title: WT_fPAR-CLIP

city: Shenzhen

first: Wee Siong Sho

last: Goh

address: Shenzhen Bay Laboratory, No. 5, Kelian Road, Goh Lab, A1819, Guangming District

institute: Shenzhen Bay Laboratory

data_processing:
Basecalling was performed using Illumina RTA, with further demultiplexing performed by Illumina bcl2fastq 2.20.
Fastq sequences were first pre-trimmed to 50bp and read1 sequences were analyzed using PARpipe14, which defines protein-RNA interaction sites using PARalyzer v1.5 on hg38 genome assembly and cutoff=0.15. The interaction sites were identified as clusters, and the distances of such clusters to their nearest TSS (within range of +/- 2kb) and PAS (within range of +/- 5kb) were recorded. Cluster density within 40nt bins were calculated and visualized as Metagene plots using Metagene2. For gene list overlap analysis, PCF11-targets were defined as genes containing PCF11 fPAR-CLIP clusters within 1kb downstream of the TSS.  AAAAAAAAAAAAAAAAAAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT.
Assembly: hg38
Supplementary files format and content: bedgraph
Library strategy: fPAR-CLIP

extract_protocol:
Cells were washed once with 1× ice-cold PBS and crosslinked with 0.4J/cm² of 365nm UV light in an Analytik Jena UVLink 3000 Crosslinker (Analytik Jena). Following crosslinking, cells were collected in PBS and centrifuged at 500g at 4°C for 5min. The cell pellet was lysed using the Nuclei EZ following the manufacturer's protocol. The nuclei were then lysed in 3x pellet volume of RIPA buffer supplemented with protease inhibitor (20mM Tris, pH 7.5, 150mM NaCl, 0.1% SDS, 0.15% sodium deoxycholate, 1% NP-40, 0.5mM DTT). The sample was sonicated for 45s (ON time) at 50% amplitude, with 10s ON and 15s OFF, using a Q800R3 Sonicator (Qsonica) and cell extract was clarified at 15,000g, 4°C for 15min.
Immunoprecipitation was performed by incubating the clarified extracts with antibodies pre-coated on Protein A&G beads (50µl of 1:1 mixture Dynabead-Protein A and Dynabead-Protein G beads per 0.5 ml of lysate). Binding was performed overnight at 4°C with gentle rotation, and the beads were washed three times with 1 ml of IP buffer (20mM Tris, pH 7.5, 150mM NaCl, 2mM EDTA, 1% NP-40. The beads were treated with 0.15 U/µl RNase I in 2x bead volume of IP buffer at 22°C for 10min. Subsequently, the beads were washed twice with 1ml of each of the following: high salt wash buffer (20mM Tris, pH 7.5, 500 mM NaCl, 2mM EDTA, 1% NP-40), RIPA buffer, and dephosphorylation buffer (50mM Tris–HCl, pH 7.5). On-bead RNA decapping was achieved using Cap-Clip™ Acid Pyrophosphatase (Cellscript, C-CC15011H) according to the manufacturer's instructions, adjusted to 1x beads volume. The reaction was performed at 37°C for 60min with shaking at 1krpm. Beads were then washed twice with dephosphorylation buffer, and on-bead RNA dephosphorylation was achieved using Quick CIP (NEB, M0525S) according to manufacturer’s instructions adjusted to 1x bead volume. The reaction was supplemented with 1U/µl RNase inhibitor (SUPERase•In™, Thermo Scientific, AM2694) and performed at 37°C for 10min with shaking. Finally, beads were washed twice with dephosphorylation buffer and twice with ligation buffer without DTT. RNA footprints were ligated to fluorescent 3’ barcode adapters (Table S6) using T4 Rnl2(1–249)K227Q (NEB, M0351). A volume of ligation mix (1× T4 RNA Ligase Reaction Buffer, 15% PEG8000, 25µM adapter, 10U/µl Rnl2(1–249) K227Q ligase, 1U/µl RNase inhibitor) equal to that of the beads was added to the beads, and ligation was performed at 4°C for 16h with slow tumbling rotation. The beads were then washed three times with 1ml ligation buffer without DTT. The footprints were phosphorylated using T4 PNK (NEB, M0201S) according to the manufacturer's instructions, adjusted to the volume of the beads. The reaction was supplemented with 1U/µl RNase inhibitor and performed at 37°C for 30min with shaking at 1krpm. The beads were washed twice with high salt buffer and twice with IP buffer. PCF11 was recovered with the addition of 1 bead volume of 2× SDS loading buffer (4x Laemmli Buffer (Bio-Rad  1610747) diluted 2x and supplemented with beta-mercaptoethanol) and incubated at 95°C for 5min. 10% of the eluted RNPs was used for Western blot analysis with an anti-PCF11 antibody. The remaining 90% of the eluted PCF11 was separated on a 4–12% SDS-PAGE alongside 20µl of a 1/10 dilution of Prestained Protein Ladder, 10 to 250 kDa (PageRuler Plus, Thermo Scientific, 26620). The gel was visualized directly using a Bio-Rad imaging system at the IR 660nm detection channel, and the bands corresponding to the PCF11 fluorescent 3′ adapter ligation products were excised and shredded using gel breaker tubes (IST Engineering BioTech, 3388). The shredded gel was incubated with 100µl (1× shredded gel volume) of 2× proteinase K buffer (100mM Tris-HCl, pH 7.5, 150mM NaCl, 12.5mM EDTA, 2% SDS) supplemented with 50mM DTT at 90°C for 2min. Proteolysis was achieved with three successive proteinase K treatments, performed by adding 400 µl of 2.4mg/ml proteinase K (Invitrogen AM2542) in 2× proteinase K buffer and incubating at 50°C for 30min, then adding 1.5mg/ml proteinase K in 250µl H2O and incubating at 50°C for 30min, and repeating this last step once more (the final volume is approximately 10 times that of the typical gel piece, which is around 100µl in volume). The shredded gel was removed using filter tubes (Millipore UFC30GV0S). The RNA was purified by phenol/chloroform/IAA extraction and precipitated using isopropanol and 15µg GlycoBlue™ (Thermo Fisher Scientific/Ambion, AM9516), then redissolved in 9 µl of H2O. 2µl of 10× RNA ligase buffer containing ATP (provided with Rnl1 T4 RNA ligase, Thermo Fisher Scientific, EL0021), 6µl of 50% PEG-8000, and 1 µl of 10 µM fPARclip 5′ adapter (Table S6) were added to the RNA sample. The RNA was denatured at 90°C for 1min and immediately cooled on ice for 2min. 2µl of Rnl1 T4 RNA ligase (Thermo Fisher Scientific, EL0021) was added, and the reaction mixture was incubated at 37°C for 1h. The RNA was purified using Oligo Clean & Concentrator™ (Zymo Research, D4060) and eluted with 11.5µl of H2O. cDNA was synthesized using fPARclip RT primer (Table S6) and SuperScript™ IV Reverse Transcriptase (Thermo Fisher Scientific, 18090010). The reaction mix was prepared according to the manufacturer's instructions. Briefly, 50pmol of fPAR-CLIP RT primer (Table S6) and 1µl of 10mM dNTP mix (each) were added to 11µl of adapter-ligated RNA sample, heated at 65°C for 5 min, and chilled on ice. 4µl of 5× SSIV Buffer, 1µl of 100mM DTT, 1µl of RNase inhibitor, and 1µl of SuperScript™ IV Reverse Transcriptase (200 U/µl) were added, and the reaction mix was incubated at 56°C for 30min. The reaction was inactivated by heating at 80°C for 10min and 60µl H2O was added to the reverse transcription reaction. Low-cycle PCR was performed using Platinum™ Taq DNA Polymerase (Thermo Fisher Scientific, 10966034), 5′ PCR primer short (Table S6), and fPARclip RT primer according to the manufacturer's instructions (10 µl of cDNA in 400 µl total, across 8 PCR tubes). The PCR programme was as follows: 94°C for 2min; 6 cycles of 94°C for 30s, 60°C for 30s, 72°C for 15s; 72°C for 1min. The amplified DNA was purified and concentrated using the DNA Clean & Concentrator™ kit (Zymo Research, D4013). Size selection of the amplified DNA was performed using the LightBench instrument (Yourgene Health) with a 3.0% agarose in a 12- channel in-channel filter size selection cassette (Yourgene health，CG-10600-13-300), where fragments between 78 and 100bp were extracted. The size selected sample was used as a template for a 1× 100 µl PCR reaction with 50 pmol Truseq PCR primers (Illumina) with the following PCR program: 94°C for 2min; 18 cycles of 94°C for 30s, 60°C for 30s, 72°C for 15s; 72°C for 1min. The fPAR-CLIP libraries were purified using DNA Clean & Concentrator™ kit (Zymo Research, D4013). Size selection of fPAR-CLIP sequencing libraries was performed using the LightBench instrument (Yourgene health). Briefly, libraries were run through a 3.0% agarose in a 12- channel in-channel filter size selection cassette (Yourgene health，CG-10600-13-300). Fragments between 145 and 175bp were extracted, corresponding to an expected fPAR-CLIP insert length of approximately 20 to 35 bp. The quality of the final library was assessed using the TapeStation system (Agilent). Sequencing was performed on an Illumina NovaSeq 6000 platform.

treatment_protocol: 16 hours before crosslinking, 4-thiouridine (4SU) was added to the cell culture medium at a final concentration of 100µM.

value: Human embryonic kidney

value: WT


title: DiFi_DGUC_F03

status: Public on Oct 31 2024

type: SRA

city: La Jolla

first: Louise

middle: C

last: Laurent

state: CA

address: SCRM, 2880 Torrey Pines Scenic Drive

department: Obstetrics, Gynecology, and Reproductive Sciences

country: USA

institute: UCSD

library_source: transcriptomic

data_processing:
All FASTQ files were processed uniformly through the extra-cellular RNA processing toolkit (exceRpt). Information about exceRpt is available at https://github.gersteinlab.org/exceRpt/.
Assembly: hg19
Supplementary files format and content: Each processed data file is a core results archive generated by exceRpt. The core results archive contains read counts for alignments to the different transcriptome libraries used by exceRpt (miRNA / piRNA / tRNA / GENCODE annotations / circRNA), taxonomy trees for exogenous rRNA and genomic reads aligned by exceRpt, and a variety of other files generated by exceRpt.

molecule: total RNA

organism: Homo sapiens

source_name: cell culture

extract_protocol:
RNA Isolation method was "500uL of biofluid was incubated with 100uL warmed Slurry and 900uL warmed Lysis Buffer A (containing 2-Mercaptoethanol) for 10 min at 60C. To this 1.5 mL of 100% ethanol was added and centrifuged for 2min at 1000rpm. To the pellet 300uL Lysis Buffer A was added and incubated for 10 min at 60C. 300uL 100% Ethanol was added, loaded onto the filter column and centrifuged for 30sec at 1000xg followed by 1 min at 16000xg. The column was washed thrice with 400uL Wash Solution for 1 min at 16000 xg and then centrifuged once again for 3 min to dry the membrane. RNA was eluted in 14uL water with a slow spin for 2 mins at 300 xg followed by a spin for 3 min at 16000 xg.". RNA Isolation kit was Plasma/Serum Circulating and Exosomal RNA Purification Kit(Norgen Biotek).
Library construction kit was NEBNext small RNA library prep (NEB). Samples were multiplexed. Type of Starting Material was total RNA.

tag: disease

value: Colorectal Carcinoma

tag: anatomical location

value: Colon structure

tag: rna source

value: other


title: DiFi_DGUC_F7_qEV_F13


title: HK-2 cell, GLT, 24h, rep3

city: Beijing

first: Jiayin

last: Tao

address: Chaoyang Beisanhuandong Street 11

country: China

institute: Beijing University of Chinese Medicine

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Assembly: genome-version GRCh38
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: kidney

growth_protocol: HK-2 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. HK-2 cells were cultured in a humidified atmosphere of 37℃ and 5% CO2 and used in experiments upon reaching 70–80% confluence.

extract_protocol:
For preparation of total RNA, using TRIZOL (Thermo Fisher, 15596018CN), chloroform and absolute ethanol forprecipitation.
Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations.

treatment_protocol:
1mM NaB was added into the cell culture for 24h to harvest
25 mM glucose and 250 μM PA were added into the cell culture for 24h to harvest

tag: tissue

value: kidney

tag: cell line

value: Human kidney-2 cell

tag: cell type

value: proximal tubular cell (PTC) line

tag: genotype

tag: treatment


title: DiFi_CD81_B2_1


title: SMARCB1_Dox_rep1_Ribo

city: Stanford

first: Anastasia

last: Hains

email: hainsa@uchicago.edu, hains@stanford.edu

address: 269 Campus Drive

institute: Stanford University

description: RNA associated with active ribosome

data_processing:
Aligned to hg38 genome with STAR aligner
Reads counted with HTseq
Assembly: hg38
Supplementary files format and content: tab-delimitated text file that contains raw counts for each file

growth_protocol: G401 rhabdoid kidney tumor cells were cultured in McCoy5a's medium (Sigma-Aldrich cat# 8403) supplemented with 10% FBS serum (Biowest S162TA) + 1% Pen/Strep + 1% glutamax (thermo scientific 35050061)

extract_protocol:
RNA was harvested using Ribolace kit (Immagina). 1.5ug of RNA was used to construct library
Library was prepared using Laceseq kit (Immagina) following manufactor's protocol

treatment_protocol: After 24hrs of seeding and reached 80% confluency, G401 SMARCB1-inducible or GFP-inducible cells were treated with 0.1ug/mL doxycycline or equivalent DMSO for 12hrs. Treated cells were harvested for experiment.


