title: NPM_KO_4_RPFs

city: Berlin

first: Chiara

last: Giacomelli

address: Robert-Rössle-Str. 10

institute: Max Delbrück Center for Molecular Medicine

data_processing:
Processing of all ribosome profiling data was carried out using the https://github.com/Bushell-lab/Ribo-seq GitHub package. All R scripts were carried out using R version 4.3.1.
For Ribosome Protected Fractments (RPFs): Cutadapt (version 1.18) was used to remove adaptors, trim 3’ bases with quality scores below 20 and discard reads less than 30 and more than 50 bases after trimming. Unique Molecular Indexes (UMIs)  (4nt at the start and end)  were extracted from the reads and appended to the read name using UMI tools (version 1.0.1). BBmap (version 38.18) was used to align reads to a FASTA file containing rRNA and then tRNA reads. All unaligned reads were then aligned to a filtered protein coding FASTA, containing the most abundant transcript per gene (calculated from cytoplasmic RNA samples). The resulting BAM files were sorted with samtools (version 1.9) and deduplicated using UMItools with the directional method. The number of reads in which the 5’ end is at each position of every transcript is then counted and the correct offset for each read length is calculated and applied, to collapse all reads into a single frame. Total counts across the entire CDS, excluding the first 20 and last 10 codons are then summed together.
For paired cytoplasmic RNA samples: Cutadapt (version 1.18) was used to remove adaptors, trim 3’ bases with quality scores below 20 and discard reads less than 30 bases after trimming. Unique Molecular Indexes (UMIs)  (12nt at the start)  were extracted from the reads and appended to the read name using UMI tools (version 1.0.1). Reads were aligned to the filtered protein coding transcriptome with bowtie2 (version 2.3.5.1), using the parameters recommended for use with RSEM, which are --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1. Both gene and isoform level quantification was carried out using RSEM (version 1.3.3).
Assembly: GENCODE vM27 Protein-coding transcript sequences, filtered to contain only transcripts that have both a 5’ and 3’ UTR, with a CDS equally divisible by 3 and an nUG start codon, a stop codon and that have been manually annotated by HAVANA. All PAR_Y transcripts were also removed.
Supplementary files format and content: Totals_APC-KRAS_KO_batchCorrected_normalised_counts.csv contains gene-level and transcript-level counts, normalised using the DESeq2 packge Regularized log transformation (rlog(dds)). Only genes with an average of 10 reads across all samples are included
Supplementary files format and content: RPFs_APC-KRAS_KO_batchCorrected_normalised_counts.csv contains gene-level and transcript-level counts aligned to the coding sequences and summed as described above. Normalization is done using the regularized log transformation formula (rlog(dds)) from DESeq2 package. Only genes with an average of 10 reads across all samples are included
Library strategy: Ribosome-Profiling

source_name: Small intestine epithelial extractions

growth_protocol: Mice were housed in conventional cages, in a pathogen-free facility between 19–23°C and 55 ± 10% humidity, subject to 12 hour day/night cycles and had access to standard diet and drinking water ad libitum. Experiments were performed mice of mixed genders, which had been bred on a C57BL/6 background for at least four generations (n≥4). Mice were aged between 2-6 months and were ≥20g at induction time.

