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTVN-3’) and Template switch oligo (TSO: 5’Biotin-GCTAATCATTGCACACGACGCTCT TCCGATCTrGrGrG-3’) at 42 °C for 90 min. Then RNA template was degradation by E. coli RNase H (NEB, M0297L), followed by second strand cDNA were synthesized by Q5 Hot Start High Fidelity Master Mix (NEB #M0494). The excess T7 primer was digested by exonuclease I (NEB, M0293S). The double strand cDNA was purified by Oligo Clean & Concentrator (Zymo Research，D4061). In vitro transcription was carried out with double strand cDNA by T7 High Yield RNA Transcription Kit, Vazyme. At the end of in vitro transcription, DNase I was added to digest double strand cDNA. RNA was purified by 1.8 × SPRIselect beads (Beckman, B23318). Then the RNA was subjected to reverse transcription with RT primer (sequence: 5’- ACACGACGCTCTTCCGATCT -3’) by SuperScript™ III (Thermo Fisher Scientific, cat. no.18080051). cDNA was purified with 1.8 × SPRIselect beads (Beckman, B23318). Library were constructed with single index primers (NEB, E7335S).

value: Sperm

value: Wild type


title: 5RACE_Wapl.KO_preselectionDP_Tra_rep2

description: Library name: sample12


title: shLONP1_1_1

description:
AK_1163
CD34+ cord blood cells expressing shRNA targeting LONP1.


title: WT-sperm-Uli-epic-GLORI-rep1


title: FGR-sperm-Uli-epic-GLORI-rep2

value: fetal growth restriction


title: 5RACE_Wapl.KO_preselectionDP_Tra_rep1

description: Library name: sample11


title: Helicon4, 30mpk, 48hr,rep1 [SH7081_SA91279_S41]

value: Colorectal adenocarcinoma


title: shFBXO11_9_2

description:
AK_1161
CD34+ cord blood cells expressing shRNA targeting FBXO11.

value: shRNA targeting FBXO11


title: 5RACE_Wapl.WT_preselectionDP_Tra_rep4

description: Library name: sample10

value: Wapl f/-


title: Helicon4, 30mpk, 48hr,rep2 [SH7082_SA91282_S44]


title: shFBXO11_9_3

description:
AK_1162
CD34+ cord blood cells expressing shRNA targeting FBXO11.


title: FGR-sperm-Uli-epic-GLORI-rep1


title: 5RACE_Wapl.WT_preselectionDP_Tra_rep3

description: Library name: sample9


title: Helicon4neg, 30mpk, 48hr,rep8 [SH7081_SA91277_S39]


title: shFBXO11_9_1

description:
AK_1160
CD34+ cord blood cells expressing shRNA targeting FBXO11.


title: Helicon4neg, 30mpk, 48hr,rep7 [SH7081_SA91276_S38]


title: 5RACE_Wapl.KO_preselectionDP_Tra_rep4

description: Library name: sample14


title: shCTR_3

description:
AK_1159
CD34+ cord blood cells expressing non-targeting shRNA.

value: non-targeting shRNA


title: WT-NSC-Uli-epic-GLORI-rep2

source_name: neural

value: neural

value: neural stem cells


title: Helicon4neg, 30mpk, 48hr,rep6 [SH7081_SA91274_S36]


title: shLONP1_1_3

description:
AK_1165
CD34+ cord blood cells expressing shRNA targeting LONP1.


title: 5RACE_Wapl.WT_preselectionDP_Tra_rep1

description: Library name: sample7


title: WT-NSC-Uli-epic-GLORI-rep1


title: Helicon4neg, 30mpk, 48hr,rep5 [SH7080_SA91273_S35]


title: 5RACE_WT_Tra_rep3

description: Library name: sample6

value: Thymocytes


title: shCTR_1

description:
AK_1157
CD34+ cord blood cells expressing non-targeting shRNA.


title: FGR-NSC-Uli-epic-GLORI-rep2


title: Helicon4neg, 30mpk, 48hr,rep4 [SH7080_SA91272_S34]


title: shCTR_2

description:
AK_1158
CD34+ cord blood cells expressing non-targeting shRNA.


title: 5RACE_WT_Tra_rep2

description: Library name: sample5

value: WT


title: Helicon4neg, 30mpk, 48hr,rep3 [SH7080_SA91271_S33]


title: WT-sperm-Uli-epic-BIDSeq-rep2

data_processing:
Reads were trimmed using Cutadapt
HEK293T mapped to hg38  use STAR, sperm and NSC mapped to mm10  use STAR
Assembly: hg38 or mm10
Supplementary files format and content: tab-delimited text file include Ψ sites and level for each Sample
Library strategy: Uli-Epic BID-seq