title: DiFi_CD9_FT2_3


title: HK-2 cell, GLT, 24h, rep1


title: Cytoplasm region MuV-infected A549 cells except for IB

city: Bunkyo-ku

first: Hiroshi

last: Katoh

state: Tokyo

country: Japan

institute: The University of Tokyo

data_processing:
The barcodes and unique-molecular-identifiers in the reads were extracted using UMI-tools (version 1.0.0) with the command: umi_tools extract -I r1.fastq --read2-in=r2.fastq --bc-pattern=NNNNNNCCCCCC --read2-stdout.
The reads were mapped by the aligning software HISAT2 (version 2.1.0) to the reference genome (GRCm38).
Read counts per gene was performed with featureCounts (version 1.6.4) and UMI-tools with the command: umi_tools count --method=directional --per-gene --per-cell --gene-tag=XT.
The duplicates in unique molecular identifiers were discarded.
Using the resulting counts, differentially expressed genes (those with adjusted p-values < 0.1) were extracted by the R library DESeq2 (version 1.20.0).
Assembly: hg19
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

molecule: cytoplasmic RNA

extract_protocol:
Lysis solution(0.1% Tween and 400 ug/ml of ProteinaseK in PBS) were loaded onto the specimens and incubated for 30 min at 55℃. cDNA;mRNA hybrids were purified with a Qiagen MinElute PCR Purification kit.
Library was constructed according to the CEL-seq2 method as follows: polyA RNAs were converted in situ to cDNAs using a polyT primer with a 5'-tail containing T7 promoter, 5' Illumina sequence adaptor, UMI and cell barcode sequences. The cDNAs were extracted and second strand DNAs were synthesized by nick translation method. The cDNAs were linearly amplified as RNAs (aRNAs) by IVT reaction, followed by reverse transcription using random primer with a 5'-tail containing 3' Illumina sequence adaptor.
Spatial transcriptome analysis using PIC (photo isolation chemistry).  PIC is a bulk RNA-seq technique to prepare cDNA libraries from specific regions (not cells) in tissue sections.

value: Human cell line derived from lung cancer

value: MuV infection


title: HK-2 cell, NaB, 24h, rep2


title: DiFi_CD63_B2_2


title: DiFi_CD81_B2_3


title: DiFi_CD81_FT2_2


title: Cytoplasmic region of mock-infected A549 cells

value: Mock


title: HK-2 cell, NaB, 24h, rep1


title: HONE-1 cells, siRAN 3

city: Guangzhou

first: Yang

last: Pan-Yang

address: 651 Dongfeng East Road

department: Collaborative Innovation Center of Cancer Medicine

institute: Sun Yat-sen University Cancer Center

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include raw counts for each sample
Supplementary files format and content: tab-delimited text files include FPKM values for each sample

extract_protocol:
RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: nasopharyngeal carcinoma cells

value: RAN knockdown


title: HONE-1 cells, negative control for siRAN 3


title: DiFi_Total_Input_2

value: total cell-free biofluid RNA


title: DiFi_DGUC_F04


title: HONE-1 cells, siRAN 2


title: DiFi_DGUC_F7_qEV_F01


title: HONE-1 cells, siRAN 1


title: DiFi_CD81_FT2_1


title: DiFi_DGUC_F7_qEV_F04


title: HONE-1 cells, negative control for siRAN 1


title: HK-2 cell, GLT, 24h, rep2


title: DiFi_DGUC_F06


title: HK-2 cell, NaB, 24h, rep3


title: HONE-1 cells, negative control 2


title: DiFi_DGUC_F7_qEV_F10


title: HONE-1 cells, siG3BP1 1

value: G3BP1 knockdown


title: HONE-1 cells, negative control 1


title: DiFi_CD9_FT1_3


title: DiFi_DGUC_F7_qEV_F12


title: DiFi_CD81_B2_2


title: DiFi_DGUC_F02


title: DiFi_DGUC_F7_qEV_F02


title: SUNE-1 cells RAN-INPUT

data_processing:
Illumina Bcl2FastQ  software is used for base-calling.
Adaptor and low-quality bases were trimmed with Trimmomatictools(version:0.36), and the clean reads underwent rRNA deleting through RNAcentral to get effective reads. Genomic alignment (hg19 from UCSC genome browser) was using Tophat(version:2.0.13) to get uniquely mapping reads.
RIPseeker was employed to perform peak calling. Then use Hommer(version 4.8) to annotate the Peaks. The nucleotides in the Peaks region were used for the detection of the consensus motif by DREME (version:4.11.1) and MEME(version:4.11.1). Motif central enrichment was performed by CentriMo (version:4.11.1).Differential peak enrichment was determined by Pyicoenrich(version 2.0.7).
Assembly: hg19
Supplementary files format and content: a tabular file which contains information about called peaks（except for input sample）

extract_protocol:
RNA was harvested using Trizol reagent. 1 ug of total RNA was used for the construction of sequencing libraries.
Briefly, rRNAs were removed from Total RNA usingQIAseqFastSelct-rRNA HRM KIT(QIAGEN, Germany)and fragmented to approximately 200bp. Subsequently, the purified RNA fragments were subjected to first-strand and second-strand cDNA synthesis followed by adaptor ligation and enrichment with a low-cycle according to instructions of NEBNext®Ultra™ Directional RNA Library Prep Kit for Illumina(NEB, USA)

tag: rip antibody


title: SUNE-1 cells RAN-RIP


title: DiFi_CD63_B2_1


title: HONE-1 cells, negative control for siRAN 2


title: HONE-1 cells, siG3BP1 2


title: HONE-1 cells RAN-RIP


title: DiFi_CD81_FT2_3


title: HONE-1 cells RAN-INPUT


title: DiFi_CD63_B2_3


title: DiFi_DGUC_F01


title: pooledCells_scADT_S3

city: Erlangen

first: Kilian

last: Schober

state: Bavaria

address: Maximiliansplatz 2

department: Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene

country: Germany

institute: Universitätsklinikum Erlangen

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
Assembly: GRCh38 (version 2020A, 10x Genomics), vdj-GRCh38 (version 5.0.0, 10x Genomics), barcodes.csv
The sequencing sample was processed via the cellranger (version 6.0.2, 10x Genomics) `multi`-command using the gene expression reference GRCh38 (version 2020A, 10x Genomics), the VDJ reference vdj-GRCh38 (version 5.0.0, 10x Genomics), and customized feature barcode references.
Supplementary files format and content: comma-separated value files and matrix files

source_name: PBMC

extract_protocol:
PBMCs were thawed from cryopreserved stocks and rested overnight at 1x10^6 cells/mL in cRPMI medium (RPMI 1640 Medium + 10% heat-inactivated FCS, 0.05 mM β-mercaptoethanol, 0.05 mg/mL gentamicin, 1.1915 g/L HEPES, 0.2 g/L L-glutamine, 100 U/mL Penicillin-Streptomycin). 5x10^6 PBMCs per donor and timepoint were recovered and first stained with a cocktail of surface antibodies and individual hashtag antibodies (2.5 µL per 5x10^6 PBMCs of TotalSeq-C anti-human hashtag antibodies 1–8, Biolegend, 394661, 394663, 394665, 394667, 394669, 394671, 394673, 394675) for 30 min on ice. Afterwards, up to 8 individual samples were pooled andwere later distinguished by individual CD45 color-barcoding at the sorter and by individual TotalSeq-C anti-human hashtag antibodies in the scRNAseq dataset. Meanwhile, a dextramer cocktail containing 16 different dextramers was prepared. For this cocktail, 8 µL per 40x10^6 cells of dCODE dextramers® (immudex) and 100 µM d-biotin (1/10 of total dextramer volume) were pre-mixed in FACS-buffer (PBS + 0.5% BSA) to block free binding sites. The pooled samples (40x10^6 cells) were stained with 400 µl of this dextramer cocktail for 30 min on ice. Single, live, CD19-, CD56-, CD8+, dextramer-positive lymphocytes were sorted in previously FCS-coated 1.5 mL tubes filled with FACS-buffer. Cells were sorted on a FACS Aria II cell sorter (BD). Immediately after sorting, cells were loaded to a Chromium Next GEM Chip K (10X Genomics) and Chromium Next GEM Single-Cell 5′ kits v.2 were used to generate GEX, VDJ and CITEseq libraries according to the manufacturer’s instructions (10X Genomics, 1000263, 1000256, 1000252, 1000286, 1000250, 1000215, 1000190). Libraries were sent to Novogene and sequenced on an Illumina NovaSeq platform with PE150 strategy.
Library construction was performed according to manufacturer's instructions (10x Genomics) for 5' GEX, VDJ and CITEseq, V2 chemistry dual index

value: PBMC

value: T cells

value: SARS-CoV-2 vaccination

tag: adt/hto

value: see barcodes.csv


title: DiFi_DGUC_F7_qEV_F11

value: Colorectal Carcinoma


title: pooledCells_scADT_S1

description: 10x Genomics


title: DiFi_CD9_B1_3

value: Colon structure


title: DiFi_IgG_FT1_2

value: other


title: pooledCells_scVDJ_T1

description: 10x Genomics


title: pooledCells_scVDJ_S3

description: 10x Genomics


title: DiFi_CD9_B1_4


title: DiFi_CD63_B1_4


title: DiFi_CD63_B1_6


title: pooledCells_scVDJ_S1

description: 10x Genomics


title: pooledCells_scGEX_S3

description: 10x Genomics


title: pooledCells_scGEX_T1

description: 10x Genomics


title: DiFi_CD81_FT2_6


title: DiFi_CD63_FT1_5


title: DiFi_Total_Input_7


title: pooledCells_scGEX_S1

description: 10x Genomics


title: pooledCells_scVDJ_P1

description: 10x Genomics


title: pooledCells_scGEX_P1

description: 10x Genomics


title: pooledCells_scADT_S2

description: 10x Genomics

extract_protocol:
PBMCs were thawed from cryopreserved stocks and rested overnight at 1x10^6 cells/mL in cRPMI medium (RPMI 1640 Medium + 10% heat-inactivated FCS, 0.05 mM β-mercaptoethanol, 0.05 mg/mL gentamicin, 1.1915 g/L HEPES, 0.2 g/L L-glutamine, 100 U/mL Penicillin-Streptomycin). 5x10^6 PBMCs per donor were recovered and first stained with a cocktail of surface antibodies and individual hashtag antibodies (2.5 µL per 5x10^6 PBMCs of TotalSeq-C anti-human hashtag antibodies 1–8, Biolegend, 394661, 394663, 394665, 394667, 394669, 394671, 394673, 394675) for 30 min on ice. Afterwards, 8 individual samples were pooled andwere later distinguished by individual CD45 color-barcoding at the sorter and by individual TotalSeq-C anti-human hashtag antibodies in the scRNAseq dataset. Meanwhile, a dextramer cocktail containing 16 different dextramers was prepared. For this cocktail, 8 µL per 40x10^6 cells of dCODE dextramers® (immudex) and 100 µM d-biotin (1/10 of total dextramer volume) were pre-mixed in FACS-buffer (PBS + 0.5% BSA) to block free binding sites. The pooled sample (40x10^6 cells) was stained with 400 µl of this dextramer cocktail for 30 min on ice. Single, live, CD19-, CD56-, CD8+, dextramer-positive lymphocytes were sorted in previously FCS-coated 1.5 mL tubes filled with FACS-buffer. Cells were sorted on a FACS Aria II cell sorter (BD). Immediately after sorting, cells were loaded to a Chromium Next GEM Chip K (10X Genomics) and Chromium Next GEM Single-Cell 5′ kits v.2 were used to generate GEX, VDJ and CITEseq libraries according to the manufacturer’s instructions (10X Genomics, 1000263, 1000256, 1000252, 1000286, 1000250, 1000215, 1000190). Libraries were sent to Novogene and sequenced on an Illumina NovaSeq platform with PE150 strategy.
Library construction was performed according to manufacturer's instructions (10x Genomics) for 5' GEX, VDJ and CITEseq, V2 chemistry dual index


title: DiFi_CD9_B2_6


title: DiFi_CD9_B2_4


title: DiFi_CD9_FT2_5


title: pooledCells_scADT_T1

description: 10x Genomics


title: pooledCells_scVDJ_S2

description: 10x Genomics


title: DiFi_CD81_FT1_2


title: pooledCells_scGEX_S2

description: 10x Genomics


title: pooledCells_scADT_P1

description: 10x Genomics


title: DiFi_CD63_FT1_4


title: Crosslinked, IPSC, sALS,CHMP7 IN, rep2

first: Brian

last: Yee

address: 9500 Gilman Dr

department: Health

description: size matched input controls

data_processing:
Data was processed using Skipper, available at: http://github.com/yeolab/skipper
Reads were trimmed for adapter sequences and barcode sequences (eCLIP samples) using skewer.
Unique Molecular Identifiers (UMIs) were extracted from raw sequencing reads with fastp
Extracted reads were aligned using STAR: --alignEndsType EndToEnd --genomeDir {params.star_sjdb} --genomeLoad NoSharedMemory --outBAMcompression 10 --outFileNamePrefix {params.outprefix} --winAnchorMultimapNmax 100 --outFilterMultimapNmax 100 --outFilterMultimapScoreRange 1 --outSAMmultNmax 1 --outMultimapperOrder Random --outFilterScoreMin 10 --outFilterType BySJout --limitOutSJcollapsed 5000000 --outReadsUnmapped None --outSAMattrRGline ID:{wildcards.replicate_label} --outSAMattributes All --outSAMmode Full --outSAMtype BAM Unsorted --outSAMunmapped Within --readFilesCommand zcat --outStd Log --readFilesIn {input.fq} --runMode alignReads --runThreadN {threads}
Skipper produced bigwig files
Assembly: GRCh38
Supplementary files format and content: bigwigs contain RPM-normalized read densities of uniquely-mapped reads