extract_protocol:
Intestinal crypts were isolated from the proximal part of the small intestine following removal of the villi. To achieve this, intestines were flushed with PBS, cut lengthwise and incubated at 37°C for 7 minutes with regular agitation in RPMI 1640 media (Thermo Fisher Scientific, 21875059) containing 10 mmol/L EDTA and 200 μg/mL cycloheximide. The tissue was then transferred to ice-cold PBS with 10 mmol/L EDTA and 200 μg/mL cycloheximide for 7 minutes with regular agitation. Tissue was then discarded and crypts pelleted by centrifugation for 5min at 350g at 4oC and snap froze in liquid N2 and stored at -80°C. Pellets were placed at -20°C for 1h and then on ice for 10min, prior to lysis with 500 µl ice cold lysis buffer (15 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 300 mM NaCl, 1% Triton X100, 0.05% Tween20, 2% n Dodecyl β Maltoside (Thermo Fisher 89903), 0.5 mM DDT, 100 µg/ml cycloheximide, 1X cOmplete EDTA-free Protease Inhibitor Cocktail (Merck 11836170001), 200 U/ml RiboLock RNase Inhibitor (Thermo Fisher Scientific, EO0381)) on ice for 5 min while titrating through a 21G needle with a 1ml syringe. Lysates were centrifuged at 4°C for 5 min at 12,000g and 500 µl supernatant pipetted into a fresh 1.5 ml tube. For cytoplasmic RNA sample, RNA was extracted from 25 µl lysate with 975 µl TRIzol, as per manufactures’ instructions. 20 µl lysate was also added to 5 µl 5X SDS gel loading buffer for Western blot to check for depletion of eIF4A1 or eIF4A2 in the knock-out samples. Remaining lysate was digested with 10 µl Ambion RNase I cloned, 100 U/µL (Thermo Fisher Scientific, AM2295) at 22°C for 15 min at 600 rpm in a thermo mixer. The digestion was stopped with 20 µl SUPERaseIn RNase Inhibitor (20 U/μL) (Thermo Fisher Scientific, AM2696) and samples were loaded onto a 10-50% sucrose gradient, containing 15 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 300 mM NaCl and 100 µg/ml cycloheximide, prepared with a BioComp gradient station and cooled to 4°C for at least 1 h. Samples were centrifuged in a Beckman XPN-90 Ultracentrifuge with an SW40ti rotor at 38,000 rpm for 2 h at 4°C. 1 ml fractions were collected with a Biocomp gradient station and Gilson FC 203B fraction collector. Fractions pertaining to the 80S peak were extracted with acid phenol chloroform, followed by two chloroform washes and RNA was precipitated with 2 µl glycogen (Roche 10901393001), 300 mM NaOac (pH 5.2) and an equal volume of isopropanol overnight at -20°C.
Purified RNA was dissolved in 10 µl RNase free water and diluted with 10 µl 2X urea buffer (Thermo Fisher Scientific, LC6876), heated at 80°C for 90 sec, placed immediately on ice and then loaded onto a pre-run 15% TBE-Urea gel (Thermo Fisher Scientific, EC68852BOX) and ran at 200V for 1h alongside 28nt (AGCGUGUACUCCGAAGAGGAUCCAACGU) and 34nt (GCAUUAACGCGAACUCGGCCUACAAUAGUGACGU) RNA markers. Gel was stained with 1X SYBR gold (Thermo Fisher Scientific, S11494) and imaged on a Typhoon FLA 7000. Image was printed to size to allow bands, inclusive of 28 nt and exclusive of 34 nt markers, to be cut from the gel, placed into a 1.5 ml RNA low-bind tube and crushed with scalpel. RNA was eluted from the crushed gel pieces in 500 µl extraction buffer (300 mM NaOAc (pH 5.2), 1 mM EDTA, 0.25 % SDS) overnight at 16°C at 600 rpm in a thermo-mixer. Gel pieces were filtered out with a Costar Spin-X Centrifuge Tube Filters 0.45 µm (SLS 8163) and RNA was precipitated with 2 µl glycogen and 500 µl isopropanol overnight at -20°C. Precipitated RNA was purified and dissolved in 43 µl RNase free water. RPF RNA was not rRNA depleted. RNA was heated at 80°C for 90 sec and immediately placed on ice before undergoing 5′ phosphorylation and 3′ dephosphorylation with 1 µl T4 PNK (NEB, M0201S), 5 µl 10X PNK buffer and 1 µl SUPERaseIn RNase Inhibitor (20 U/μL) (Thermo Fisher Scientific, AM2696) at 37 °C for 35 min, with 5µl 10mM ATP added for final 20 min. RNA was extracted with acid phenol chloroform and isopropanol precipitated. Purified RNA was quantified on a qubit with the high-sensitivity RNA kit (Q32852) and ran on an agilent bioanalyser to check for correct length. 10 ng RNA was input into the Bioscientific Nextflex small RNA v3 kit (NOVA-5132-06), using the alternative step F bead clean up, 11 PCR cycles and gel extraction option. Cytoplamsic RNA samples were run on an agilent tapestation to check for integrity and measured on a nanodrop to quantify and check for purity. rRNA was depleted from 1 µg cytoplasmic RNA with the RiboCop v2 and sequencing libraries were prepared from the rRNA depleted RNA with the Corall Total RNA-Seq Library Prep Kit (Lexogen Cat. 095.96) with 13 PCR cycles. Final cytoplamsic RNA and RPF libraries were quantified on an agilent tapestation with a High Sensitivity D1000 tape and sequenced single-end on an Illumina NextSeq500 with a 75 cycles high output kit.

treatment_protocol: Tamoxifen (Sigma, #T5648) suspended in corn oil (Sigma, #C8267) was administered by intraperitoneal injection (IP) at 80mg/kg to induce genetic recombination. Mice were given a single dose of tamoxifen and sampled 3 days later.