extract_protocol:
HEK293T total RNA was extracted by Trizol in accordance with the manufacturer’s protocol. mRNA were purified by VAHTS mRNA capture beads (Vazyme).    Sperm were collected from the cauda epididymis of 10-12 week old mice and released into 2 mL of phosphate-buffered saline (PBS) maintained at 37°C for a 20-minute incubation. Following incubation, the suspension was filtered through a 40 µm cell strainer to remove tissue debris. Subsequently, the sperm were treated with 10 mL somatic cell lysis buffer (0.1% SDS, 0.5% Triton X-100) on ice for 40 minutes to eliminate somatic cell contamination. The purity of the sperm sample was confirmed via microscopic examination. The sperm pellet was washed twice in 5 mL of PBS, followed by centrifugation at 1000g for 5 minutes. The sperm pellet was resuspended in TRIzol reagent (Invitrogen), homogenized, and subjected to RNA extraction. The hippocampi were isolated from embryonic 17.5 Nestin-GFP mice and dissociated using the Neural Tissue Dissociation Kit P (Miltenyi Biotec, 130-092-628). Following enzymatic digestion, the solution was diluted by adding 9 mL of Hibernate™-A medium (Invitrogen, A1247501) and centrifuged at 250 × g for 8 minutes. The cell pellets were then resuspended in 0.5 mL of 1× red blood cell lysis buffer (Biogems, 64010-00-100) and incubated for 1 minute. Subsequently, 5 mL of Hibernate™-A medium was added, and the mixture was centrifuged again at 250 × g for 6 minutes. The cells were resuspended in FACS sorting medium (0.5% BSA in Hibernate™-A), incubated with DAPI (Solarbio, C0060) at a 1:1,000 dilution for 5 minutes to label dead cells, and passed through a cell strainer. Finally, the cells were sorted using a BD FACS Aria II cell sorter. Total RNAs were isolated from sorting cells by using TRIzol reagent (Invitrogen) and MaXtractTM High Density (Qiagen).
Uli-Epic BID-seq steps are combined steps of our uli-epic and reported bulk BID-seq. 1 ng or 100 pg 293T mRNA and were fragmented using 0.1M NaHCO3 pH 9.2 and incubate at 95 °C for 5 min, then add 3M NaOAc pH 5.2 to neutralize pH. RNA was 3’ end-repaired by T4 polynucleotide kinase (PNK) (catalog no. M0201, NEB). Then add poly (A) tailing to the 3’ end of RNA by E. coli Poly(A) Polymerase (NEB, M0276L). Carrier DNA was added to poly (A) RNA and was purified by Oligo Clean & Concentrator（Zymo Research，D4061), eluting with 7 µl RNase-free water. A 1.5 µl aliquot of the purified RNA was saved for ‘Input’ library construction, 5 µl was subjected to BID-seq optimized bisulfite treatment, as the ‘Treated’ sample. The 5 µl RNA was mixed with 45 µl freshly prepared BID-seq BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, prepared by dissolving 270 mg Na2SO3 and 34 mg NaHSO3 in 850 µl RNase-free water) and incubated at 70 °C for 3 h. Then, 100 µl RNase-free H2O, 250 µl RNA binding buffer (RNA Clean and Concentrator), and 400 µl ethanol were added to the reaction mixture, which was mixed well and loaded on a RNA Clean and Concentrator-5 column. After spinning and washing once with 200 µl RNA wash buffer (RNA Clean and Concentrator), 200 µl RNA Desulphonation Buffer (catalog no. R5001-3-40, Zymo Research) was added to the column and incubated at room temperature for 1 h. This was followed by spinning and washing twice with 700 µl RNA wash buffer (RNA Clean and Concentrator), followed by eluting RNA with 10.5 µl RNase-free water. Reverse transcription and template switching were performed with template switching RT enzyme mix (NEB, M0466L)、 T7 primer (sequence: 5’-GCCGCGAAATTAATACGACTCACTATAGGGATAATGAGCAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTVN-3’) and Template switch oligo (TSO: 5’Biotin-GCTAATCATTGCACACGACGCTCTTCCGATCTrGrGrG-3’) at 42 °C for 90 min. Then RNA template was degradation by E. coli RNase H (NEB, M0297L), followed by second strand cDNA were synthesized by Q5 Hot Start High Fidelity Master Mix (NEB #M0494). The excess T7 primer was digested by exonuclease I (NEB, M0293S). The double strand cDNA was purified by Oligo Clean & Concentrator (Zymo Research，D4061). In vitro transcription was carried out with double strand cDNA by T7 High Yield RNA Transcription Kit, vazyme. At the end of in vitro transcription, DNase I was added to digest double strand cDNA. RNA was purified by 1.8 × SPRIselect beads (Beckman, B23318). Then the RNA was subjected to reverse transcription with RT primer (sequence: 5’- ACACGACGCTCTTCCGATCT -3’) by SuperScript™ III (Thermo Fisher Scientific, cat. no.18080051). cDNA was purified with 1.8 × SPRIselect beads (Beckman, B23318). Library were constructed with single index primers (NEB, E7335S).


title: FGR-NSC-Uli-epic-GLORI-rep1


title: Helicon4neg, 30mpk, 48hr,rep2 [SH7080_SA91270_S32]


title: 5RACE_WT_Tra_rep1

description: Library name: sample4


title: 5RACE_Wapl.WT_preselectionDP_Tra_rep2

description: Library name: sample8


title: 5RACE_WT_Trb_rep3

description: Library name: sample3


title: sperm-Uli-epic-BIDSeq-input-rep1


title: Helicon4neg, 30mpk, 48hr,rep1 [SH7080_SA91269_S31]


title: 5RACE_WT_Trb_rep2

description: Library name: sample2


title: FGR-sperm-Uli-epic-BIDSeq-rep2


title: Western Normal, rep5

type: RNA

city: Guangzhou

first: YAO

last: LUO

address: Dong Feng East Road

institute: SYSUCC

hyb_protocol: Hybridization of bio-labeled RNA samples was performed according to the manufacturers instructions for the Affymetrix GeneChip miRNA 3.0 Array with GeneChip Hybridization Wash and Stain kit.

scan_protocol: Detection was done in Affymetrix scanning machine GeneChip™ Scanner 3000 7G (Affymetrix [part of ThermoFisher Scientific company]).

data_processing: The Affymetrix raw data were normalised using  the robust multichip average algorithm from ‘oligo’ Bioconductor package in R version 3.0.1. Normalized miRNA expression data were evaluated using Bioconductor LIMMA differential expression analysis.