source_name: IPSC

extract_protocol:
Cells were lysed and and sonicated in eCLIP lysis buffer, and RNA was fragmented using RNase I (Ambion)
RNA was reverse transcripted with Superscript III, and adaptor was ligated with T4 RNA Ligase 1, then libraries were constructed with Q5 High-Fidelity 2x Master Mix (NEB)

value: IPSC


title: DiFi_CD81_B1_5


title: Crosslinked, IPSC, sALS,CHMP7 IN, rep1

description: size matched input controls


title: Crosslinked, IPSC, fALS,CHMP7 IN, rep2

description: size matched input controls


title: DiFi_Total_Input_6


title: Crosslinked, IPSC, fALS,CHMP7 IN, rep1

description: size matched input controls


title: DiFi_CD9_FT2_4


title: DiFi_CD9_FT2_6


title: DiFi_IgG_B2_3


title: Crosslinked, IPSC, control,CHMP7 IN, rep2

description: size matched input controls


title: DiFi_CD81_B2_6


title: DiFi_CD63_B1_5


title: Crosslinked, IPSC, control,CHMP7 IN, rep1

description: size matched input controls


title: Crosslinked, IPSC, sALS,CHMP7 IP, rep1


title: Crosslinked, IPSC, sALS,CHMP7 IP, rep2


title: DiFi_IgG_B2_2


title: Crosslinked, IPSC, fALS,CHMP7 IP, rep2


title: DiFi_CD63_B2_5


title: DiFi_CD9_B2_5


title: Crosslinked, IPSC, fALS,CHMP7 IP, rep1


title: DiFi_CD81_FT2_4


title: Crosslinked, IPSC, control,CHMP7 IP, rep1


title: DiFi_IgG_B1_3


title: DiFi_CD63_FT2_6


title: Crosslinked, IPSC, control,CHMP7 IP, rep2


title: Hela,GFP_helix5,rep2

data_processing:
After acquiring wildtype and mutant sequences, reads were aligned using STAR: ultrafast universal RNA-seq aligner (Dobin et al., 2013) to the reference GRCh38 annotation (GENCODE release 42).
Once genes were aligned, reads aligned to genomic features were counted using featureCounts (Liao et al., 2014) and the same GRCh38 genome annotation file to quantify the number of reads per gene in the data.
Differential expression analysis was done using DESeq2 v1.39.3 (Love et al., 2014) in R v4.1.3.
The differentially expressed genes (DEGs) were further analyzed in python to produce a volcano plot with a Benjamini-Hochberg adjusted p-value cut-off of 0.05.
Gene ontology analysis was completed using GOrilla (Eden et al., 2009), using pvalue below 0.05 from most significant
Assembly: GRCh38
Supplementary files format and content: up and down regulated gene

molecule: polyA RNA

source_name: HeLa

extract_protocol:
Total RNA was isolated for hela cells, RNeasy Mini kit (QIAGEN) with DNase I treatment,
indexed cDNA libraries were prepared using the TruSeq stranded mRNA Library Preparation kit (Illumina) and sequenced using a NovaSeq6000 (Illumina)

value: HeLa


title: Hela,GFP_helix5,rep1


title: DiFi_CD63_FT2_5


title: Hela,GFP_Control,rep2


title: DiFi_CD9_FT1_5


title: T Cells,CHRONIC exhaustion, EED226-treated, 11032023 sequenced, rep 3

city: Philadelphia

first: Gregory

last: Donahue

state: PA

address: 3400 Civic Center Blvd, Bldg 421

department: Cell & Developmental Biology

institute: The University of Pennsylvania

data_processing:
Alignment: reads were aligned to the mm10 reference genome using STAR (v2.7.1a)
Normalization: aligned files were normalized using PORT gene-based normalization (https://github.com/itmat/Normalization) with default parameters.
Differential gene expression: performed using DESeq2 (v1.38.3) where genes expressing less than 10 reads across all samples were removed.
Assembly: mm10 / GRCm38
Supplementary files format and content: Supplemental table has gene counts per sample after re-sampling with PORT, and should be treated as normalized for use with DESeq2

organism: Mus musculus

source_name: P14 CD8+ T cells transgenic for a TCR recognizing the DbGP33-41 epitope of LCMV

growth_protocol: Mice were housed at the University of Pennsylvania in a specific pathogen–free facility and experiments and procedures were performed in accordance with Institutional Animal Care and Use Committee. For the in vitro model, P14 transgenic mice expressing a TCR specific for the LCMV peptide DbGP33–41 were backcrossed to C57BL/6 mice (Charles River), transferred from the lab of John Wherry, and bred in house. Mice were between 8 and 24 weeks old, randomly assigned to experimental groups, and sexes were alternated between experiments.

extract_protocol:
RNA was extracted using TRIzol reagent according to the manufacturer’s instructions.
RNA-seq libraries were constructed using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760L). The NEBNext® Library Quant Kit for Illumina® (E7630L) and bioanalyzer were used to assess library quality and concentration then libraries were sequenced using the Illumina® NextSeq 500 and NextSeq 1000.

treatment_protocol: Complete RPMI (cRPMI) in this protocol is RPMI 1640 (Corning Cellgro) supplemented with 10% fetal bovine serum, 50 μM β-mercaptoethanol, 2 mM glutamine (Gibco), penicillin (100 U/ml; Sigma-Aldrich), streptomycin (100 μg/ml; Sigma-Aldrich), 100 μM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), and 20 mM Hepes buffer (Invitrogen). CD8 T cells were isolated from spleens of naïve P14 transgenic mice by homogenizing against a 70-μm cell strainer, washing with cRPMI, passing through a 70-μm cell strainer again, and then isolation via a negative selection kit (STEMCELL Technologies) per the manufacturer’s instructions. DCs were isolated from spleens of naïve C57BL/6 mice by cutting samples into small pieces, incubating at 37°C for 30-45 min with deoxyribonuclease I (1 U/ml; Roche) and collagenase D (10 U/ml; Roche) in cRPMI, then homogenization against a 70-μm cell strainer. Red blood cells were lysed with ammonium-chloride-potassium (ACK) lysis buffer (Gibco) for 5 min, the cell suspension was quenched and washed with phosphate-buffered saline (PBS) and then passed through a 70-μm cell strainer again. DCs were isolated with CD11c+ Microbeads (Miltenyi) per the manufacturer’s instructions and then pulsed with DbGP33–41 peptide (0.1 μM, GenScript) in cRPMI for 30 min-1 hour at 37°C. CD8 P14 cells and DCs were then cocultured in cRPMI at a 1:1 ratio (105 DCs + 105 P14 cells per well) in a flat-bottom 96-well plate. At day 2, 4, and 6 of coculture, P14 cells were counted via BD Accuri and normalized to a concentration of 0.5 × 106 P14 cells/ml (105 P14 cells per well) to be replated into in vitro culture. Chronically stimulated P14 cells received DbGP33–41 peptide (1 nM) at day 2, 4, and 6 and both in vitro conditions (chronic and acute) received recombinant human IL-2 (10 U/ml; PeproTech) at day 2, 4, and 6. For EED inhibition experiments, EED226 (#22031) was bought from Cayman Chemical, resuspended in DMSO to make a stock concentration of 10mM and added to in vitro cell culture media at a concentration of 4uM. Control conditions were given an equivalent amount of DMSO.

value: Clone 13 strain of the LCMV

tag: strain

tag: date sequenced

tag: exhaustion

value: CHRONIC

tag: replicate


title: Hela,GFP_Control,rep1


title: DiFi_CD81_B2_4


title: PT4,C3D1

city: Crawley

first: Wee Loong

last: Chin

state: Western Australia

address: 35 Stirling Hwy

country: Australia

institute: University of Western Australia

description: 10X Genomics

data_processing:
Alignment was performed using Cell Ranger on default parameters. The count matrix and metadata were stored in an Anndata object for downstream analysis.
Label transfer was applied using Azimuth for cell labelling
Assembly: GRCh38 human genome
Supplementary files format and content: single_cell_DREAM.h5ad, AnnData object

source_name: Peripheral blood

growth_protocol: Not applicable

extract_protocol:
PBMCs were thawed by immersing the cryotube in a 37°C water bath until a small ice shard remained. The cryotube contents were then mixed using a 1 mL pipette and transferred to a 50 mL centrifuge tube. Additionally, 1 mL of media was mixed with the cryovial and then transferred to the same 50 mL tube. Four sequential dilutions were performed by incrementally adding media to the centrifuge tube while gently agitating the mixture. Specifically, 2 mL, 4 mL, 8 mL, and 16 mL were added in succession, with a 30-second interval between each step. The PBMCs were subsequently centrifuged at 400g for 5 minutes. The supernatant was removed, leaving approximately 1 mL, in which the cells were resuspended. An additional 9 mL of media was added, followed by another 5-minute centrifugation at 400g. The supernatant was once again removed and discarded. The cells were finally resuspended in 1xDPBS+0.04% BSA. Cell count and viability were assessed using a Cellometer K2 and Acridine Orange/Propidium Iodide (AO/PI) staining. Single cell libraries were then constructed using the 10x Genomics chromium 5′ v2 workflow according to the manufacturer’s protocol. All experiments and conditions aimed for a capture of approximately 6000 cells. Libraries were quantified with Qubit and checked for fragment size using the TapeStation D1000 kit (Agilent). Libraries were pooled in equimolar concentration for a total pooled concentration of 1.8 nM. The libraries were sequenced using S4 (200 cycle) sequencing kits on the Illumina NovaSeq 6000 with a read one length of 100 cycles, and a read two length of 100 cycles.
Illumina NovaSeq 6000 with a read one length of 100 cycles, and a read two length of 100 cycles

treatment_protocol: Not applicable

value: Peripheral blood

value: Peripheral blood mononuclear cells

value: Achieved PFS6M

tag: patient


title: PT7,C2D1

description: 10X Genomics


title: PT46,Baseline

description: 10X Genomics

value: Baseline


title: DiFi_IgG_B1_2


title: T Cells,CHRONIC exhaustion, EED226-treated, 11032023 sequenced, rep 2

value: P14 CD8+ T cells transgenic for a TCR recognizing the DbGP33-41 epitope of LCMV


title: T Cells,CHRONIC exhaustion, EED226-treated, 11032023 sequenced, rep 1


title: PT9,Baseline

description: 10X Genomics


title: PT7,C3D1

description: 10X Genomics


title: DiFi_CD9_FT1_6


title: T Cells,CHRONIC exhaustion, DMSO-treated, 11032023 sequenced, rep 3

value: DMSO


title: PT10,C3D1

description: 10X Genomics


title: DiFi_CD81_B2_5


title: PT51,C2D1

description: 10X Genomics


title: PT55,Baseline

description: 10X Genomics


title: T Cells,CHRONIC exhaustion, DMSO-treated, 11032023 sequenced, rep 2


title: PT15,C3D1

description: 10X Genomics

value: Did not achieve PFS6M


title: DiFi_IgG_B2_1


title: T Cells,CHRONIC exhaustion, DMSO-treated, 11032023 sequenced, rep 1


title: PT35,C2D1

description: 10X Genomics


title: PT45,Baseline

description: 10X Genomics


title: T Cells,ACUTE exhaustion, EED226-treated, 11032023 sequenced, rep 2

value: Armstrong strain of the LCMV

value: ACUTE


title: DiFi_CD9_B1_6


title: PT47,C3D1

description: 10X Genomics


title: PT26,C3D1

description: 10X Genomics


title: T Cells,ACUTE exhaustion, DMSO-treated, 11032023 sequenced, rep 3


title: DiFi_CD63_B2_6


title: PT10,C2D1

description: 10X Genomics


title: PT7,Baseline

description: 10X Genomics


title: T Cells,ACUTE exhaustion, DMSO-treated, 11032023 sequenced, rep 2


title: PT24,C3D1

description: 10X Genomics


title: PT37,C2D1

description: 10X Genomics


title: PT54,Baseline

description: 10X Genomics


title: DIFi_CD81_FT1_5


title: GFP_Dox_rep2_Ribo

description: RNA associated with active ribosome


title: DiFi_IgG_FT2_1


title: PT42,C2D1

description: 10X Genomics


title: DMS-MaP, wild type ModT long, deproteinated cell lysate 24h, control

city: Würzburg

first: Johannes

last: Suzer

address: Josef Schneider Straße D15

department: Institute of Molecular Infection Biology Würzburg

institute: Universität Würzburg

library_source: other

data_processing:
DMS-MaP data was trimmed using cutadapt v 1.18 (Kechin et al., 2017) and aligned to the reference sequence using bowtie2 (Langmead and Salzberg, 2012).  cutadapt parameters were ‘–nextseq-trim 25 –maxn 0 -g gtgatatttaggtctgtg -G gatatttagctgacctgg’.  bowtie2 parameters were ‘-D 20 -R 3 -N 1 -L 15 -i S,1,0.50’.  Further analysis was carried out using the rf-count and rfnorm modules of RNA Framework package v2.7.2 (Incarnato et al., 2018).  rfcount parameters were ‘--count-mutations –only-mut G>Y;A>B;C>D;T>V -q 30’ and a sample specific mask file was included to ignore counting at primer binding sites.  rf-norm parameters were ‘-rb AC -sm 3 -nm 2 –remap-reactivities’, meaning that DMS reactivities were calculated by subtracting background mutations in the untreated sample and normalized using 90% Winsorizing.
Assembly: GTGATATTTAGGTCTGTGGTTGAAAGTCCAAGCCAGTCGCAGGCAAAGGGATCCACGTAAGTTAACACTAAAAACTGGGTTAATGATCATGGTGCGGCTTAGAAGTAAGACCTACCGATAATAAAATCGAGAGAGAGTAGTAGTGCGGCGAACCCAAGCGAACCCTCCAGTAGGCGAGTGTGGGGTCAAAAACCAGGTCAGCTAAATATCATATTAAAAGAGAGCTAGCATACCTTTATGTATAGCTCTTTTTTATAT
Supplementary files format and content: rc files, RC files are binary files storing the transcript’s sequence, per-base RT-stop/mutation counts per-base read coverage, and total number of mapped reads.
Supplementary files format and content: xml

molecule: other

organism: Clostridioides difficile

growth_protocol: For in vitro experiments, the short and the long isoform of ModT were transcribed with the T7 Megascript Kit (Thermo Fisher) according to the Manufacturer's instructions, using a DNA template consisting of either C. difficile ModT isoform fused to a T7 promoter. Template was removed by Turbo DNase I digestion (Thermo Fisher). The RNA was column purified and eluted in RNase free water and the purity was assessed on a denaturing (7M Urea) polyacrylamide gel. For in vivo samples form native cells and deproteinated cell lysates, Trypton-Yeast medium (TY) was inoculated to an OD of 0.05 from exponentially growing  day cultures of C. difficile 630 with a time offset, so that all cultures reached the respective sampling growth phase (3h - exponential phase, 9h - transition to stationary phase, and 24h - stationary phase) simultaneously.