value: Small intestine epithelial extractions

tag: mouse id

tag: Sex

value: M

value: Villin-CreER-T2 Apc fl/fl Kras G12D/+ Npm1 fl/fl

tag: time

value: 3 days post-induction

value: Tamoxifen (80mg/kg)

tag: fraction

value: Ribosome protected fragments


title: WT Run 1 input replicate 1

status: Public on Feb 15 2024

type: SRA

city: Bethesda

first: Pedro

middle: J

last: Batista

state: MD

address: 37 Convent Street Bldg 37

department: National Cancer Institute

country: USA

institute: National Institutes of Health

library_source: transcriptomic

data_processing:
Following sequencing, read quality was assessed with FastQC. Barcode split, read collapse, and adaptor trimming was performed using scripts from the icSHAPE pipeline. Trimmed reads were first mapped to a costume geneme that includes rRNA and repeat genome sequences. Reads that did not map to the costume genome were then aligned to hg38 using STAR (v/2.7.6.a). Peak analysis was performed using MACS2. Peaks were required to appear in at least 2 replicates for further analysis. Peak intersection with the hg38 longest transcript isoforms was performed using bedtools (v/2.30.0). Peak changes were identified in DESeq2 or edgeR.
Assembly: hg38
Supplementary files format and content: count matrix

molecule: total RNA

organism: Homo sapiens

extract_protocol:
Total RNA was collected using Trizol reagent according to the manufacturer’s protocol. Following separation of the aqueous and organic phases, the aqueous phase was removed and purified with a Qiagen RNA kit according to the manufacturer’s protocol.
mRNA was polyA selected through two rounds of selection with oligo dT magnetic beads. Following polyA selection, mRNA was fragmented using ZnCl2 before selection with m6A-specific antibody. 3' adaptors sequencing adaptors were added with RNA ligase and reactions were reverse-transcribed with SuperScript IV. cDNA was circularized with circLigase and libraries were PCR amplified to add Illumina sequencing primers. Libraries were sequenced on a NextSeq 550 instrument.

tag: cell line

tag: cell type

value: renal cell carcinoma

tag: genotype

value: WT

tag: treatment

value: input


title: AD3_D60A

city: Newcastle Upon Tyne

first: Majlinda

last: Lako

address: International Centre for Life, Central Parkway

department: Institute of Genetic Medicine and Institute for Ageing

country: United Kingdom

institute: Newcastle University

data_processing:
Spaceranger version 1.0 was used to demulitplex the data, generate gene expression matrices by aligning the FASTQs to GRCH38.
The pipeline was also used to calculate spot co-ordinates using fiducial detection and to identify the spots covered by the tissue. The results from Spaceranger were imported into R using Spaniel (version 1.12). The data from each sample, which was derived from 4 consecutive sections within the tissue, was normalised and clustered, with a resolution of 0.5, using Seurat as described in the scRNA analysis section.
Supplementary files format and content: csvFile containing filtered count matrix for each cell
Supplementary files format and content: csv with metadata for each cell, contains clusters
Supplementary files format and content: Scaling factors from spaceranger
Supplementary files format and content: Image file processed by space ranger - 4,000 pixels in its largest dimension
Library strategy: Spatial Transcriptomics

molecule: polyA RNA

source_name: organoid

extract_protocol:
Fresh frozen sections of four fetal retina samples of 8, 10, 11 and 13PCW were used for the spatial transcriptome analyses performed with the Visium Spatial Gene Expression kit from 10XGenomics. First the tissue optimisation was performed defining 30 minutes as the most optimal permeabilization time window. The spatial transcriptomic procedure was performed according to manufacturer’s instructions. Four tissue sections from each sample were carefully placed into the four capture areas, fixed, haematoxylin and eosin stained and images in order to preserve histological information. This makes it possible to overlay the cell tissue image and the gene expression data in a later step. After permeabilization, reverse transcription reagents were added on top of the tissues. The tissues were subsequently removed, leaving the cDNA coupled to the arrayed oligonucleotides on the slide.
The cDNA-RNA hybrids were cleaved off the chip and the sequencing libraries were prepared. The sequencing depth varied between 25,000,000 and 150,000,000 million reads.

tag: tissue

value: organoid

tag: developmental stage

tag: section

value: A


title: CC044-F4-Tu1_S45

city: Nedlands

first: Scott

last: Fisher

state: Western Australia

address: 35 Stirling Highway, Nedlands

department: Institute for Respiratory Health

country: Australia

institute: The University of Western Australia

data_processing:
Raw sequencing files were aligned against the murine reference genome (GRCm38) using Kallisto (v0.46.1).
Transcript abundance was converted into gene-level estimate using Tximport package (v1.22) in Rstudio.
Gene counts data was normalized by DEseq2 package and filtered for low-expressed genes in Rstudio.
Assembly: GRCm38
Supplementary files format and content: DESeq normalized gene count data

organism: Mus musculus

source_name: Asbesos induced tumor

growth_protocol: Male mice from different CC strains were mated with female MexTAg mice to generate CC-MexTAg mouse model.

extract_protocol:
Whole tumors were harvested and stored in RNAlater (Thermo fisher) at -80C. Tumours were homogenized with tissue ruptor (Qiagen) and total RNA was extracted using Rneasy mini kit (Qiagen) and 500 ng of total RNA was used to construct sequenting libraries.
Sureselect strand specific mRNA library prep kit (Agilent technology australia) was used for RNA library prepration at Genomics WA (Perth, Australia).