label: biotin

source_name: CD19 positive cells of healthy donors

label_protocol: The method used oligonucleotide probes attached to glass matrix, organized into a specific pattern. At the beginning of the experiment, each oligonucleotide in the sample was enriched with the polyA tail that was connected with modified biotin containing polyT.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: PBMCs from peripheral blood  were isolated using Ficoll-PaqueTM PLUS (GE Healthcare Bio-Sciences, Uppsala, Sweden) gradient centrifugation, and normal CD19-positive B cell were purified using CD19 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany).

tag: population

value: Western

tag: disease

value: normal


title: vehicle, , 48hr,rep9 [SH7080_SA91267_S29]

tag: treatment

value: vehicle


title: vehicle, , 48hr,rep10 [SH7080_SA91268_S30]


title: Western Normal, rep4


title: Wapl.KO_preselectionDP_HTGTS_TCR_seq_Trav_mix_rep4

description: Library name: sample28

data_processing:
For Tcra and Tcrb gene rearrangement analysis, sequencing reads were aligned to the mm10 reference genome for mouse T cells and to the hg38 reference genome for human T cells. All duplicate junctions were retained in the analyses. To quantify the utilization level of V, D and J segments in TCR α and β gene rearrangements, we defined “on-target” sequences as the 40-bp regions flanking the recombination signal sequences (RSS) (RSS ± 40 bp). which were then used to determine the usage of V(D)J segments. To quantify TCR repertoires, we employed a pipeline called "HTGTSrep" to identify the productive and non-productive V(D)J rearrangements, as well as to analyze the motifs and length distribution of CDR3 regions.
Assembly: mm10
Supplementary files format and content: txt

value: Thymus

value: Preselection DP thymocytes


title: 5RACE_WT_Trb_rep1

description: Library name: sample1


title: WT-NSC-Uli-epic-BIDSeq-rep2


title: Western Normal, rep2


title: Wapl.KO_preselectionDP_HTGTS_TCR_seq_Trav_mix_rep3

description: Library name: sample27

value: Cd4-cre Wapl f/-


title: Western Normal, rep3


title: vehicle, , 48hr,rep7 [SH7079_SA91265_S27]


title: WT-sperm-Uli-epic-BIDSeq-rep1

value: Sperm


title: WT-NSC-Uli-epic-BIDSeq-rep1

value: Wild type


title: Wapl.KO_preselectionDP_HTGTS_TCR_seq_Trav_mix_rep2

description: Library name: sample26


title: vehicle, , 48hr,rep6 [SH7079_SA91264_S26]


title: Western Normal, rep1


title: Western Normal, rep6


title: NSC-Uli-epic-BIDSeq-input-rep1


title: Wapl.KO_preselectionDP_HTGTS_TCR_seq_Trav_mix_rep1

description: Library name: sample25


title: Asian Normal, rep8

value: Asian


title: Wapl.WT_preselectionDP_HTGTS_TCR_seq_Trav_mix_rep4

description: Library name: sample24


title: FGR-sperm-Uli-epic-BIDSeq-rep1

value: fetal growth restriction


title: Asian Normal, rep7


title: Asian Normal, rep6


title: FGR-NSC-Uli-epic-BIDSeq-rep1


title: FGR-NSC-Uli-epic-BIDSeq-rep2


title: vehicle, , 48hr,rep3 [SH7079_SA91261_S23]

value: Colorectal adenocarcinoma


title: vehicle, , 48hr,rep8 [SH7079_SA91266_S28]


title: Wapl.WT_preselectionDP_HTGTS_TCR_seq_Trav_mix_rep3

description: Library name: sample23

value: Wapl f/-


title: Alveolar macrophages, HCoV-OC43, Day5, rep3

city: Beijing

first: Chen

last: Dong

address: Haidian District

institute: Tsinghua University

description:
Library name: Sample 9
HCoV-OC43_Day5_3

data_processing:
Briefly, the reads were aligned to mm10 by HISAT2 v.2.2.1 (Kim et al., 2019) with default parameters and the uniquely mapping reads were summarized by feature Counts (from Subread v.2.0.1) (Liao et al., 2014).
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

source_name: Alveolar macrophages

extract_protocol:
Total cellular RNA was extracted from cells with the Magzol reagent (Magen).
Library preparation is performed using Optimal Dual-mode mRNA Library Prep Kit (BGI-Shenzhen, China).

value: Alveolar macrophages


title: Wapl.WT_preselectionDP_HTGTS_TCR_seq_Trav_mix_rep2

description: Library name: sample22


title: vehicle, , 48hr,rep4 [SH7079_SA91262_S24]


title: Alveolar macrophages, HCoV-OC43, Day5, rep2

description:
Library name: Sample 8
HCoV-OC43_Day5_2


title: Asian Normal, rep9


title: Wapl.WT_preselectionDP_HTGTS_TCR_seq_Trav_mix_rep1

description: Library name: sample21


title: vehicle, , 48hr,rep1 [SH7078_SA91259_S21]


title: Asian Normal, rep4


title: Alveolar macrophages, HCoV-OC43, Day5, rep1

description:
Library name: Sample 7
HCoV-OC43_Day5_1


title: Alveolar macrophages, HCoV-OC43, Day3, rep3

description:
Library name: Sample 6
HCoV-OC43_Day3_3


title: Asian Normal, rep5


title: Asian Normal, rep3


title: vehicle, , 48hr,rep5 [SH7079_SA91263_S25]