extract_protocol:
RNA was extracted according to the manufacturer’s instuctions. Briefly, 0.2 volumes of chloroform was added, followed by thorough mixing. Samples were centrifuged at 12000 x g for 15 minutes, 4 °C and the aqueous phase was transferred to a new tube in which the RNA was precipitated by adding 1 volume of isopropanol and 1 µl of Glycoblue (Invitrogen) followed by a 30 minutes incubation at -80°C. RNA was centrifuged at 12000 x g, 4 °C for 30 minutes and the pellets were washed twice with 1 ml of 75% Ethanol, air dried, and eluted in 15 µl water.
For cDNA synthesis half the volume of RNA (7.5 µl) was heated to 65 °C together with 1.5 µl 100 µM primer (LongModT_Rv for the long ModT isoform, and ShortModT_Rv for short ModT isoform) for 5 minutes and used for reverse transcription with the Marathon-RT (Zhao et al., 2018) in RT-buffer (50 mM Tris–HCl pH 8.3, 200 mM KCl, 5 mM DTT, 20% glycerol, 1 mM MnCl2, 0.5 mM dNTP and 8 U RNaseIn) for 8 hours at 42 °C.  cDNAs were diluted to 1/8 with nuclease-free water. 5 µl the diluted cDNA was used as template in 50 µl PCR reaction using the Q5 High-Fidelity DNA Polymerase (NEB) and the primers HF_ModT_Fw (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGATATTTAGGTCTGTG) and HR_LongModT_Rv (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATATAAAAAAGAGCTATAC) for the long ModT isoform, and HF_ModT_Fw and HR_ShortModT_Rv (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATATTTAGCTGACCTGG)  the  short ModT isoform, appending Nextera sequencing adapter sequences HF and HR to the amplicons extremities. Cycling conditions were 2 min at 98°C then 25 cycles of 10 s at 98°C, 15 s at 55°C and 15 s at 72°C then 72°C for 5 min. 5 µl of PCR products were verified on a 1.5% agarose gel and the rest was column purified using NucleoSpin Gel and PCR Clean-up kits, Macherey-Nagel according to the manufacturer’s instructions. A final indexing PCR was carried out using Illumina Nextera DNA CD indexes (96 Indexes, 96 Samples, Illumina). Reaction conditions were 80 ng of purified PCR product, 1 × Q5 reaction buffer, 0.2 mM dNTPs, 2.5 µl of indexing primer, 0.02 U/µl of Q5 polymerase in a final volume of 14 µl. Indexed PCR products were verified on a 1.5% agarose gel, pooled together in an equimolar ratio, before final purification on a 1.5% agarose gel. The pooled indexed sequencing library was quantified using the NEBNext library Quant Kit for Illumina and paired-end PE150 sequencing was carried out on an Illumina Novaseq instrument (Novogene).

treatment_protocol: For DMS-MaP in vitro, the in vitro transcribed RNA stocks were denatured for 2 minutes at 95 °C and subsequently chilled on ice for 5 minutes. 50 ng of each RNA transcript was allowed to fold for 15 minutes at 37 °C in a “physiological buffer” (20 mM HEPES-NaOH pH 7.5, 140 mM KCl, 2 mM MgCl2) in a volume of 30 µl. DMS diluted in Ethanol was added to the folded RNAs (final concentration 85 mM) and incubated at 37 °C for 6 minutes. For each condition, an Ethanol-only control was included. Reactions were quenched on ice by adding β-mercaptoethanol (Sigma-Aldrich) to final concentration of 200 mM and 3 volumes of TRI reagent LS (Sigma Aldrich). For in vivo DMS-MaP in native cells, 4.5 ml of each culture was mixed with 225 µl of 3.4  M DMS diluted in Ethanol. As a control, a culture treated with 225 µl of Ethanol was included. All samples were incubated at 37°C for 6 minutes. The reaction was stopped by transferring the samples to ice and immediate addition of 150 µl of β-mercaptoethanol, followed by inversion.Cells were centrifuged at 5000 x g for 10 minutes, 4 °C and the pellet was dissolved in 1 ml of TRI reagent (Sigma Aldrich). To support lysis, the ice-cold samples were transferred into tubes containing 500 mg of glass beads (0.1 mm diameter) and subjected to 3 grinding cycles (30 sec, 6 m/s) in a FastPrep (MP Biomedicals) beadbeater with cooling on ice for 2 minutes between each cycle. Samples were centrifuged for 10 minutes at 10000 x g, 4 °C. The bead-free supernatant was transferred to a new tube. For DMS-MaP in deproteinated cell lysate, 6 ml of the bacterial culture (~2-5 ODs) was centrifuged at 5000 x g for 15 minutes, 4 °C and the bacterial pellet was dissolved in 1 ml of lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM KCl, 1 mM MgCl2, 0.2 % Triton X100, 200 U/ml RNaseIn). Lysis was achieved by grinding the ice-cold samples with 500 mg of glass beads (0.1 mm diameter) in a FastPrep (MP Biomedicals) beadbeater with 3 grinding cycles (30 sec, 6 m/s), cooling the samples on ice for 2 minutes between each cycle. The lysate was clarified by centrifugation for 5 min at 10.000 xg, 4°C, transferred to a new tube and adjusted to 1 ml with lysis buffer. 110 µl of  10% SDS and 50 µl of Proteinase K (NEB) were added to 1mL of clear lysate and the mixture was incubated at 37°C for 30 min with gentle shaking. The digested lysate was split equally in two microtubes, one added with 50 µl of DMS (final concentration 85 mM) and one added with 50 µl of EtOH, and the incubation was prolonged at 37°C for 6 min. The reaction was quenched by adding 50 µl β-mercaptoethanol and transferred on ice followed by addition of 1.5 ml of TRI reagent LS.

value: wild type

tag: isoform

value: ModT long

tag: growth stage

value: stationary phase

tag: treatment condition

value: Proteinase K digested cell lysate

value: Ethanol


title: DMS-MaP, wild type ModT short, deproteinated cell lysate 24h, control

value: ModT short


title: PT29,C3D1

description: 10X Genomics


title: DMS-MaP, wild type ModT long, deproteinated cell lysate 9h, control

value: transition to stationary phase


title: PT21,Baseline

description: 10X Genomics


title: DiFi_IgG_FT2_3

tag: disease

value: Colorectal Carcinoma

tag: anatomical location

value: Colon structure

value: other


title: DiFi_CD9_B1_5

tag: rna source


title: DMS-MaP, wild type ModT short, deproteinated cell lysate 9h, control


title: DiFi_CD63_B2_4


title: PT54,C2D1

description: 10X Genomics

tag: tissue

value: Peripheral blood

tag: cell line

value: Peripheral blood mononuclear cells

value: Achieved PFS6M


title: DMS-MaP, wild type ModT long, deproteinated cell lysate 3h, control

tag: genotype

value: exponential phase


title: PT51,Baseline

description: 10X Genomics

tag: treatment

value: Baseline


title: DiFi_CD9_FT1_4


title: PT13,C3D1

description: 10X Genomics


title: DiFi_CD63_FT1_6


title: PT41,Baseline

description: 10X Genomics


title: PT33,C2D1

description: 10X Genomics

value: Did not achieve PFS6M


title: DMS-MaP, wild type ModT short, deproteinated cell lysate 3h, control


title: PT5,Baseline

description: 10X Genomics


title: PT28,C3D1

description: 10X Genomics


title: PT18,C2D1

description: 10X Genomics


title: DMS-MaP, wild type ModT long, in vivo, TY medium, 24h, control

value: native


title: PT28,C2D1

description: 10X Genomics


title: DiFi_Total_Input_8

value: total cell-free biofluid RNA


title: PT36,C3D1

description: 10X Genomics


title: DMS-MaP, wild type ModT long, in vivo, TY medium, 9h, control

value: wild type

value: ModT long


title: DMS-MaP, wild type ModT short, in vivo, TY medium, 24h, control

value: stationary phase


title: DiFi_IgG_B1_1


title: PT2,C2D1

description: 10X Genomics


title: PT2,C3D1

description: 10X Genomics


title: PT55,C3D1

description: 10X Genomics


title: PT44,C2D1

description: 10X Genomics


title: PT13,Baseline

description: 10X Genomics


title: DiFi_CD81_B1_6


title: PT4,Baseline

description: 10X Genomics


title: DiFi_CD81_B1_4


title: DMS-MaP, wild type ModT long, in vivo, TY medium, 3h, control

value: Ethanol


title: DiFi_IgG_FT1_1


title: PT32,C2D1

description: 10X Genomics


title: DMS-MaP, wild type ModT short, in vivo, TY medium, 9h, control

value: ModT short


title: DiFi_CD63_FT2_4


title: PT6,Baseline

description: 10X Genomics


title: DiFi_IgG_FT1_3


title: DMS-MaP, wild type ModT short, in vivo, TY medium, 3h, control


title: PT42,C3D1

description: 10X Genomics


title: PT35,C3D1

description: 10X Genomics


title: DMS-MaP, wild type ModT long, in vitro, control

value: in vitro


title: PT34,C2D1

description: 10X Genomics


title: PT33,Baseline

description: 10X Genomics


title: DMS-MaP, wild type ModT short, in vitro, control


title: PT8,Baseline

description: 10X Genomics


title: DiFi_CD81_FT1_4

value: Colorectal Carcinoma


title: DiFi_CD81_FT1_6

value: Colon structure


title: PT33,C3D1

description: 10X Genomics


title: DMS-MaP, wild type ModT long, deproteinated cell lysate 24h

value: Proteinase K digested cell lysate

value: DMS


title: PT27,Baseline

description: 10X Genomics

value: Peripheral blood

value: Peripheral blood mononuclear cells


title: PT28,Baseline

description: 10X Genomics


title: PT4,C2D1

description: 10X Genomics

value: Achieved PFS6M


title: DiFi_CD81_FT2_5

value: other


title: DiFi_IgG_FT2_2


title: DMS-MaP, wild type ModT short, deproteinated cell lysate 24h


title: PT45,C2D1

description: 10X Genomics


title: DMS-MaP, wild type ModT long, deproteinated cell lysate 9h

value: transition to stationary phase


title: iPSCCM_CD9_FT1_2

source_name: induced pluripotent stem cell

value: Healthy Subject

value: Entire mesoderm


title: DMS-MaP, wild type ModT short, deproteinated cell lysate 9h

value: wild type


title: PT49,C3D1

description: 10X Genomics

value: Did not achieve PFS6M


title: iPSCCM_CD9_FT2_3


title: iPSCCM_CD63_B1_1


title: PT42,Baseline

description: 10X Genomics

value: Baseline


title: DMS-MaP, wild type ModT long, deproteinated cell lysate 3h

value: ModT long

value: exponential phase


title: DMS-MaP, wild type ModT short, deproteinated cell lysate 3h

value: ModT short


title: PT24,C2D1

description: 10X Genomics


title: iPSCCM_CD63_FT2_2


title: iPSCCM_CD81_B1_1


title: PT5,C3D1

description: 10X Genomics


title: meso_tumor_Patient21

type: RNA

hyb_protocol: hybridisation with codeset DNA probes was performed in accordance with manufacturers instructions https://nanostring.com/wp-content/uploads/MAN-10056-06-Gene-Expression-Panels-and-Custom-CodeSet-User-Manual.pdf

scan_protocol: plates were run on an nCounter Sprint platform in accordance with manufacturers instructions

data_processing: Raw data generated by the nCounter Sprint platform were processed in nSolver 4.0 software by the following steps, as described in the nSolver 4.0 Analysis Software User Manual (MAN-C0019-08) : Firstly, background thresholding was performed by subtracting the mean value of the negative controls included by the manufacturer in the IO360 codeset. Subsequently, dataset normalisation was performed by the combination of: 1) Positive Control Normalisation, using positive controls spiked into every sample by the manufacturer, and 2) CodeSet Content Normalisation, where 20 housekeeping genes are included in the CodeSet by the manufacturer.