treatment_protocol: The resultant CC‑MexTAg progeny were given two intraperitoneal injections of 3 mg Wittenoom crocidolite (blue) asbestos delivered one month apart.

tag: survival

value: Above median survival

value: Asbesos induced tumor

tag: strain

tag: disease

value: Asbestos related disease (mesothelioma)

value: No treatment


title: AD3_D20C

value: C


title: CC044-F2_S69

value: Below median survival


title: CTRL2

city: Derio

first: Ana

middle: Maria

last: Aransay

state: BIZKAIA

address: Parque tecnologico de Bizkaia, Building 801-A

department: Genome Analysis Platform

country: Spain

institute: CIC bioGUNE

data_processing:
Using STAR software [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530905/], FASTQ files were aligned to Genome data base (Homo_sapiens.GRCh38.94.gtf) and read counts obtained for each gene/transcript/feature
For the detection of diﬀerentially expressed genes, three different methods have been applied:  a) DESeq2 [https://www.ncbi.nlm.nih.gov/pubmed/25516281]: a shrinkage estimation has been applied for dispersions and calculation of expression fold changes, to improve stability and interpretability of estimates  b) edgeR [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2796818/]: An overdispersed Poisson model is used to account for both biological and technical variability, and Empirical Bayes methods are used to moderate the degree of overdispersion across transcripts.  c) Limma & Voom: a linear model was ﬁtted to the data that has been prepared with Voom [https://www.ncbi.nlm.nih.gov/pubmed/24485249], and empirical Bayes moderated t-statistics were calculated using the limma package [https://www.ncbi.nlm.nih.gov/pubmed/16646809] from Bioconductor.  Adjustment of p-values was done by the determination of false discovery rates (FDR) using Benjamini- Hochberg procedure.
Assembly: GRCh38.94
Supplementary files format and content: A matrix is generated as a plain text file (*.txt), in which each column is a sample, each row is a gene (ensembl_ID), and in each cell indicates the number of raw counts obtained by STAR per sample and gene.

source_name: human epithelial renal cell line (HK2)

growth_protocol: human epithelial renal cell line (HK2) (CRL-2190; ATCC) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin, 1% insulin transferrin selenite  and 5ng/ml hydrocortisone.

extract_protocol:
PureLink TM RNA Mini kit (Invitrogen), with Dnase treatment. Total RNA was eluted in RNase-free water
Sequencing libraries were prepared following the protocol "TruSeq Stranded mRNA Sample Preparation Guide(Part#15031047Rev.E)" with the “TruSeq® Stranded mRNA Library Prep” kit  (Illumina Inc. Cat. # 20020594) and TruSeq RNA Single Indexes (Illumina Inc. Cat. # 20020492 and 20020493)
mRNA stranded library sequencing

treatment_protocol: Cell were stimulated with thapsigargin (4 μM) in absence or presence  of JQ1 (500nM) for 24h

value: human epithelial renal cell line


title: TG2

source_name: human epithelial renal cell line (HK2) under thapsigargin treatment


title: AD3_D20A


title: AD3_D20B

value: B


title: CTRL1


title: CC044-D3-Tu2_S12


title: NPM_KO_3_RPFs

country: Germany

description: RPFs_APC-KRAS_KO_batchCorrected_normalised_counts.csv

value: Small intestine epithelial extractions

value: F

value: Villin-CreER-T2 Apc fl/fl Kras G12D/+ Npm1 fl/fl


title: NPM_KO_2_RPFs

value: M

value: 3 days post-induction

value: Tamoxifen (80mg/kg)

value: Ribosome protected fragments


title: CC044-D3-Tu1_S11


title: 7085

city: Ramat Gan

first: Tzipi

last: Braun

address: Derech Sheba 2

country: Israel

institute: Sheba Medical Center at Tel Hashomer

data_processing:
Reads were quantified by kallisto version 42.5, using Gencode v24 as the reference genome
Assembly: Gencode v24
Supplementary files format and content: tab-delimited text files include estimates counts values for each Sample

source_name: Caco-2 human colon carcinoma cell line

growth_protocol: Caco-2 human colon carcinoma cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in standard culture conditions in DMEM (GIBCO 41965-039, Scotland) containing 20% (v/v) heat-inactivated fetal bovine serum (GIBCO 12657-029, Scotland).

extract_protocol:
Total RNA was isolated using Tri Reagent-LS (Sigma, T9424, Saint Louis, MO, USA).
Lexogen QuantSeq 3' mRNA-Seq libraries and single-end was performed

treatment_protocol: LHFPL3-AS2 stable knockdown was achieved by infecting Caco-2 cells with CRISPR/dCAS9-KRAB inactivator plasmid that was packaged into virions generated in HEK293T cells using lentiviral vector system, monoclonal clones were selected with Puromycin. Then the pKLV-gRNAs clones which contained the U6 promoter and hygromycin gene (hygro) were packaged into virions and generated in HEK293T cells using lentiviral vector system. Afterwards, a stable clone of Caco-2 expressing CRISPRi plasmid was infected by a lentivirus carrying the specific gRNA or with an empty plasmid that is used as a control (gCtl).