title: Wapl.KO_preselectionDP_HTGTS_TCR_seq_Traj_mix_rep4

description: Library name: sample20


title: Wapl.KO_preselectionDP_HTGTS_TCR_seq_Traj_mix_rep3

description: Library name: sample19


title: Asian Normal, rep2


title: Asian Normal, rep1


title: Wapl.KO_preselectionDP_HTGTS_TCR_seq_Traj_mix_rep2

description: Library name: sample18


title: Wapl.KO_preselectionDP_HTGTS_TCR_seq_Traj_mix_rep1

description: Library name: sample17


title: Western CLL,rep9

source_name: CD19 positive cells of CLL

value: CLL


title: Alveolar macrophages, HCoV-OC43, Day0, rep3

description:
Library name: Sample 3
HCoV-OC43_Day0_3


title: vehicle, , 48hr,rep2 [SH7079_SA91260_S22]


title: OPCs_MSC-EVs_rep3

city: Milano

first: Stefano

last: Raffaele

state: -- Please Select (only U.S. / Can / Aus)

address: Via Balzaretti 9

department: Department of Pharmaceutical Sciences

country: Italy

institute: Università degli Studi di Milano

description: Gene expression data from OPCs treated with EVs derived from microglia co-cultured with mesenchymal stem cells in presence of pro-inflammatory cytokines

hyb_protocol: 5.5 µg of cDNA were hybridized for 16 hr at 45°C on Clariom™ D mouse arrays. Arrays were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: Scanning of the arrays was conducted using a GeneChip® Scanner 3000 7G (Thermo Fischer Scientific, USA).

data_processing: The images were scanned by ThermoFisher GeneChip Command Console (AGCC) and analyzed with the ThermoFisher GeneChip Expression Console.

source_name: OPCs treated with MSC-EVs

label_protocol: 100 ng of RNA was used for the preparation of targets for Clariom™ D mouse arrays according to the GeneChip™ WT Plus Reagent Kit manual.

growth_protocol: OPCs were isolated from P2 mouse brain using anti-CD140a microbeads and MACS sorting, plated on poly-ornithine coated 6-well multiwells at a density of 300.000 cells/well, expanded in proliferation medium (including PDGFAA and bFGF) for 48h, then switched to differentiation medium (with T3, without PDGFA and bFGF) for 24h.

extract_protocol: Total RNA extraction was performed using the RNeasy Micro kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

treatment_protocol: After approximately 4 hours from the switch in differentiation medium, microglia-derived EVs (i-EVs, IL4-EVs, or MSC-EVs) resuspended in 50 μl of medium or medium alone (CTRL) were added to the OPC culture (1:2 recipient OPCs-donor microglia ratio).

tag: strain

tag: cells

value: anti-CD140a MACS-sorted primary cells

tag: density

value: 300.000 cells/well


title: Alveolar macrophages, HCoV-OC43, Day0, rep2

description:
Library name: Sample 2
HCoV-OC43_Day0_2

value: WT


title: Western CLL,rep8

value: Western


title: Alveolar macrophages, HCoV-OC43, Day0, rep1

description:
Library name: Sample 1
HCoV-OC43_Day0_1


title: Wapl.WT_preselectionDP_HTGTS_TCR_seq_Traj_mix_rep2

description: Library name: sample14

tag: cell line


title: Wapl.WT_preselectionDP_HTGTS_TCR_seq_Traj_mix_rep3

description: Library name: sample15


title: Wapl.WT_preselectionDP_HTGTS_TCR_seq_Traj_mix_rep1

description: Library name: sample13


title: 5530

city: Asheville

first: Melissa

middle: Marie

last: Heintz

state: NC

address: 31 College Place, Suite B118

institute: ToxStrategies

description: Library name: Sample 72

data_processing:
Sequencing reads are demultiplexed using the standard sequencing instrument software and sample-specific barcodes to give a FASTQ file for each sample
bcl2fastq2 v2.20 for demultiplexing
no adapter trimming for TempO-Seq fastqs, default settings for clusters passing filters
fastq files were aligned to a pseudo transcriptome using Bowtie allowing up to 2 mismatches
Assembly: templated oligo sequencing
Supplementary files format and content: comma-separated text file includes raw count values for each gene_probe

source_name: liver

extract_protocol:
Mounted, unstained FFPE liver samples were scraped from the slides and processed according to the TempO-Seq® protocol by BioSpyder Technologies
TempO-Seq® protocol by BioSpyder Technologies

treatment_protocol: Female B6C3F1 mice within, 9-11 weeks of age with a weight range of 15-25 g were used in these studies. Animals received a subcutaneously implanted electronic identification chip and assigned to exposure groups by a stratified randomization scheme designed to achieve similar group mean body weights. Following randomization, mice were single- housed to ensure accurate water consumption data on a per-animal basis. Nine mice per group were exposed to TBA in drinking water at concentrations of 0, 10, 20, and 40 mg/mL for 14 or 28 days of consecutive exposure. Following the last day of exposure, mice were euthanized by CO2 anesthesia and exsanguination. Livers were fixed in 10% neutral-buffered formalin, embedded in paraffin (FFPE), and sections approximately 4 – 6 µm in thickness were mounted to glass slides.