source_name: mesothelioma

extract_protocol: RNA was retrieved from FFPE sections on glass slides as follows. Slides were placed in two sequential washes of D-limonene then one wash of 100% EtOH; 2 mins each, with 10-15 seconds gentle agitation at the start of each wash. Slides were then air-dried for 5-10 mins. Tissue was rehydrated in 3% glycerol (Sigma-Aldrich, #G5519) one slide at a time, excess glycerol carefully removed from slide by paper towel, and tissue gently scraped off using a razor blade and collected in 1.5 ml microfuge tube. Samples were then processed using Roche High Pure FFPET RNA Isolation kit (Roche, #06650775001) as per manufacturer instructions. RNA concentration and quality was checked using a Bioanalyzer (Agilent)

tag: batch

tag: lane

value: mesothelioma

tag: gender

value: Male

tag: histology

value: Epithelioid

tag: progression-free at 6 months

value: Yes


title: meso_tumor_Patient39

value: Biphasic

value: No


title: DMS-MaP, wild type ModT long, in vivo, TY medium, 24h

tag: strain

value: stationary phase

value: native

value: DMS


title: DMS-MaP, wild type ModT short, in vivo, TY medium, 24h


title: PT37,C3D1

description: 10X Genomics

value: Peripheral blood

value: Peripheral blood mononuclear cells

value: Achieved PFS6M

tag: patient


title: meso_tumor_Patient05


title: meso_tumor_Patient45


title: meso_tumor_Patient27


title: PT47,C2D1

description: 10X Genomics

value: Did not achieve PFS6M


title: iPSCCM_CD63_FT1_2

value: Healthy Subject

value: Entire mesoderm

value: other


title: iPSCCM_CD63_FT2_1


title: meso_tumor_Patient48


title: DMS-MaP, wild type ModT short, in vivo, TY medium, 9h

value: wild type

tag: isoform

value: ModT short

value: transition to stationary phase


title: PT47,Baseline

description: 10X Genomics

value: Baseline


title: meso_tumor_Patient13

value: mesothelioma

value: Female

value: Epithelioid

value: Yes


title: PT44,C3D1

description: 10X Genomics


title: iPSCCM_CD81_FT1_3


title: PT41,C2D1

description: 10X Genomics


title: meso_tumor_Patient02

value: Male


title: DMS-MaP, wild type ModT long, in vivo, TY medium, 3h

value: ModT long

tag: growth stage

value: exponential phase

value: native

value: DMS


title: DMS-MaP, wild type ModT short, in vivo, TY medium, 3h

tag: treatment condition


title: iPSCCM_Total_Input_1

tag: disease

value: total cell-free biofluid RNA


title: PT49,C2D1

description: 10X Genomics

value: Peripheral blood

value: Peripheral blood mononuclear cells

value: Did not achieve PFS6M


title: meso_tumor_Patient12


title: PT22,Baseline

description: 10X Genomics

value: Achieved PFS6M


title: DMS-MaP, wild type ModT long, in vitro

value: wild type

value: in vitro


title: iPSCCM_CD9_FT1_1

value: Healthy Subject

tag: anatomical location

value: Entire mesoderm

value: other


title: PT46,C3D1

description: 10X Genomics


title: DMS-MaP, wild type ModT short, in vitro

value: ModT short


title: meso_tumor_Patient22

value: mesothelioma

value: Epithelioid

value: Yes


title: DMS-MaP, wild type ModT long, in vivo, TY medium, 9h

value: ModT long

value: transition to stationary phase


title: meso_tumor_Patient32

value: Female


title: iPSCCM_Total_Input_3

tag: rna source

value: total cell-free biofluid RNA


title: iPSCCM_CD9_FT1_3


title: meso_tumor_Patient43

tag: tissue

value: Male

value: No


title: PT29,C2D1

description: 10X Genomics

value: Peripheral blood

tag: cell line

value: Peripheral blood mononuclear cells


title: hVIC, OST, H3K4me3 antibody, Sample 2

city: Pittsburgh

first: Rolando

last: Cuevas

address: 200 LOTHROP ST

institute: University of Pittsburgh

description: hVICs, OST

library_source: genomic

data_processing:
Calls were obtained with CLC Genomic Workbench, version 24.0.1, from Qiagen.
CUT&RUN reads were aligned to the UCSC hg38 genome using CLC Genomic Workbench, version 24.0.1, from Qiagen.
Assembly: hg38
Supplementary files format and content: Comma delimited CSV file containing chromosome coorddinates, lengths, scores, p-values, and annotations.
Library strategy: CUT&RUN

molecule: genomic DNA

source_name: Aortic valve

growth_protocol: Human VICs were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1x P/S in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
Chromatin fragments were isolated using CUTANA CUT&RUN Assay Kit version 3  from Epicypher following the manufacturer’s instructions.
DNA was  indexed with CUTANA™ CUT&RUN Library Prep Kit from EpiCypher.

treatment_protocol: MEM alpha with nucleosides, 10% FBS, 1X P/S, 10 mM glycerol phosphate, 50 μM ascorbic acid 2-phosphate, and 100 nM dexamethasone.

value: Aortic valve

value: Human valve interstitial cell, primary line

tag: cell type

value: VIC

tag: genotype

value: Wild-type

value: Osteogenic differentiation media (OST)


title: hVIC, IgG control, IgG antibody, Sample 3

description: hVICs, IgG

tag: treatment

value: No treatment control (NT)


title: PT29,Baseline

description: 10X Genomics

value: Achieved PFS6M

value: Baseline


title: hVIC, NT, H3K4me3 antibody, Sample 1

description: hVICs, NT


title: meso_tumor_Patient49

tag: batch

tag: lane

value: mesothelioma

value: Sarcomatoid


title: meso_tumor_Patient08

tag: gender

tag: histology

value: Epithelioid


title: iPSCCM_CD81_FT2_2

value: Healthy Subject

value: Entire mesoderm

value: other


title: iPSCCM_CD9_B2_1


title: PT37,Baseline

description: 10X Genomics


title: iPSCCM_CD81_B2_3


title: meso_tumor_Patient29

value: Male

tag: progression-free at 6 months

value: Yes


title: PT22,C3D1

description: 10X Genomics

value: Peripheral blood

value: Peripheral blood mononuclear cells

tag: patient


title: meso_tumor_Patient42

value: No


title: meso_tumor_Patient36


title: PT24,Baseline

description: 10X Genomics

value: Did not achieve PFS6M


title: PT13,C2D1

description: 10X Genomics


title: PT21,C2D1

description: 10X Genomics


title: PT54,C3D1

description: 10X Genomics


title: iPSCCM_CD63_FT1_3


title: iPSCCM_CD81_FT1_1


title: iPSCCM_CD63_B2_2


title: meso_tumor_Patient23


title: iPSCCM_CD81_B1_3


title: meso_tumor_Patient34


title: meso_tumor_Patient09

value: Female


title: PT46,C2D1

description: 10X Genomics


title: iPSCCM_CD81_B2_2


title: meso_tumor_Patient17


title: iPSCCM_CD81_FT1_2


title: PT26,Baseline

description: 10X Genomics


title: iPSCCM_CD9_B1_1


title: PT9,C3D1

description: 10X Genomics


title: iPSCCM_CD9_B2_2


title: meso_tumor_Patient53


title: iPSCCM_CD9_B2_3


title: iPSCCM_CD9_B1_3


title: meso_tumor_Patient06


title: iPSCCM_CD9_FT2_1


title: meso_tumor_Patient52


title: PT2,Baseline

description: 10X Genomics


title: meso_tumor_Patient40

value: Biphasic


title: PT27,C3D1

description: 10X Genomics


title: meso_tumor_Patient18


title: meso_tumor_Patient30


title: iPSCCM_CD63_B1_2


title: PT55,C2D1

description: 10X Genomics


title: PT6,C3D1

description: 10X Genomics


title: meso_tumor_Patient28


title: meso_tumor_Patient10


title: PT32,Baseline

description: 10X Genomics


title: iPSCCM_CD63_B1_3


title: meso_tumor_Patient31


title: PT9,C2D1

description: 10X Genomics


title: PT34,C3D1

description: 10X Genomics


title: meso_tumor_Patient51


title: iPSCCM_CD63_FT1_1


title: PT15,C2D1

description: 10X Genomics


title: meso_tumor_Patient04


title: iPSCCM_CD63_FT2_3


title: PT44,Baseline

description: 10X Genomics


title: iPSCCM_CD9_FT2_2


title: PT15,Baseline

description: 10X Genomics


title: iPSCCM_CD81_B1_2


title: iPSCCM_CD81_B2_1


title: iPSCCM_CD63_B2_1


title: PT51,C3D1

description: 10X Genomics


title: meso_tumor_Patient46


title: meso_tumor_Patient20


title: PT49,Baseline

description: 10X Genomics


title: iPSCCM_CD81_FT2_1


title: meso_tumor_Patient19


title: meso_tumor_Patient50


title: iPSCCM_CD9_B1_2


title: iPSCCM_CD63_B2_3


title: PT6,C2D1

description: 10X Genomics


title: PT18,C3D1

description: 10X Genomics


title: meso_tumor_Patient37


title: iPSCCM_CD81_FT2_3


title: PT26,C2D1

description: 10X Genomics


title: iPSCCM_Total_Input_2

value: total cell-free biofluid RNA


title: PT41,C3D1

description: 10X Genomics

value: Achieved PFS6M


title: iPSCCM_DGUC_F07_1

tag: rna isolation kit

value: Plasma/Serum Circulating and Exosomal RNA Purification Kit(Norgen Biotek)


title: meso_tumor_Patient54

value: mesothelioma

value: Desmoplastic


title: meso_tumor_Patient03

value: Epithelioid


title: PT22,C2D1

description: 10X Genomics


title: iPSCCM_DGUC_F8_qEV_F01_5

value: Healthy Subject


title: iPSCCM_DGUC_F7_qEV_F08_5

value: Entire mesoderm


title: meso_tumor_Patient24


title: PT43,Baseline

description: 10X Genomics

value: Baseline


title: meso_tumor_Patient38

value: Male


title: iPSCCM_DGUC_F7_qEV_F13_6

value: other


title: PT32,C3D1

description: 10X Genomics

value: Peripheral blood


title: PT5,C2D1

description: 10X Genomics

value: Peripheral blood mononuclear cells


title: meso_tumor_Patient47

value: No


title: 902_ovary

city: Oxford

first: Cecilia

last: Lindren

address: Old Road Campus

department: Big Data Institute

country: United Kingdom

institute: University of Oxford

description: small piece of tissue taken from ovary

data_processing:
merged fastq files were processed using Cellranger 6.1.1 count function
Assembly: refdata-gex-GRCh38-2020-A
Supplementary files format and content: .hd5 filtered

molecule: nuclear RNA

source_name: ovary

growth_protocol: tissue biopsy

extract_protocol:
125mg was used as input. The pieces were sliced into smaller pieces in a precooled petri dish kept on dry ice. The sample was transferred into a 5ml tube and lysed using a lysis buffer while constantly mixing in a cold room. The lysate was passed through a 40μm mini cell strainer and collected into a clean pre-labelled 5.0 mL Eppendorf tube. 1 mL of PBS + 1% BSA was added to the tube to further dilute the sample and then centrifuged to pellet the nuclei. The supernatant was decanted, leaving behind a small volume. The nuclei were resuspended in the remaining supernatant. An aliquot of resuspended nuclei was taken and stained with DAPI at a final DAPI concentration of 1:1000. Nuclei were counted using an Neubauer Improved haemocytometer.
We used 10x Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1(10x Genomics, Pleasanton, CA) for single nuclei library preparation. The libraries were prepared using the Chromium Single Cell Controller (10x Genomics, Pleasanton, CA) using the Chromium Single Cell 3′ Library & Gel Bead v2 kit. Briefly, nuclei suspensions were diluted in nuclease-free water according to manufacturer instructions to obtain a target nuclei recovery of 10,000 nuclei. Sample encapsulation was performed using The 10x  Chromium Controller. The rest of the library preparation was carried out according to the manufacturer’s instructions. Twelve cycles of cDNA amplification were performed and 13 cycles were used for sample indexing. Final libraries were pooled and sequenced on one flow cell of an Illumina NextSeq 500 (Illumina, San Diego) with a read 1: 28 cycles (cell barcode and unique molecule identifier (UMI)), i7 index: 10 cycles i5 index: 10 cycles, and read 2 90 cycles (RNA read).
snRNA-seq

value: ovary


title: PT27,C2D1

description: 10X Genomics

value: Did not achieve PFS6M


title: meso_tumor_Patient11


title: meso_tumor_Patient33

value: Biphasic


title: PT10,Baseline

description: 10X Genomics


title: iPSCCM_DGUC_F02_5


title: meso_tumor_Patient35


title: Th17-1 rep2

first: Xiao

last: Ruipei

address: Guangzhou North Stress

institute: Southern Medical University

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts.
Hisat2 tools soft were used to map with reference genome.
Quantification of gene expression levelsGene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped.
Differential expression analysis of two conditions/groups was performed using the DESeq2.
Assembly: mm11
Supplementary files format and content: TXT files include FPKM values for each Sample

source_name: T cell

growth_protocol: RPMI 1640, 10% fetal bovine serum，1%Penicillin-Streptomycin，1×glutamine,1×β-Mercaptoethanol，cells were cultured at 37°C at 5%CO2.