tag: group

value: gCtl


title: 7089


title: NPM_KO_1_RPFs


title: NPM_WT_3_RPFs

value: Villin-CreER-T2 Apc fl/fl Kras G12D/+


title: CC040-H1_S154


title: 7084


title: AD3_D150B

value: organoid


title: AD3_D150D

value: D


title: NPM_WT_4_RPFs


title: AD3_D150A

value: A


title: AD3_D150C


title: CC036-F1-Tu2_E3_S57

value: Above median survival

value: Asbesos induced tumor

value: Asbestos related disease (mesothelioma)

value: No treatment


title: CC040-E4-_S48


title: NPM_WT_1_RPFs


title: 7083


title: CC040-E1--Tu2_S15


title: NPM_WT_2_RPFs


title: 7080


title: CC040-D3_S148


title: AD3_D10D


title: CC036-F1-Tu1_D3_S56


title: NPM_KO_4_Totals

description: Totals_APC-KRAS_KO_batchCorrected_normalised_counts.csv

value: Total Cytoplasmic RNA from undigested lysate


title: 7082


title: CC036-C3_S54


title: NPM_KO_3_Totals

value: Small intestine epithelial extractions


title: AD3_D10C

value: C


title: 7078


title: AD3_D10A


title: AD3_D10B

value: B


title: 7079


title: 7077


title: CC036-C1_S55


title: NPM_KO_2_Totals

value: M

value: Villin-CreER-T2 Apc fl/fl Kras G12D/+ Npm1 fl/fl


title: NPM_WT_4_Totals

value: 3 days post-induction

value: Tamoxifen (80mg/kg)


title: CC035-H2-tu_S15


title: CC035-H1-tu3_S18


title: NPM_WT_3_Totals

value: F


title: CB003

city: Montreal

first: Daniel

last: Sinnett

state: QC

address: 2900 Edouard Montpetit Blvd

country: Canada

institute: University of Montreal

data_processing:
Raw reads were aligned to the GRCh37 genome reference using STAR v2.5.3a.
CircRNA expression counts were obtained using CIRCexplorer3.
Assembly: grch37
Supplementary files format and content: circrna expression counts

source_name: Pediatric acute lymphoblastic leukemia

extract_protocol:
Mini AllPrep DNA/RNA kits from Qiagen
Ribo-Zero Gold Kit according to Illumina's protocol

tag: diagnosis

value: B cell control

value: Cord blood CD10+/CD19+

tag: gender

value: normal-B

tag: cohort

value: discovery


title: NPM_KO_1_Totals

tag: mouse id

tag: Sex


title: NPM_WT_2_Totals

value: Villin-CreER-T2 Apc fl/fl Kras G12D/+

tag: time

tag: fraction


title: 7076

tag: cell line

value: gCtl


title: CB002


title: CC035-H1-tu2_S17

value: Above median survival

value: Asbesos induced tumor

value: Asbestos related disease (mesothelioma)


title: CB001


title: NPM_WT_1_Totals

value: Small intestine epithelial extractions

tag: genotype

value: Total Cytoplasmic RNA from undigested lysate


title: CC035-D3-pins_S14

tag: treatment

value: No treatment


title: CC021-C4_S51

tag: tissue


title: CC021-C2_S53

value: Below median survival


title: DFAM-61345_Quizartinib_Spleen ZQSN03

city: Singapore

first: Shruti

last: Bhatt

state: Singapore

address: Blk S7 level 2,  Science Drive 3, Room 02-05

department: Pharmacy

country: Singapore

institute: National University of Singapore

data_processing:
Paired-end RNA-seq samples were matched to the human (hg19) and mouse (mm9) genomes with the first STAR.
Each pair of BAM files were then passed onto Xenofilter to eliminate mouse reads, and resulting BAM files were converted back to fastq files.
Transcript expression was quantified using Salmon and was summarized to gene-level. Genes with transcripts smaller than 300bp were ignored.
The count data matrix obtained from Salmon was imported into R using tximport (R package), and then Limma was applied to perform differential analysis.
The output from Limma was then used for gene set enrichment analysis (GSEA), using clusterProfiler to calculate normalized enrichment score (NES) and false discovery rate (FDR).
Assembly: hg19 and mm9
Supplementary files format and content: comma separated file including raw counts of all sample.
Supplementary files format and content: comma separated file including raw counts of all sample.