value: liver

tag: Sex

value: female

tag: exposure route

value: drinking water

tag: exposure duration

value: 40 mg/mL


title: 5528

description: Library name: Sample 71


title: Alveolar macrophages, HCoV-OC43, Day3, rep2

description:
Library name: Sample 5
HCoV-OC43_Day3_2


title: Alveolar macrophages, HCoV-OC43, Day3, rep1

description:
Library name: Sample 4
HCoV-OC43_Day3_1


title: Western CLL,rep5


title: Western CLL,rep6


title: Aged.Splenic.CD8_HTGTS_TCR_seq_Trabj_mix_rep3

description: Library name: sample12

source_name: Aged Spleen

value: Aged Spleen

value: Splenic CD8+ T cells


title: Western CLL,rep7


title: Western CLL,rep4


title: 5527

description: Library name: Sample 70


title: Aged.Splenic.CD8_HTGTS_TCR_seq_Trabj_mix_rep2

description: Library name: sample11


title: Wapl.WT_preselectionDP_HTGTS_TCR_seq_Traj_mix_rep4

description: Library name: sample16

value: Thymus

value: Preselection DP thymocytes


title: Western CLL,rep3


title: Aged.Splenic.CD8_HTGTS_TCR_seq_Trabj_mix_rep1

description: Library name: sample10


title: 5524

description: Library name: Sample 69


title: 5523

description: Library name: Sample 68


title: 5522

description: Library name: Sample 67


title: Western CLL,rep2


title: Aged.Splenic.CD4_HTGTS_TCR_seq_Trabj_mix_rep3

description: Library name: sample9

value: Splenic CD4+ T cells


title: Western CLL,rep1


title: OPCs_MSC-EVs_rep2


title: OPCs_IL4-EVs_rep2

description: Gene expression data from OPCs treated with EVs derived from microglia stimulated with interleukin 4

source_name: OPCs treated with IL4-EVs


title: OPCs_IL4-EVs_rep1


title: OPCs_i-EVs_rep2

description: Gene expression data from OPCs treated with EVs derived from microglia stimulated with pro-inflammatory cytokines

source_name: OPCs treated with i-EVs


title: Aged.Splenic.CD4_HTGTS_TCR_seq_Trabj_mix_rep2

description: Library name: sample8


title: RSCV_1, stationary, rep3

city: Toledo

last: Prestwich

state: Ohio

address: 2801 W. Bancroft St.

department: Medicinal/Biological Chemistry

institute: University of Toledo

description: Library name: RSCV_1-3

data_processing:
Quality control and adapter trimming was performed with the bcl2fastq tool with default parameters. Read mapping was performed using hisat2. Read quantification was performed using Subread's featureCounts functionality.
Assembly: Assembly Accession GCF_000014625.1, NCBI BioSample Accession SAMN02603591, NCBI BioProject PRJNA386 (Lee et al., 2006)
Supplementary files format and content: csv file of the normalized raw counts for each sample

organism: Pseudomonas aeruginosa

growth_protocol: RSCV_1, RSCV_3, and WT PA14 cells were grown to stationary phase at 37 °C with orbital shaking for 16 to 18 hours. Cell pellets were washed with sterile saline water (0.9% NaCl) prior to RNA extraction.

extract_protocol:
RNA was harvested using a modified CTAB isolation protocol with phenol:chlorofom:isoamyl alcohol at a pH of 6.7. Isolated RNA was purified through proteinase K and DNAse treatment prior to ethanol precipitation.
RNA libraries were prepared using Illumina's Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit

value: Rugose small colony variant


title: OPCs_MSC-EVs_rep1


title: OPCs_IL4-EVs_rep3


title: OPCs_CTRL_rep3

description: Gene expression data from OPCs treated with control medium

source_name: OPCs treated with CTRL


title: OPCs_CTRL_rep2


title: Asian CLL,rep6

value: Asian


title: Aged.Splenic.CD4_HTGTS_TCR_seq_Trabj_mix_rep1

description: Library name: sample7


title: OPCs_CTRL_rep1


title: 5518

description: Library name: Sample 65


title: Splenic.CD3.T_2ug_HTGTS_TCR_seq_Trabj_mix1_rep3

description: Library name: sample6

source_name: Spleen

value: Spleen

value: Splenic CD3+ T cells


title: Asian CLL,rep5

value: CLL


title: RSCV_1, stationary, rep1

description: Library name: RSCV_1-1


title: RSCV_1, stationary, rep2

description: Library name: RSCV_1-2


title: OPCs_i-EVs_rep3

value: anti-CD140a MACS-sorted primary cells

value: 300.000 cells/well


title: Asian CLL,rep4


title: 5515

description: Library name: Sample 63


title: RSCV_3, stationary, rep3

description: Library name: RSCV_3-3


title: Asian CLL,rep3


title: Splenic.CD3.T_2ug_HTGTS_TCR_seq_Trabj_mix1_rep1

description: Library name: sample4

value: WT


title: 5519

description: Library name: Sample 66

value: liver


title: Splenic.CD3.T_2ug_HTGTS_TCR_seq_Trabj_mix1_rep2

description: Library name: sample5


title: OPCs_i-EVs_rep1


title: Asian CLL,rep2


title: Asian CLL,rep1


title: RSCV_3, stationary, rep2

description: Library name: RSCV_3-2


title: Splenic.CD3.T_100ng_HTGTS_TCR_seq_Trabj_mix1_rep2

description: Library name: sample2


title: Splenic.CD3.T_100ng_HTGTS_TCR_seq_Trabj_mix1_rep3

description: Library name: sample3


title: WT, stationary, rep2

description: Library name: WT 2


title: 5511

description: Library name: Sample 61

value: female


title: 5505

description: Library name: Sample 57

value: drinking water


title: WT, stationary, rep3

description: Library name: WT 3


title: 5510

description: Library name: Sample 60

value: 40 mg/mL


title: Splenic.CD3.T_100ng_HTGTS_TCR_seq_Trabj_mix1_rep1

description: Library name: sample1


title: 4529

description: Library name: Sample 54

value: 20 mg/mL


title: 5514

description: Library name: Sample 62


title: RSCV_3, stationary, rep1

description: Library name: RSCV_3-1


title: CB15p-shSCR

description:
AK01
CD34+ cord blood cells expressing non-targeting shRNA.

hyb_protocol: 16-18hr at 45C, FS450_0001 fluidics protocol for washing.

scan_protocol: Affymetrix GeneChip Scanner 3000.