extract_protocol:
Total RNA of of T cells  was extract using RNAex Pro RNA Reagent(AG,AG21102)
A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology (Shanghai) Co., Ltd.) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample.

treatment_protocol: T cells were souted from the spleens of C57 and cultured with the presence of anti-CD3(4ug/mL),anti-CD28(1ug/mL) for 3 days.The cluture of Th1/Th17 hybrid cells were complished.  Total RNA of of Th1/Th17 cells  was prepared using RNAex Pro RNA Reagent(AG,AG21102) on fourth day .The Th17-1 and pTh17-1 cells were re-stimulated with anti-CD3(0.05ug/mL),anti-CD28(0.05ug/mL) ,IL2(100U/mL) on the seventh day. Total RNA of T cells was prepared using RNAex Pro RNA Reagent(AG,AG21102) on the tenth day. Among them, all cytokines are used in the following concentrations：IL-6：20 ng/mL, TGF-β：1 ng/mL, IL-1β：10 ng/mL, IL-12: 10 ng/mL, anti IL-4: 5ug/mL, anti IFNg: 10ug/mL, IL23:10ng/mL.

value: T cell

value: WT

value: Th17-1 differentiation


title: iPSCCM_DGUC_F7_qEV_F04_5

extract_protocol:
RNA Isolation method was "500uL of biofluid was incubated with 100uL warmed Slurry and 900uL warmed Lysis Buffer A (containing 2-Mercaptoethanol) for 10 min at 60C. To this 1.5 mL of 100% ethanol was added and centrifuged for 2min at 1000rpm. To the pellet 300uL Lysis Buffer A was added and incubated for 10 min at 60C. 300uL 100% Ethanol was added, loaded onto the filter column and centrifuged for 30sec at 1000xg followed by 1 min at 16000xg. The column was washed thrice with 400uL Wash Solution for 1 min at 16000 xg and then centrifuged once again for 3 min to dry the membrane. RNA was eluted in 14uL water with a slow spin for 2 mins at 300 xg followed by a spin for 3 min at 16000 xg.". RNA Isolation kit was Plasma/Serum Circulating and Exosomal RNA Purification Kit(Norgen Biotek).
Library construction kit was NEBNext small RNA library prep (NEB). Samples were multiplexed. Type of Starting Material was total RNA.


title: Th17-1 rep3


title: PT18,Baseline

description: 10X Genomics


title: siPcif1 m6ACE-input 3

state: Guangdong

department: Institute of Molecular Physiology

data_processing:
Basecalling is performed using Illumina RTA, with further demultiplexing performed by Illumina bcl2fastq 2.20.
Fastq sequences were first filtered for a quality score of 20, then trimmed of 5′ and 3′ adapter sequences and poly(A) tails using Cutadapt 3.7 -q 20 -m 30 -a TGGAATTCTCGGGTGCCAAGG -A GATCGTCGGACTGTAGAACTCTGAAC.
The 8-mer UMI located at the first 8 nucleotides of read 1 was registered and trimmed. Any complementary UMI sequence in read 2 was also trimmed.
Reads were mapped to the methylated spike-in using Bowtie2, or to the hg38 assembly transcriptome (Gencode v28 comprehensive gene annotations) using STAR. Aligned pairs that had the same mapping coordinates and UMIs were filtered out as PCR duplicates.
Read-start coordinates in hg38-mapped reads that began with an adenosine nucleotide, and had a minimum mean read count of 1 across the triplicate samples were collated. m6A or m6Am sites were identified as read starts that were at least 2-fold enriched in m6ACE libraries than in the corresponding input libraries. This enrichment was calculated using DESeq2 performed on A-only sites across triplicate pairs of m6ACE and corresponding input libraries (FDR < 0.1, padj < 0.05). Identified sites that were 1–4 nucleotides upstream of another identified significant Rm6AC site or sites found within clustered read-starts were filtered out.
To calculate the relative methylation level (RML) of each site in each sample: The read-start counts at positions –4 to 0 of each site in the m6ACE library were summed and divided by the read-start counts at positions –51 to 0 of the same site in the input library to give ‘X’. Similarly, the read-start counts at positions -4 to 0 of the spike-in m6A site in the m6ACE library were summed and divided by the read-start counts at positions –21 to 0 of the same spike-in m6A site in the input library to give ‘Y’. X was normalized to Y to give RML. RML values of each site was averaged across triplicates for each sample condition.
Assembly: hg38
Supplementary files format and content: bedgraph

growth_protocol: ATCC BE(2)-C CRL-2268 cells were cultivated in a sterile 5% CO2 incubator at 37°C, and in DMEM supplemented with 10% FBS. Cells within passage 3–20 were used for experiments. Cells were regularly subjected to MycoAlert Plus Mycoplasma kit (Lonza LT07) to verify that they were mycoplasma-free.

extract_protocol:
Cells were washed twice with cold PBS and RNA extracted using Trizol LS (Ambion 10296) according to manufacturer’s protocol with an additional ethanol precipitation step.
Library was prepared using m6ACE

treatment_protocol: BE(2)-C cells were seeded into T75 flasks and transfected with siRNA against Pcif1 (Silencer Select S34301) or against scrambled control (Silencer Select 4390843) for 24hr using Lipofectamine RNAimax (Invitrogen 13778030) as per manufacturer's protocol. For differentiation by ATRA treatment, BE(2)-C cells were subsequently treated with 10µM ATRA for 4day with ATRA-containing media replaced freshly every 24hr.

value: Human neuroblastoma cell line


title: iPSCCM_DGUC_F10_1

data_processing:
All FASTQ files were processed uniformly through the extra-cellular RNA processing toolkit (exceRpt). Information about exceRpt is available at https://github.gersteinlab.org/exceRpt/.
Assembly: hg19
Supplementary files format and content: Each processed data file is a core results archive generated by exceRpt. The core results archive contains read counts for alignments to the different transcriptome libraries used by exceRpt (miRNA / piRNA / tRNA / GENCODE annotations / circRNA), taxonomy trees for exogenous rRNA and genomic reads aligned by exceRpt, and a variety of other files generated by exceRpt.

value: Plasma/Serum Circulating and Exosomal RNA Purification Kit(Norgen Biotek)


title: Th17-1 rep1


title: iPSCCM_DGUC_F03_1


title: meso_tumor_Patient14

value: mesothelioma

value: Female

value: Epithelioid


title: siPcif1 m6ACE-input 2


title: pTh17-1 rep3

value: pTh17-1 differentiation


title: iPSCCM_DGUC_F7_qEV_F03_6

value: Healthy Subject

value: Entire mesoderm


title: iPSCCM_DGUC_F8_qEV_F06_6

value: other


title: PT36,C2D1

description: 10X Genomics

value: Peripheral blood

value: Peripheral blood mononuclear cells


title: pTh17-1 rep2


title: iPSCCM_DGUC_F7_qEV_F09_6


title: siScram m6ACE-input 3

value: siScram


title: PT35,Baseline

description: 10X Genomics

value: Did not achieve PFS6M

value: Baseline


title: iPSCCM_DGUC_F09_5


title: PT34,Baseline

description: 10X Genomics


title: PT36,Baseline

description: 10X Genomics


title: siScram m6ACE-input 2


title: iPSCCM_IgG_B2_2


title: PT54, C3D1

data_processing:
Alignment was performed on paired-read samples using Kallisto (v0.43.0) for transcript abundance estimation.
sleuth_to_matrix was used to generate count and transcripts
Assembly: Gencode v34 reference.
Supplementary files format and content: csv file of counts (gzipped)
Supplementary files format and content: csv file of TPM (gzipped)

extract_protocol:
Peripheral blood was collected, processed and frozen at hospital sites within 90 minutes of phlebotomy as follows: (1) collected in PAXgene blood collection tubes containing RNA stabilizers and frozen upright at ‐70°C (or lower); (2) collected in CPT blood collection tubes to recover PBMCs; 1st centrifugation at 1500g, 30 minutes, 18‐25°C, no brake applied; buffy coat was transferred to a fresh centrifuge tube and volume brought to 15ml with PBS; 2nd centrifugation at 300g at 15 minutes, 18‐25°C, no brake applied; PBS was aspirated and pellet resuspended in fresh 10ml PBS, 3rd centrifugation 300g at 15 minutes, 18‐25°C, no brake applied; PBS was aspirated, cell pellet resuspended in freezing medium (RPMI, 10% FCS (v/v), 10% DMSO (v/v) on ice) at 2x105 cells/ml; frozen slowly at ‐70°C (or lower), then transferred into liquid nitrogen storage within 2 weeks.
Illumina NovaSeq S4, 300 cycles (150 bp paired-end reads). Samples were processed with Illumina’s TruSeq Stranded Total RNA protocol and sequenced across three lanes of the NovaSeq 6000 (NovaSeq S4 300 cycle reagent kit).

tag: response (pfs)

value: Achieved PFS6M


title: siScram m6ACE-input 1

value: WT


title: Th1/Th17 rep3

value: T cell

value: Th1/Th17 differentiation


title: iPSCCM_DGUC_F8_qEV_F11_5

value: Plasma/Serum Circulating and Exosomal RNA Purification Kit(Norgen Biotek)


title: Th1/Th17 rep2


title: 903_ovary

description: small piece of tissue taken from ovary

value: ovary


title: PT21,C3D1

description: 10X Genomics


title: PT54, C2D1


title: siPcif1 m6ACE 3

value: Human neuroblastoma cell line


title: Th1/Th17 rep1


title: PT54, Baseline


title: iPSCCM_DGUC_F06_5


title: siPcif1 m6ACE-input 1


title: siPcif1 m6ACE 2


title: iPSCCM_DGUC_F7_qEV_F02_5


title: siPcif1 m6ACE 1


title: PT53, C3D1


title: PT53, C2D1


title: siScram m6ACE 3


title: iPSCCM_DGUC_F7_qEV_F13_5


title: pTh17-1 rep1


title: cKO-IP2

city: Nanjing City

first: Haifeng

last: Sun

state: JiangSu province

address: Tianyuan East Road 818

institute: Nanjing Medical University

data_processing:
Reads were quality controlled using FastQC (v0.11.5) and TrimGalore (v0.6.0) with default parameters.
Removal of rRNA reads was achieved using Bowtie2 (v2.3.4.1) with the '--un-conc-gz' parameter.
Filtered reads were aligned to GRCm38 using HISAT2 (v2.2.1) with the '--rna-strandness RF' parameter.
All BAM files were converted to bigWigs for visualization in Integrative Genomics Viewer (IGV, v2.8.2) by using the bamCoverage command in deepTools (v3.5.1).
Assembly: GRCm38
Supplementary files format and content: bigwig files for each Sample

source_name: mouse developing forebrain (E13.5)

extract_protocol:
Total RNAs were extracted from the forebrain of E13.5 mice using Trizol reagent. To ensure accurate quantification, 1 μL of spike-in control RNA was added to 5 μg of total RNA, and the RNAs were treated with RNase-free DNase I for 20 minutes at 37℃ to remove DNA contamination. Next, the RNAs were fragmented by adding the RNA fragmentation buffer and incubating for 5 minutes at 70℃. The fragmented RNAs were then recovered using the RNA Clean & Concentrator-5 kit (Zymo Research, R1014) and subjected to immunoprecipitation (IP) with an anti-m6A antibody (Millipore, ABE572) and protein-A&G magnetic beads in IP buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% IGEPAL CA-630, supplemented with RNase inhibitor) for 2 hours at 4℃. One-tenth of the fragmented RNAs were saved as "input". After the IP, the beads were washed in IP buffer, and the RNAs were eluted using m6A monophosphate solution. The eluted RNAs, along with the input RNAs, were recovered using the RNA Clean & Concentrator-5 kit and used for library preparation.
RNA libraries were prepared using the SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio, 634413)

value: mouse developing forebrain (E13.5)

value: cKO


title: cKO-IP3


title: siScram m6ACE 2


title: PT53, Baseline


title: iPSCCM_DGUC_F06_1

value: Healthy Subject


title: cKO-IP4


title: cKO-IP1


title: PT51, C3D1


title: siScram m6ACE 1


title: PT51, C2D1


title: PT51, Baseline


title: WT-IP4


title: iPSCCM_DGUC_F04_5

value: Entire mesoderm


title: iPSCCM_DGUC_F8_qEV_F04_6

value: other


title: mitoRPF_2_Input_RNAseq

city: New York

first: Caleb

last: Lareau

state: New York

address: 417 E 68th St, Zuckerman - ZRC 1132

institute: Memorial Sloan Kettering

data_processing:
Raw sequence reads were created using Illumina bcl2fastq
Raw sequencing reads were trimmed using cutadapt
Trimmed reads were aligned via bwa v0.6
mtDNA reads were extracted via samtools v1.10
Assembly: hg38
Supplementary files format and content: Abundance of each allele between m.7076A and G for
Library strategy: mitoRibo-seq