source_name: Spleen

extract_protocol:
mRNA was extracted from fresh or frozen samples with Qiagen’s RNeasy Mini Kit following manufacturer’s instructions.
Non-stranded RNA-seq libraries were generated using the True-SeqRNA Sample Prep kit (Illumina) on a Sciclone platform (Perkin Elmer). mRNA underwent fragmentation, cDNA synthesis, and nextgeneration library synthesis via exome capture and PCR amplification

value: Spleen


title: DFAM-61345_Quizartinib_Spleen ZQSN02


title: B051

value: bone marrow

value: M


title: B030


title: B031

value: F

value: HHD


title: DFAM-61345_Quizartinib_Bonemarrow ZQBN04

source_name: Bone marrow

value: Bone marrow


title: CC017-I4_S47


title: CC021-C1_S52


title: B018


title: DFAM-61345_Quizartinib_Bonemarrow ZQBN03


title: CC017-H2-tu2_S13


title: B012


title: DFAM-61345_Quizartinib_Bonemarrow ZQBN02


title: DFAM-61345_Venetoclax_Spleen ZASN02


title: CC035-H1-tu1_S16


title: B016


title: B068


title: DFAM-61345_Venetoclax_Bonemarrow ZABN01


title: CC001-D1-Tu2_G3_S59


title: DFAM-61345_Venetoclax_Spleen ZASN01


title: CC001-H2-tu2_S30


title: DFAM-15354_Birinapant_Spleen XTSN04


title: B009


title: B007


title: Boon-I4-Tu2_S68

value: BoonxMexTAg


title: CC001-H2-tu1_S70


title: CC001-H3_S60


title: DFAM-15354_Birinapant_Spleen XTSN01


title: DFAM-15354_Birinapant_Spleen XTSN03


title: Pooled SHARE-seq sublibraries from human kidney tissues (ATAC-seq Novaseq S4 2nd run)

city: New Haven

first: Haikuo

last: Li

state: CT

address: 55 Prospect St

institute: Washington University in St. Louis

description: snATAC-seq, paired with Novaseq_S4_2_RNA

library_source: genomic single cell

data_processing:
bowtie2 (ATAC) or STAR (RNA) alignment
demultiplexing with customized scripts
duplicate removal with Picard
read annotation with featurecounts
UMI-Tools for matrix creation (Overall, using codes from either https://github.com/masai1116/SHARE-seq-alignment or https://github.com/masai1116/SHARE-seq-alignmentV2/
Assembly: hg19
Supplementary files format and content: h5 or h5ad file: count matrix for snRNA-seq
Supplementary files format and content: csv.gz file: cell-by-gene count matrix for snRNA-seq
Supplementary files format and content: bed.gz or tsv.gz file: fragment profile for snATAC-seq
Supplementary files format and content: Two extra supp files (primer_sequence.py, P5_primer_ID.txt). They clarify the barcode sequences and the specific P5 primers of use. Users should be able to perform all analysis with this information.

molecule: genomic DNA

source_name: kidney

extract_protocol:
Nuclei isolation from human kidney tissues
An optimized SHARE-seq protocol
snRNA-seq and snATAC-seq in the same cells

value: kidney


title: Boon-H2-Tu3_S23


title: CC001-D1-Tu1_F3_S58


title: Pooled SHARE-seq sublibraries from human kidney tissues (RNA-seq Novaseq S4 2nd run, lane4)

description: snRNA-seq, paired with Novaseq_S4_2_ATAC

library_source: transcriptomic single cell


title: DFAM-61345_Quizartinib_Spleen ZQSN04


title: B011

value: discovery


title: DFAM-15354_Birinapant_Bonemarrow XTBN04


title: B003


title: Boon-H2-Tu1_S53


title: DFAM-15354_Birinapant_Bonemarrow XTBN03


title: frog_embryo_mRNA_stage_10_PAL_seq_v4

city: Cambridge

first: Kehui

last: Xiang

state: MA

address: 455 Main Street

institute: Whitehead Institute

description: Endognous mRNA, library made with PAL-seq v4

data_processing:
For gene-specific tail-seq of mRNA reporters, data were processed with a custom script available at https://github.com/coffeebond/MPRA_tail_seq.
For PAL-seq v3 or v4, reads were trimmed with cutadapt v3.7 with the parameters “-m 15 --quality-base=64 -q 20,20 --match-read-wildcards -e 0.05 -a NNNNATCTCGTATGCCGTCTTCTGCTTG -O 7”. The trimmed reads were mapped using STAR (v2.7.1a) to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained, the genomic sequences of humans or fish, depending on which spike-in RNAs were used, and the sequences of the poly(A) standards generated previously, with the parameters “--runThreadN 16 --runMode alignReads --outFilterMultimapNmax 1 --outReadsUnmapped Fastx --outFilterType BySJout --outSAMattributes All --outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL-seq.
Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4, primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly.
Supplementary files format and content: For gene-specific tail-seq of mRNA reporters, tab-delimited text files with columns indicating 1) sequence of the variable region; 2) median tail length; 3) number of reads; 4–7) tail lengths at quantile 10, 25, 75, and 90.
Supplementary files format and content: For PAL-seq v3 or v4, tab-delimited files with columns indicating 1) gene ID or poly(A) site ID; 2) read cluster ID; 3) tail length
Library strategy: PAL-seq v4