data_processing: CEL files were imported into R and the probe expression values were normalized using the Robust Multichip Average (RMA) method from the 'oligo' (v.1.50.0) package. Probes that were lowly expressed, defined as not having normalized expression values greater than 4 in at least 3 samples, were filtered out. Probes that ambiguously mapped to multiple genes were filtered out.

source_name: Human cord-blood derived CD34+ cells

label_protocol: Biotin allonamide triphosphate.

growth_protocol: CD34+ cells were cultured in StemSpan SFEM media (Catalog # 09650, STEMCELL Technologies) supplemented with 20ng/mL CC100 cocktail (Catalog # 02690, STEMCELL Technologies) and 50ug/mL hTPO (Catalogue # 597402, BioLegend).

extract_protocol: Total RNA was harvested from transduced cells using TRIzol RNA extraction.

treatment_protocol: CD34+ cells were lentivirally transduced to express non-targeting shRNA or with two different shRNAs targeting FBXO11.

value: CD34+ cord blood cells

value: non-targeting shRNA


title: PreselectionDP_HTGTS_TCR_seq_Trbj_mix1_rep1

description: Library name: sample61

value: Thymus

value: Preselection DP thymocytes


title: C9337p-shFBXO11-10

description:
AK09
CD34+ cord blood cells expressing shRNA targeting FBXO11.

value: shRNA targeting FBXO11


title: 5517

description: Library name: Sample 64


title: C9337p-shFBXO11-9

description:
AK08
CD34+ cord blood cells expressing shRNA targeting FBXO11.


title: Control rep1

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: Tebiro 6-10-1

department: New Projects Development Division

country: Japan

institute: Toray Industries,Inc.

hyb_protocol: Hybridized for 16 h at 37 C with rotary shake (250 rpm). Hybridization buffer and washing protocol was followed by the protocol supplied by TORAY Industries, Inc..

scan_protocol: 3D-Gene Scanner ((Toray Industries Inc., Tokyo, Japan) was used for scanning. Images were quantified using Extraction(Toray Industries Inc., Tokyo, Japan).

data_processing: The raw data of each spot was normalized by substitution with a mean intensity of the background signal determined by all blank spots’ signal intensities of 95% confidence intervals.

source_name: Control virus-infected astrocytes derived from P0 Chd7flox/flox mouse cortex, cultured with 10% FBS, FGF2, EGF, and B-27 supplement

label_protocol: Amino Allyl aRNA was synthesis by Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion#1753). CyDye Coupling and fragmentation were performed as the protocol supplied by TORAY Industries, Inc..

growth_protocol: Astrocytes derived from P0 Chd7flox/flox mouse cortex were maintained in the presence of 10% FBS, FGF2, EGF, and B-27 supplement.

extract_protocol: Total RNA was isolated using an RNeasy Kit (Qiagen) according to the manufacturer's instructions.

tag: developmental stage

value: brain

value: astrocytes

tag: infection

value: control lentivirus


title: 5509

description: Library name: Sample 59


title: IVT_RNA, PyBo, rep2

description: Library name: IVT_RNA_4

data_processing:
Raw read data were quality-assessed by FastQC (v.0.11.9, https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Alignment to the reference genome was performed using STAR (v. 2.7.10a) allowing soft-clipping with non-default parameters: --outFilterScoreMinOverLread 0.33, --outFilterMatchNminOverLread 0.33 and --outFilterMismatchNoverReadLmax 0.02, that ensure mapping of overlapping reads and allowed for up to 2% of mismatches. The reference (GRCm39.dna_sm.primary_assembly.fa) was taken from https://ftp.ensembl.org/pub/release-107 and the genome index was generated using M.mus.GRCm39.107.gtf for annotation and --sjdbOverhang 99 (read length - 1).
Rsamtools (v2.16.0) was used to quantify how many C-to-T sites identified in cellular RNA were detected in the modification-free RNA, applying the following parameters : min_base_quality = 30 and min_mapq = 10. The average C-to-T signal was calculated as mean of two PyBo-treated replicates. If one replicate had insufficient coverage (<5 reads), the value from the replicate with adequate coverage was reported.
Assembly: GRCm39 (mm39)
Supplementary files format and content: bedGraph; PyBo-induced C-to-T signal (average) observed in IVT transcriptome which are shared with CTRL samples from cellular RNA

source_name: mouse embryonic stem cells

extract_protocol:
Total RNA was isolated from cultured cells using QIAzol Lysis Reagent (Qiagen) according to the manufacturer’s protocol followed by TURBO DNase (Invitrogen) treatment and another round of QIAzol purification. Poly(A)+ RNA was purified from total RNA using Oligo d(T)25 Magnetic Beads (Thermo Fisher Scientific), following the manufacturer’s protocol, with three consecutive rounds of selection.
IVT RNA generation: 400 ng poly(A)+ RNA isolated from mESCs was incubated with 1.67 µM oligo(dT)-VN RT primer and 1.67 mM dNTPs for 5 min at 75°C. For RT reaction, template switching RT enzyme mix and buffer (NEB) and 3.75 µM template switching oligo was added and incubated for 90 min at 42°C and 5 min at 85°C. 2nd strand cDNA synthesis was performed with Q5 Hot Start High-Fidelity DNA Polymerase (NEB), RNase H and 0.2 µM T7 extension primer for 15 min at 37°C, 1 min at 95°C and 15 min at 65°C. Double-stranded DNA was purified with HighPrep PCR beads (0.8x beads, MagBio) and used for in vitro transcription with the HiScribe T7 High Yield RNA Synthesis Kit (NEB) and canonical NTPs. Synthesized RNA was purified using DNase I (NEB) and the RNA Clean & Concentrator kit (Zymo research) following the manufacturer’s protocol. IVT RNA was size selected with HighPrep RNA Elite beads (0.6x beads, MagBio).
PyBo treatment: IVT RNA was incubated in 900 mM pyridine borane in 600 mM sodium acetate (pH 5.2) at 37°C for 24 hours. For mock treated samples, water was added instead of chemicals. RNA was purified using the RNA Clean & Concentrator kit (Zymo research).
RNA library preparation was performed using the TruSeq Stranded mRNA Library Prep kit (20020594, Illumina), entering the protocol at the RNA fragmentation step and excluding the poly(A) enrichment. 20 ng RNA was used as starting amount. Libraries were size-selected for >100 bp fragments.