source_name: peripheral blood mononuclear cells

extract_protocol:
The protocol for Mitochondrial ribosome enrichment was adapted from Hsin-Jung Li et al., 2021 (PMID: 33953394). Briefly, 20×106 PBMC cells were washed with PBS containing 100 μg/mL chloramphenicol and 100 μg/mL cycloheximide, pelleted and flash-frozen in liquid nitrogen. The cell pellet was resuspended in 1 mL of lysis buffer (20 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM MgCl2, 100 μg/mL chloramphenicol, 100 μg/mL cycloheximide, 1 mM DTT, 1% (vol/vol) Triton X-100, 0.1% (vol/vol) NP-40, 1× cOmplete protease inhibitor cocktail, 20 U/mL RNasin, 20 U/mL DNase I) and incubated for 10 min on ice. The lysate was homogenized by passing it 3 times through a 26 G needle. Cell debris were removed by centrifugation at 16,000g for 10 min (4 °C) and 100 µL of clarified lysate was kept aside for preparation of RNA-seq libraries. Ribosome protected fragments (RPFs) were generated by nuclease digestion. To this end, 3.75 U/µL of micrococcal nuclease (NEB) and 5 mM CaCl2 were added to the remaining cell lysate; the incubation was performed at room temperature with gentle shaking for 1 h. The reaction was quenched by adding 6 mM of EGTA. The digested lysate was loaded on top of a linear 5-45 % sucrose gradient containing 20 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM MgCl2, 100 μg/mL chloramphenicol, 100 μg/mL cycloheximide and 1 mM DTT and centrifuged at 35,000 rpm, 4 °C for 2 hr using a Beckmann SW40 rotor. The gradient was then fractionated into 20 fractions of 0.57 mL using a Biocomp gradient station fractionator, which allowed recording a UV absorbance profile at 254 nm.
Ribosome protected fragments were isolated using Phenol/Chloroform extraction. To that end, 300 µl of the corresponding gradient fractions were mixed with equal amounts of Phenol:Chloroform:IAA (25:24:1, pH 6.6), transferred to a PhaseLock tube (VWR International) and centrifuged at 15,000g for 5 min. A second clean-up step was performed using the isolated aqueous phase and adding equal amounts of Chloroform. After centrifugation, the aqueous phase was isolated and purified using the RNA Clean & Concentrator kit (Zymo Research) by following the manufacturer’s instructions for small RNAs. Subsequent library preparation was performed as previously described (PMID: 31959994) with the following modification: ribosomal RNA depletion was skipped for RPF libraries due to low amount of starting material.    For preparation of matched RNA-seq libraries, total RNA was extracted from 40 µL of clarified input lysate using the Direct-zol RNA MiniPrep Kit (Zymo Research). Subsequently, rRNA was depleted using riboPOOLs (siTOOLs Biotech) and following the manufacturer’s instructions. Subsequently, RNA-seq libraries were generated as described previously (PMID: 31959994).

treatment_protocol: Thawed cryopreserved PBMCs were resuspended to 1M/ml density in either complete medium supplemented with   complete medium supplemented with IL2 and ImmunoCult Human CD3/CD28 T cell activation then profiled 7 days after the initial culture.

value: peripheral blood mononuclear cells

tag: sorted cell type

value: T cells

tag: fraction

value: input


title: iPSCCM_IgG_B2_1

tag: disease

tag: anatomical location


title: WT-IP3

tag: tissue


title: mitoRPF_2_fraction_8_RPF


title: PT50, C3D1

value: Peripheral blood

value: Peripheral blood mononuclear cells

value: Did not achieved PFS6M


title: iPSCCM_DGUC_F8_qEV_F04_5

tag: rna source


title: iPSCCM_DGUC_F7_qEV_F07_6


title: WT-IP1

value: WT


title: mitoRPF_2_fraction_7_RPF

tag: cell type


title: mitoRPF_2_fraction_9_RPF


title: WT-IP2

tag: genotype


title: PT50, C2D1


title: iPSCCM_IgG_B1_1


title: PT50, Baseline


title: mitoRPF_2_fraction_6_RPF


title: cKO-Input4


title: PT49, C3D1


title: iPSCCM_IgG_FT2_3


title: cKO-Input3


title: mitoRPF_2_fraction_5_RPF


title: PT49, C2D1


title: cKO-Input2


title: iPSCCM_DGUC_F7_qEV_F11_6


title: iPSCCM_DGUC_F8_qEV_F08_6


title: mitoRPF_1_Input_RNAseq


title: cKO-Input1


title: PT49, Baseline


title: iPSCCM_DGUC_F7_qEV_F01_6


title: WT-Input4


title: mitoRPF_1_fraction_9_RPF


title: PT48, C3D1

value: Achieved PFS6M


title: PT48, C2D1


title: WT-Input3


title: iPSCCM_DGUC_F8_qEV_F08_5


title: iPSCCM_DGUC_F8_qEV_F02_6


title: WT-Input2


title: iPSCCM_DGUC_F7_qEV_F11_5


title: mitoRPF_1_fraction_7_RPF


title: mitoRPF_1_fraction_6_RPF


title: PT48, Baseline


title: WT-Input1


title: mitoRPF_1_fraction_5_RPF


title: PT47, C3D1


title: iPSCCM_DGUC_F7_qEV_F05_5


title: PT47, C2D1


title: PT47, Baseline


title: Bulk RNA-seq, F51, no treatment

city: Atlanta

first: Steven

last: Sloan

state: Georgia

address: 615 Michael Street, Room 375

institute: Emory University

description:
Unk
20 million PE reads
D2HG_feature_counts.xlsx
F51_no_treat

data_processing:
Fastq files were first trimmed using Trimmomatic and mapped using STAR aligner with the paired end option selected (hg38). We used FeatureCounts to assemble transcripts and generate raw count matrices.
Assembly: hg38
Supplementary files format and content: xlsx count matrix

source_name: human fetal cortex

growth_protocol: Human cortical organoids: Following 3D formation, spheroids were treated daily in neural induction media (DMEM/F12, KSR, NEAA, Glutamax, Pen/Strep, Beta-mercaptoethanol) supplemented with Dorsomorphin (Sigma, Cat. 142 P5499-25MG, 5 μM) and SB-431542 (Selleck Chemicals, Cat. S1067, 10 μM) for 6 days.  Following this treatment, organoids were treated daily with neural media supplemented with EGF (R&D Systems, Cat. 236-EG-01M, 20 ng/mL) and FGF2 (R&D Systems, Cat. 233-FB-01M, 20 ng/mL) for 10 days, and every other day for days 16-24. At day 25, organoids were treated every other day with neural media supplemented with BDNF (PeproTech, Cat. 450-02- 1mg, 20 ng/mL) and NT-3 (R&D Systems, Cat. 267-N3-005/CF, 20 ng/mL) to promote differentiation of progenitors. From day 43 onwards, organoids were fed every 3 days with neural media only.

extract_protocol:
RNA was harvested using miRneasy kit (Qiagen).
Libraries were prepared with Takara Bio SMART-Seq v4 Ultra Low Input RNA Kit and NEBNext Ultra II kit.

treatment_protocol: Addition of D2HG or DMSO for several primary human fetal astrocyte samples

value: human fetal cortex

value: cd49f+ cells

tag: treatment

value: no treatment


title: Bulk RNA-seq, F51, 0.1% D2HG

description:
Unk
20 million PE reads
D2HG_feature_counts.xlsx
F51_D2HG_0.1


title: iPSCCM_DGUC_F7_qEV_F01_5

value: Plasma/Serum Circulating and Exosomal RNA Purification Kit(Norgen Biotek)


title: iPSCCM_DGUC_F04_1


title: iPSCCM_DGUC_F7_qEV_F05_6


title: mitoRPF_1_fraction_8_RPF


title: Liver, TAnn3

first: Jie

last: Long

address: No.106 Zhongshan 2nd Road

institute: Guangdong Provincial People's Hospital

description:
TA_ctrl3
merged_rpkm.txt

data_processing:
STAR-2.5.2b software was used to map reads to the reference genome.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include RPKM values for bulk RNA-seq Samples

source_name: murine liver tissue

extract_protocol:
Total RNA was extracted utilizing RNA iso Plus (TaKaRa, 9109) from liver tissues
RNA libraries were prepared for sequencing using standard Illumina protocols

value: murine liver tissue

tag: age

value: 2 weeks

tag: strain

value: TG Aire-/-

value: liver total cells


title: Liver, CART2

description:
TA_CART2
merged_rpkm.txt

value: 5 weeks

value: CART treated


title: Liver, CART3

description:
TA_CART3
merged_rpkm.txt


title: Liver, CART1

description:
TA_CART1
merged_rpkm.txt


title: PT46, C2D1


title: PT46, C3D1


title: Bulk RNA-seq, F52, 0.1% DMSO

description:
Unk
20 million PE reads
D2HG_feature_counts.xlsx
F52_DMSO_0.1

value: 0.1% DMSO


title: Liver, TAnn1

description:
TA_ctrl1
merged_rpkm.txt


title: Liver, TAnn2

description:
TA_ctrl2
merged_rpkm.txt


title: iPSCCM_DGUC_F03_5

first: Louise

last: Laurent

value: Healthy Subject

value: Entire mesoderm

value: other


title: Bulk RNA-seq, F52, no treatment

description:
Unk
20 million PE reads
D2HG_feature_counts.xlsx
F52_no_treat


title: iPSCCM_IgG_FT1_2


title: Liver, WT2

description:
WT2
merged_rpkm.txt

value: Aire+/+


title: iPSCCM_IgG_FT2_2


title: Bulk RNA-seq, F53, 0.1% D2HG

description:
20 million PE reads
D2HG_feature_counts.xlsx
F53_D2HG_0.1

tag: Sex

value: male


title: PT44, C3D1

value: Peripheral blood

value: Peripheral blood mononuclear cells

value: Achieved PFS6M


title: PT44, C2D1


title: PT46, Baseline


title: iPSCCM_DGUC_F11_5


title: Liver, WT1

description:
WT1
merged_rpkm.txt

value: no treatment


title: Bulk RNA-seq, F52, 0.1% D2HG

description:
Unk
20 million PE reads
D2HG_feature_counts.xlsx
F52_D2HG_0.1

value: human fetal cortex

value: cd49f+ cells


title: PT44, Baseline


title: cd200-high capillaries (CD200HiSca1+CD31+EMCN-CD45-) replicate3 sample

city: Boston

first: Mahmoud

middle: Adel

last: Bassal

state: Massachusetts

address: 3 Blackfan Circle

department: Hematology and Oncology

institute: Beth Israel Deaconess Medical Center

data_processing:
Raw fastq files first had optical duplicates removed using clumpify from the BBTools suite using the flag “dedupe spany addcount”.  Next, adaptor trimming was performed using bbduk, also from the BBTools suite using the flag “ktrim=l hdist=2”.  Next, reads were quality trimmed with Trimmomatic in paired end mode with the flags “ILLUMINACLIP: <adapter_path> Truseq3.PE.fa:2:15:4:4:true LEADING:28 TRAILING:28 SLIDINGWINDOW:4:26 MINLEN:20”.
Alignment was performed using STAR with the flags “--outFilterScoreMinOverLread 0.25 --outFilterMatchNminOverLread 0.25 --outFilterMultimapNmax 5“ with the resultant bam files sorted and indexed using samtools.  BamCoverage was used to generate coverage maps using default parameters. FeatureCounts was used to generate gene counts values using the flags “-g gene_id --extraAttributes gene_name -p -M -O -J -f -d 20”.
Differential gene expression analysis was performed following an available tutorial (https://combine-australia.github.io/RNAseq-R/06-rnaseq-day1.html) in R v4.3 (https://www.R-project.org/).
Assembly: hg38
Supplementary files format and content: rnaSeq_normalized_counts_collated.txt - tab-delimited text file containing the normalized tmm counts for each sample

source_name: bone marrow (crushed tibia, femur, and iliac)

growth_protocol: B6 mice were housed under pathogen free conditions.

extract_protocol:
BM cells were obtained by crushing bone (tibia, femur, and iliac) using molars and pestles.
RNA was isolated from sorted cells lysed in TRIzol. Biological triplicates were prepared using six B6 mice. RNA quality was assessed using Agilent Bioanalyzer RNA-Pico kit (Agilent Technologies, USA). Libraries were prepared using the Clontech Ultra Low v4 kit (Takara Bio) for cDNA synthesis, followed by NexteraXT (Illumina, USA), and sequenced on an Illumina NovaSeq6000

treatment_protocol: No special treatment was administered to the mice

value: B6 mouse


title: iPSCCM_DGUC_F8_qEV_F13_6

address: SCRM, 2880 Torrey Pines Scenic Drive

institute: UCSD

value: Plasma/Serum Circulating and Exosomal RNA Purification Kit(Norgen Biotek)


title: PT42, C3D1

value: Did not achieved PFS6M


title: Bulk RNA-seq, F50, no treatment

description:
20 million PE reads
D2HG_feature_counts.xlsx
F50_no_treat

value: female


title: iPSCCM_DGUC_F8_qEV_F06_5


title: cd200-high capillaries (CD200HiSca1+CD31+EMCN-CD45-) replicate1 sample

value: bone marrow (crushed tibia, femur, and iliac)