organism: Xenopus laevis

source_name: embryo

extract_protocol:
Frog oocytes and embryos were lysed in ice-cold buffer RL (20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1% [v/v] Triton X-100, 100 µg/ml cycloheximide, cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer], and 200 units/ml SUPERase•In) in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri-reagent for RNA isolation. Fish embryos were de-chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. After removing all E3 medium, Tri-Reagent was added to the embryos for RNA isolation. Mouse GV-oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus-oocyte-complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG-induced mice (16 hr), denuded with 3 mg/ml hyaluronidase for 2 min, washed, and then collected in Tri-reagent for RNA isolation.
For gene-specific tail-seq of reporter libraries, total RNA with reporter mRNA libraries was ligated to a pre-adenylated 3ʹ adapter directly in most cases, but for the N60 library injected into fish embryos and frog oocytes, the N37-PAS-N17 library injected into fish embryos and frog oocytes, and the CPEmos-N60 and N60(LC)-PASmos libraries injected into frog oocytes, reporter library mRNAs were enriched by anti-sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009, 8 pmol KXSH010, and 2x SSC (0.3 M NaCl, 30 mM sodium citrate pH 7.0) in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads (Thermo Fisher, 65002) and incubated for 20 min at 23°C on a thermal mixer, shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl) and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined, precipitated with ethanol, and resuspended in 6.5 µl water.
The anti-sense oligo-enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre-adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330, 50 mM HEPES pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol, 1 unit/µl T4 RNA ligase 1 (New England Biolabs, M0204S). The ligation reaction was incubated at 23°C for 150 min. After ligation, RNA was extracted with phenol/chloroform, precipitated with ethanol, and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl, incubated at 65°C for 5 min, and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First-Strand Buffer, 500 µM dNTPs, 5 mM dithiothreitol, 1 unit/µl SUPERase•In, and 200 units SuperScript III (Thermo Fisher, 18080044) at 50°C for 1 hr. After reverse transcription, RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min, followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio-Spin P-30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer (Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR-amplified library was cleaned up twice with AMPure XP beads (Beckman Coulter, A63881) with a beads-to-sample ratio of 1.2.
For sequencing of endogenous mRNA poly(A)-tail length, libraries were prepared with PAL-seq v3 (for frog oocytes) or PAL-seq v4 (for frog embryos, fish embryos, and mouse oocytes) as described previously (PMID: 34213414). When preparing the sequencing libraries of mRNAs from fish embryos, a different 3ʹ adapter (KXS013) was used for 3ʹ-end ligation, and poly(A)-selected mRNA from HeLa cells was used as spike-in, replacing poly(A)-selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer, shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined, precipitated with ethanol, and resuspended in 30 µl water. The oligo-depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample after monitoring consistency.

value: embryo


title: frog_embryo_mRNA_stage_9_PAL_seq_v4


title: Pooled SHARE-seq sublibraries from human kidney tissues (RNA-seq Novaseq S4 2nd run, lane3)


title: frog_embryo_mRNA_stage_8_PAL_seq_v4


title: DFAM-15354_Birinapant_Bonemarrow XTBN01-re


title: Boon-D1-Tu2_S52

value: Above median survival


title: frog_embryo_mRNA_stage_7_PAL_seq_v4


title: frog_embryo_mRNA_stage_12_PAL_seq_v4


title: DFAM-15354_Quizartinib_Spleen XQSN03


title: Pooled SHARE-seq sublibraries from human kidney tissues (RNA-seq Novaseq S4 1st run)

description: snRNA-seq, paired with Novaseq_S4_1_ATAC


title: frog_embryo_mRNA_stage_6_PAL_seq_v4


title: Pooled SHARE-seq sublibraries from human kidney tissues (ATAC-seq Novaseq S2)

description: snATAC-seq, paired with Novaseq_S2_RNA


title: frog_embryo_mRNA_stage_3_PAL_seq_v4


title: Boon-H2-Tu2_S1

value: Asbesos induced tumor


title: DFAM-15354_Quizartinib_Bonemarrow XQBN03


title: BOON-I2_S150

value: Asbestos related disease (mesothelioma)


title: Boon-D1-Tu1_S51


title: D687_TI2

data_processing:
Reads were quantified by kallisto version 42.5, using Gencode v24 as the reference genome
Data was filtered to includ mRNAs  with TPM above 1 in 20% of the samples.
Assembly: Gencode v24
Supplementary files format and content: tab-delimited text files include TPM values for each Sample

source_name: Terminal ileum

extract_protocol:
mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.
The strand-specific cDNA sequencing libraries were generated using NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® (NEB, USA), and index codes were added. Samples were purified (AMPure XP system) and clustering of the index-coded samples was performed on a cBot Cluster Generation System using HiSeq 4000 PE Cluster Kit (Illumina, NEB, USA). After cluster generation, the stranded, poly-A selected libraries were sequenced on an Illumina NovaSeq 6000 platform by Novogene Bioinformation Technology.