tag: cell type

value: mouse embryonic stem cells

tag: genotype

value: Tet1/2/3 wildtype


title: 5506

description: Library name: Sample 58

tag: tissue


title: WT, stationary, rep1

first: Erin

description: Library name: WT 1


title: C9337p-shSCR

description:
AK07
CD34+ cord blood cells expressing non-targeting shRNA.

tag: shRNA


title: C9252p-shFBXO11-10

description:
AK06
CD34+ cord blood cells expressing shRNA targeting FBXO11.


title: Lung, PBS, WT, rep2

description: Library name: Sample 11

source_name: Lung

extract_protocol:
Total cellular RNA was extracted with the Magzol reagent (Magen).
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: Lung


title: NRP1-SUM159-siNT-RNA.3

city: Brisbane

first: Colleen

last: Nelson

state: Queensland

address: 199 Ipswich Road

department: Institute of Health and Biomedical Innovation; Vancouver Prostate Centre

institute: Queensland University of Technology; University of British Columbia

data_processing:
Paired-end sequencing on the MGI DNBSEQ-G400 sequencer at QUT Central Analytical Research Facility (CARF), Brisbane, Queensland, Australia with a read length of 150 bp and ~40M read pairs per sample.
Quality control of the raw reads was performed using FastQC (v.0.11.9).
Raw sequencing reads were trimmed for quality control using TrimGalore (v.0.6.5).
Trimmed sequence reads were mapped to the human genome and transcriptome using the STAR (v.2.7.2b) aligner.
Read quantification was performed using RSEM (v.1.3.3).
Assembly: GRCh38 / hg38 and Ensembl.v.114 / Gencode.v.48 (May-2025).
Supplementary files format and content: Tab separated TXT files with raw COUNTS, TPM and ISOPCT (Isoform Percentage) values for human genes and transcript variants, respectively, for each sample as generated by RSEM.

source_name: Breast Cancer cell line

growth_protocol: MDA-MB-231, HS578T and SUM159PT claudin-low breast cancer cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM), High Glucose, Pyruvate supplemented with 10% Fetal Bovine Serum (FBS) both from Gibco. HEK293T cells were cultured in DMEM, High Glucose, Pyruvate supplemented with 5% FBS (Gibco, Thermofisher Scientific). All cells were maintained in 37 degrees celcius, 5% CO2 incubators.

extract_protocol:
Cells were lysed with TriZol reagent (Invitrogen, Thermofisher Scientific). Total RNA isolation was performed using the Direct-zol RNA Miniprep Plus kit (Zymo Research) as per manufacturer’s instructions. The 2100 Bioanalyzer (Agilent Technologies) was used to determine the RNA integrity number (RIN), with the RNA 6000 Nano Kit (Agilent Technologies) according to manufacturer’s instructions used for sample preparation. Samples with a RIN >8 were submitted for sequencing.
RNA libraries for RNA-seq were prepared using the Illumina TruSeq Stranded mRNA Sample Prep Kit (strand-specific, polyA enrichment), no ERCC controls. Input for each sample was 500 ng.

treatment_protocol: siRNA reverse transfection: 20 nM siRNA was reverse transfected into 0.7 x 106 SUM159PT, MDA-MB-231 and HS578T cells using 1:500 Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen, Thermofisher Scientific) and 1:10 Opti-MEM™ Reduced Serum Medium (Gibco, Thermofisher Scientific) to DMEM supplemented with 10% FBS (Gibco, Thermofisher Scientific). RNA was extracted from the cells at 72hrs post-transfection. A predesigned Silencer™ Select negative control (Negative Control siRNA No. 1, #4390844, Thermofisher Scientific) was used in addition to two custom Silencer™ Select NRP1-targeting siRNAs (Thermofisher Scientific) which are siNRP1 3 and siNRP1 5. siNRP1 3 targeted transmembrane-NRP1 only, whereas siNRP1 5 targeted total NRP1. Lentiviral transduction of shRNA: 1.5 x 10^6 HEK293T cells per shRNA were seeded and cultured in 10 mL of DMEM, High Glucose, Pyruvate supplemented with 10% heat inactivated FBS (Gibco, Thermofisher Scientific). FBS heat inactivation was done by a 30-minute incubation in a 56 degrees cecius water bath. At 50-60% confluency, the HEK293T cells were transfected with 200 µL of lentiviral transfection mix. Transfection mix was prepared as follows: serum-free DMEM (Gibco), 12 µL X-tremeGENE™ HP DNA Transfection Reagent (Sigma Aldrich), 1.8 µg pCMV Delta 8.2 R (Addgene), 0.2 µg pCMV-VSV-G (Addgene) and 2.0 µg of shRNA cloned to the pLKO.1 lentiviral vector (Addgene) was combined to a 200 µL final volume and incubated for 15 minutes at room temperature. After an overnight incubation at 37oC with 5% CO2, all transfection media was aspirated from the HEK293T cells and replaced with 7 mL of regular culture media. Viral supernatant was then harvested at 48 hrs and 72 hrs post-transfection. For transduction of the shRNA, 0.7 x 106 HS578T, MDA-MB-231 and SUM159PT cells per shRNA were cultured to 30-50% confluency. The cells were then incubated at 37 degrees celcius overnight in 4.6 mL of previously harvested lentiviral supernatant with 6 µg/mL of protamine sulfate (Sigma Aldrich). Successfully transduced cells were selected with 1 µg/mL puromycin (Gibco, Thermofisher Scientific) for a minimum of three days for before use and maintained in regular culture media. Cells were seeded at 0.7 x 10^6 and RNA was extracted at 80-90% confluency. The shRNAs used were a non-targeting shRNA (Thermofisher Scientific), and two custom NRP1-targeting shRNA sequences obtained from Thermofisher Scientific, which are shNRP1 3 and shNRP1 5. shNRP1 3 targets transmembrane-NRP1 only, whereas shNRP1 5 targets total NRP1.