title: Bulk RNA-seq, F50, 0.1% DMSO

description:
20 million PE reads
D2HG_feature_counts.xlsx
F50_DMSO_0.1

value: 0.1% DMSO


title: cd200-high capillaries (CD200HiSca1+CD31+EMCN-CD45-) replicate2 sample


title: iPSCCM_DGUC_F8_qEV_F13_5

value: Healthy Subject


title: PT42, C2D1


title: Bulk RNA-seq, F46, 0.1% D2HG

description:
Unk
20 million PE reads
D2HG_feature_counts.xlsx
F46_D2HG_0.1


title: PT42, Baseline


title: PT40, C3D1


title: Bulk RNA-seq, F53, no treatment

description:
20 million PE reads
D2HG_feature_counts.xlsx
F53_no_treat


title: arterial (CD200-Sca1+CD31+CD45-) replicate3 sample


title: iPSCCM_DGUC_F09_1

value: Entire mesoderm


title: arterial (CD200-Sca1+CD31+CD45-) replicate2 sample


title: PT40, C2D1


title: Bulk RNA-seq, F53, 0.1% DMSO

description:
20 million PE reads
D2HG_feature_counts.xlsx
F53_DMSO_0.1


title: PT40, Baseline


title: iPSCCM_DGUC_F8_qEV_F05_6

value: other


title: arterial (CD200-Sca1+CD31+CD45-) replicate1 sample


title: Bulk RNA-seq, F46, 0.1% DMSO

description:
Unk
20 million PE reads
D2HG_feature_counts.xlsx
F46_DMSO_0.1


title: Bulk RNA-seq, F50, 0.1% D2HG

description:
20 million PE reads
D2HG_feature_counts.xlsx
F50_D2HG_0.1


title: iPSCCM_DGUC_F02_1


title: PT39, C3D1


title: sinusoidal (CD31+Sca1+CD45-) replicate3 sample


title: Bulk RNA-seq, F100, Doxycycline control

description:
Unk
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F100_Doxpos

value: HepaCAM+ cells

value: Doxycycline


title: iPSCCM_DGUC_F8_qEV_F02_5


title: Bulk RNA-seq, F100, ContGFP, GFPneg, rep2

description:
Unk
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F100_ContGFP_rep2_GFPneg

value: GFP- cells

value: Control GFP virus


title: iPSCCM_DGUC_F08_1


title: sinusoidal (CD31+Sca1+CD45-) replicate2 sample


title: PT39, C2D1

value: Peripheral blood

value: Peripheral blood mononuclear cells


title: Bulk RNA-seq, F100, ContGFP, GFPpos, rep2

description:
Unk
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F100_ContGFP_rep2_GFPpos

value: human fetal cortex

value: GFP+ cells


title: Bulk RNA-seq, F100, ContGFP, GFPneg, rep1

description:
Unk
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F100_ContGFP_rep1_GFPneg


title: sinusoidal (CD31+Sca1+CD45-) replicate1 sample

value: B6 mouse


title: iPSCCM_DGUC_F7_qEV_F03_5

value: Plasma/Serum Circulating and Exosomal RNA Purification Kit(Norgen Biotek)


title: type h vessel (EMCN+Sca1+CD31+CD45-) replicate3 sample


title: PT38, C3D1

value: Did not achieved PFS6M


title: Bulk RNA-seq, F100, ContGFP, GFPpos, rep1

description:
Unk
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F100_ContGFP_rep1_GFPpos


title: PT39, Baseline


title: iPSCCM_DGUC_F7_qEV_F08_6


title: type h vessel (EMCN+Sca1+CD31+CD45-) replicate2 sample


title: PT38, Baseline


title: iPSCCM_IgG_FT1_1


title: PT38, C2D1


title: PT37, Baseline

value: Achieved PFS6M


title: Bulk RNA-seq, F100, ASCL1, GFPneg, rep2

description:
Unk
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F100_ASCL1_rep2_GFPneg

value: TurboGFP:p2A:ASCL1 virus


title: iPSCCM_DGUC_F8_qEV_F01_6


title: Bulk RNA-seq, F100, ASCL1, GFPpos, rep2

description:
Unk
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F100_ASCL1_rep2_GFPpos


title: PT37, C3D1


title: Elf5-Cre; Slc25a1f/f scRNAseq

city: Nanjing

first: WENLI

last: FAN

state: Jiangsu

address: Hankou road 22

institute: Nanjing university

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software.Based on indicators such as the number of gene detections and the proportion of mitochondrial UMI, cells are re-filtered, and DoubletFinder is used to predict doublets in scRNA-sequencing data. Multicellular cells in the dataset are removed to obtain the filtered matrix.
Assembly: mm10
Supplementary files format and content: matrix files

source_name: placenta

extract_protocol:
Euthanize the pregnant mice, harvest the placentas, remove the decidua and umbilical cord, and submerge the remaining placental tissues in tissue preservation solution.
Raw reads were demultiplexed and mapped to the reference genome using the 10X Genomics Cell Ranger pipeline with default parameters. All downstream single-cell analyses were performed using Cell Ranger and Seurat. Briefly, unique molecule identifiers (UMIs) for each gene and each cell barcode (filtered by Cell Ranger) were counted to construct digital expression matrices. During secondary filtration by Seurat, a gene was considered expressed if it appeared in more than three cells, and each cell was required to have at least 200 expressed genes.

value: placenta

value: Elf5-Cre; Slc25a1f/f

value: not applicable


title: control, scRNAseq

description: 10x Genomics

value: wide-type


title: iPSCCM_DGUC_F11_1

value: Healthy Subject

value: Entire mesoderm

value: other


title: PT35, C3D1


title: PT37, C2D1


title: Yolk Sac, E9.5, WT, Rep4

first: Alena

last: Yermalovich

address: 450 Brookline Avenue

institute: Dana-Farber Cancer Institute

description:
Processed data for all E9.5 Yolk Sac samples -- column headers: gene_id, Cmtr2WT1, Cmtr2WT2, Cmtr2WT3, Cmtr2WT4, Cmtr2KO1, Cmtr2KO2, Cmtr2KO3, Cmtr2KO4
E9.5_Yolksac_fpkm_sample.xlsx
E9.5_Yolksac_Cmtr2WT4

data_processing:
Differential expression analysis was performed using the DESeq2 R package (Anders et al. 2015). Genes with an adjusted p-value <0.05 found by DESeq2 were assigned as differentially expressed.
GO enrichment analysis of DEGs was implemented by the clusterProfiler R package (Yu et al. 2012). GO terms with corrected p-value <0.05 were considered significantly enriched by differentially expressed genes.
KEGG Pathway enrichment analysis used the R package clusterProfiler to test the statistical enrichment of differential expression genes in KEGG pathways (Kanehisa and Goto 2000). KEGG terms with adjusted corrected p=value <0.05 were considered significantly enriched.
Assembly: GRCm38/mm10
Supplementary files format and content: FPKM values per gene for each sample

source_name: yolk sac

extract_protocol:
Total RNA was isolated by TRIzol from embryos using Direct-zol RNA Micro Prep Kit and treated with DNase
Library construction and sequencing was performed using Novogene Co., Ltd. sequencing service. Briefly, sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina

tag: developmental stage

value: yolk sac

value: Cmtr2 KO


title: Bulk RNA-seq, F100, ASCL1, GFPneg, rep1

description:
Unk
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F100_ASCL1_rep1_GFPneg

value: HepaCAM+ cells

value: GFP- cells


title: iPSCCM_DGUC_F10_5


title: Bulk RNA-seq, F100, ASCL1, GFPpos, rep1

description:
unk
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F100_ASCL1_rep1_GFPpos

value: human fetal cortex

value: GFP+ cells


title: Yolk Sac, E9.5, WT, Rep3

description:
Processed data for all E9.5 Yolk Sac samples -- column headers: gene_id, Cmtr2WT1, Cmtr2WT2, Cmtr2WT3, Cmtr2WT4, Cmtr2KO1, Cmtr2KO2, Cmtr2KO3, Cmtr2KO4
E9.5_Yolksac_fpkm_sample.xlsx
E9.5_Yolksac_Cmtr2WT3


title: iPSCCM_IgG_B2_3

value: Plasma/Serum Circulating and Exosomal RNA Purification Kit(Norgen Biotek)


title: Bulk RNA-seq, F99, no treatment

description:
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F99_notreat

value: female

value: no treatment


title: Bulk RNA-seq, F99, Doxycycline control

description:
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F99_Doxpos

value: Doxycycline


title: iPSCCM_DGUC_F7_qEV_F09_5


title: Yolk Sac, E9.5, WT, Rep2

description:
Processed data for all E9.5 Yolk Sac samples -- column headers: gene_id, Cmtr2WT1, Cmtr2WT2, Cmtr2WT3, Cmtr2WT4, Cmtr2KO1, Cmtr2KO2, Cmtr2KO3, Cmtr2KO4
E9.5_Yolksac_fpkm_sample.xlsx
E9.5_Yolksac_Cmtr2WT2


title: type h vessel (EMCN+Sca1+CD31+CD45-) replicate1 sample

value: bone marrow (crushed tibia, femur, and iliac)

value: B6 mouse


title: iPSCCM_DGUC_F7_qEV_F07_5


title: Yolk Sac, E9.5, WT, Rep1

description:
Processed data for all E9.5 Yolk Sac samples -- column headers: gene_id, Cmtr2WT1, Cmtr2WT2, Cmtr2WT3, Cmtr2WT4, Cmtr2KO1, Cmtr2KO2, Cmtr2KO3, Cmtr2KO4
E9.5_Yolksac_fpkm_sample.xlsx
E9.5_Yolksac_Cmtr2WT1


title: iPSCCM_DGUC_F7_qEV_F04_6


title: Bulk RNA-seq, F99, ContGFP, GFPpos, rep3

description:
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F99_ContGFP_rep3_GFPpos

value: Control GFP virus


title: PT35, Baseline

value: Peripheral blood

value: Peripheral blood mononuclear cells

value: Did not achieved PFS6M


title: iPSCCM_DGUC_F8_qEV_F10_5

value: Healthy Subject

value: Entire mesoderm

value: other


title: Bulk RNA-seq, F99, ContGFP, GFPneg, rep3

description:
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F99_ContGFP_rep3_GFPneg

value: HepaCAM+ cells


title: PT35, C2D1


title: Bulk RNA-seq, F99, ContGFP, GFPpos, rep2

description:
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F99_ContGFP_rep2_GFPpos


title: iPSCCM_DGUC_F8_qEV_F05_5


title: Yolk Sac, E9.5, KO, Rep4

description:
Processed data for all E9.5 Yolk Sac samples -- column headers: gene_id, Cmtr2WT1, Cmtr2WT2, Cmtr2WT3, Cmtr2WT4, Cmtr2KO1, Cmtr2KO2, Cmtr2KO3, Cmtr2KO4
E9.5_Yolksac_fpkm_sample.xlsx
E9.5_Yolksac_Cmtr2KO4

value: yolk sac

value: Cmtr2 WT


title: Yolk Sac, E9.5, KO, Rep3

description:
Processed data for all E9.5 Yolk Sac samples -- column headers: gene_id, Cmtr2WT1, Cmtr2WT2, Cmtr2WT3, Cmtr2WT4, Cmtr2KO1, Cmtr2KO2, Cmtr2KO3, Cmtr2KO4
E9.5_Yolksac_fpkm_sample.xlsx
E9.5_Yolksac_Cmtr2KO3


title: iPSCCM_DGUC_F05_5


title: Bulk RNA-seq, F99, ContGFP, GFPneg, rep2

description:
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F99_ContGFP_rep2_GFPneg

value: human fetal cortex

value: GFP- cells


title: iPSCCM_DGUC_F05_1

value: Plasma/Serum Circulating and Exosomal RNA Purification Kit(Norgen Biotek)


title: Embryo, E9.5, WT, Rep4

description:
Processed data for all E9.5 Embryo samples -- column headers: gene_id, Cmtr2WT1, Cmtr2WT2, Cmtr2WT3, Cmtr2WT4, Cmtr2KO1, Cmtr2KO2, Cmtr2KO3, Cmtr2KO4
E9.5_Embryo_fpkm_sample.xlsx
E9.5_Embryo_Cmtr2WT4

source_name: embryo

value: embryo

value: Cmtr2 KO


title: PT33, Baseline


title: PT33, C3D1


title: Yolk Sac, E9.5, KO, Rep2

description:
Processed data for all E9.5 Yolk Sac samples -- column headers: gene_id, Cmtr2WT1, Cmtr2WT2, Cmtr2WT3, Cmtr2WT4, Cmtr2KO1, Cmtr2KO2, Cmtr2KO3, Cmtr2KO4
E9.5_Yolksac_fpkm_sample.xlsx
E9.5_Yolksac_Cmtr2KO2


title: Bulk RNA-seq, F99, ContGFP, GFPneg, rep1

description:
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F99_ContGFP_rep1_GFPneg

value: female


title: PT33, C2D1


title: iPSCCM_DGUC_F7_qEV_F12_6


title: Bulk RNA-seq, F99, ASCL1, GFPneg, rep3

description:
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F99_ASCL1_rep3_GFPneg

value: TurboGFP:p2A:ASCL1 virus


title: Bulk RNA-seq, F99, ContGFP, GFPpos, rep1

description:
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F99_ContGFP_rep1_GFPpos

value: GFP+ cells

value: Control GFP virus


title: PT34, Baseline

value: Peripheral blood

value: Peripheral blood mononuclear cells


title: Embryo, E9.5, WT, Rep3

description:
Processed data for all E9.5 Embryo samples -- column headers: gene_id, Cmtr2WT1, Cmtr2WT2, Cmtr2WT3, Cmtr2WT4, Cmtr2KO1, Cmtr2KO2, Cmtr2KO3, Cmtr2KO4
E9.5_Embryo_fpkm_sample.xlsx
E9.5_Embryo_Cmtr2WT3


title: iPSCCM_DGUC_F01_5

value: Healthy Subject


title: Bulk RNA-seq, F99, ASCL1, GFPpos, rep3

description:
20 million PE reads
ASCL1_fetal_feature_counts.xlsx
F99_ASCL1_rep3_GFPpos


title: PT34, C3D1

value: Did not achieved PFS6M


title: PT34, C2D1