value: Breast Cancer cell line

value: siNT


title: Chd7 KD rep3

source_name: Cre virus-infected astrocytes derived from P0 Chd7flox/flox mouse cortex, cultured with 10% FBS, FGF2, EGF, and B-27 supplement

value: cre lentivirus


title: IVT_RNA, mock, rep2

description: Library name: IVT_RNA_2

value: mock


title: C9252p-shFBXO11-9

description:
AK05
CD34+ cord blood cells expressing shRNA targeting FBXO11.


title: 5504

description: Library name: Sample 56

value: liver

value: female

value: drinking water

value: 40 mg/mL


title: IVT_RNA, mock, rep1

description: Library name: IVT_RNA_1


title: Chd7 KD rep2


title: 4524

description: Library name: Sample 50

value: 20 mg/mL


title: Splenic.CD8_HTGTS_TCR_seq_TcrV_mix_rep1

description: Library name: sample52

value: Spleen

value: Splenic CD8+ T cells

value: WT


title: C9252p-shSCR

description:
AK04
CD34+ cord blood cells expressing non-targeting shRNA.

value: CD34+ cord blood cells

value: non-targeting shRNA


title: CB15p-shFBXO11-10

description:
AK03
CD34+ cord blood cells expressing shRNA targeting FBXO11.

value: shRNA targeting FBXO11


title: IVT_RNA, PyBo, rep1

description: Library name: IVT_RNA_3

value: mouse embryonic stem cells

value: Tet1/2/3 wildtype


title: Lung, HCoV-OC43, WT, rep3

description: Library name: Sample 9

value: Lung


title: NRP1-SUM159-siNT-RNA.1

value: Breast Cancer cell line

value: siNT


title: 4521

description: Library name: Sample 49


title: 5502

description: Library name: Sample 55


title: Chd7 KD rep1

value: brain

value: astrocytes

value: cre lentivirus


title: PreselectionDP_HTGTS_TCR_seq_Trbj_mix1_rep3

description: Library name: sample63

source_name: Thymus

value: Thymus

value: Preselection DP thymocytes


title: NRP1-SUM159-siNRP1.5-RNA.3

value: siNRP1 5


title: 4519

description: Library name: Sample 48

value: liver

value: female

value: drinking water

value: 20 mg/mL


title: Splenic.CD4_HTGTS_TCR_seq_TcrV_mix_rep2

description: Library name: sample50

value: Spleen

value: Splenic CD4+ T cells

value: WT


title: Lung, HCoV-OC43, WT, rep1

description: Library name: Sample 7


title: Control rep3

value: control lentivirus


title: PreselectionDP_HTGTS_TCR_seq_Trbj_mix1_rep2

description: Library name: sample62


title: CB15p-shFBXO11-9

description:
AK02
CD34+ cord blood cells expressing shRNA targeting FBXO11.

value: CD34+ cord blood cells

value: shRNA targeting FBXO11


title: Lung, PBS, WT, rep3

description: Library name: Sample 12

value: Lung


title: 4528

description: Library name: Sample 53


title: 4526

description: Library name: Sample 52


title: NRP1-SUM159-siNRP1.3-RNA.3

value: Breast Cancer cell line

value: siNRP1 3


title: Control rep2

value: brain

value: astrocytes


title: 4516

description: Library name: Sample 46


title: Splenic.CD8_HTGTS_TCR_seq_TcrV_mix_rep3

description: Library name: sample54

value: Splenic CD8+ T cells


title: NRP1-SUM159-siNRP1.3-RNA.2


title: 4525

description: Library name: Sample 51


title: Lung, PBS, WT, rep1

description: Library name: Sample 10


title: Splenic.CD8_HTGTS_TCR_seq_TcrV_mix_rep2

description: Library name: sample53


title: NRP1-SUM159-siNT-RNA.2

value: siNT


title: Lung, HCoV-OC43, WT, rep2

description: Library name: Sample 8


title: NRP1-SUM159-siNRP1.5-RNA.2

value: siNRP1 5


title: Splenic.CD8_HTGTS_TCR_seq_TcrJ_mix_rep1

description: Library name: sample46


title: 4518

description: Library name: Sample 47

value: liver

value: female


title: Splenic.CD4_HTGTS_TCR_seq_TcrV_mix_rep3

description: Library name: sample51


title: Lung, HCoV-OC43, Csf2rb KO, rep2

description: Library name: Sample 2

value: Csf2rb KO


title: Lung, PBS, Csf2rb KO, rep3

description: Library name: Sample 6


title: Splenic.CD4_HTGTS_TCR_seq_TcrV_mix_rep1

description: Library name: sample49

value: Spleen


title: Splenic.CD8_HTGTS_TCR_seq_TcrJ_mix_rep3

description: Library name: sample48


title: 4513

description: Library name: Sample 45

value: drinking water

value: 20 mg/mL