tag: patient id

tag: country

value: China

tag: diagosis

value: CD

tag: patient group

value: male

tag: age

tag: bmi

tag: crp mg_l


title: BOM-L1_S36

tag: survival

tag: strain

value: BOMxMexTAg

tag: disease

value: No treatment


title: 8156-MH-8

city: Nashville

first: Bruce

last: Carter

state: TN

address: 2215 Garland Ave

department: Biochemistry

institute: Vanderbilt University

data_processing:
Paired-end RNA sequencing reads (150nt long) were trimmed and filtered for quality using Trimgalore v0.6.7 (Krueger et al, 2021). Trimmed reads were aligned and counted using Spliced Transcripts Alignment to a Reference (STAR) (Dobin et al., 2013) v2.7.9a with the –quantMode GeneCounts parameter against the GRCm38 (mm10) mouse genome.
Pre-processed reads were aligned using TopHat (v2.0.11) and differential gene expression was determined using Cuffdiff (v.2.1.1)
Assembly: GRCm38 (mm10) mouse genome.
Supplementary files format and content: Excel workbook, differential expression analysis and normalized counts for region and genotype comparisons.

source_name: Primary Microglia

growth_protocol: Jedi knockout (JKO) mice were Pear1tm1a(KOMP)Wtsimice derived from embryonic stem cells provided by the International Mouse Phenotype Consortium (IMPC, Cat # CSD31459_C05). Control mice were wild-type (WT) C57/BL6 mice obtained from Jackson Labs and then maintained by our laboratory (catalog no. 000664).

extract_protocol:
Microdissected Cortex and V-SVZ tissue samples from WT and JKO mice were dissociated using the Papain Dissociation System (see ‘Primary microglia cultures’), and microglia were then isolated from the cell suspension by Magnetic Activated Cell Sorting using Cd11b microbeads per manufacturer instructions with MS columns and MiniMACs separator (Miltenyi Biotec, Cat # 130-093-634).
RNA was harvested using Rneasy micro plus kit (Qiagen)
RNA samples were submitted to Vanderbilt University Medical Center VANTAGE Core for poly-A enrichment-based library preparation and sequencing on the Illumina NovaSeq (PE-100).

value: Primary Microglia

tag: cell type

value: Microglia

value: JKO

tag: region

value: V-SVZ


title: Pooled SHARE-seq sublibraries from human kidney tissues (RNA-seq Novaseq S2)

description: snRNA-seq, paired with Novaseq_S2_ATAC


title: DFAM-15354_Quizartinib_Spleen XQSN02

value: Spleen


title: DFAM-15354_Quizartinib_Spleen XQSN01


title: frog_oocyte_mRNA_progesterone_9hr_PAL_seq_v3

description: Endognous mRNA, library made with PAL-seq v3

data_processing:
For gene-specific tail-seq of mRNA reporters, data were processed with a custom script available at https://github.com/coffeebond/MPRA_tail_seq.
For PAL-seq v3 or v4, reads were trimmed with cutadapt v3.7 with the parameters “-m 15 --quality-base=64 -q 20,20 --match-read-wildcards -e 0.05 -a NNNNATCTCGTATGCCGTCTTCTGCTTG -O 7”. The trimmed reads were mapped using STAR (v2.7.1a) to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained, the genomic sequences of humans or fish, depending on which spike-in RNAs were used, and the sequences of the poly(A) standards generated previously, with the parameters “--runThreadN 16 --runMode alignReads --outFilterMultimapNmax 1 --outReadsUnmapped Fastx --outFilterType BySJout --outSAMattributes All --outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL-seq.
Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4, primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly.
Supplementary files format and content: For gene-specific tail-seq of mRNA reporters, tab-delimited text files with columns indicating 1) sequence of the variable region; 2) median tail length; 3) number of reads; 4–7) tail lengths at quantile 10, 25, 75, and 90.
Supplementary files format and content: For PAL-seq v3 or v4, tab-delimited files with columns indicating 1) gene ID or poly(A) site ID; 2) read cluster ID; 3) tail length
Library strategy: PAL-seq v3

source_name: oocyte

value: oocyte

value: 10 uM progesterone 9 hours


title: frog_oocyte_mRNA_progesterone_7hr_PAL_seq_v3

value: 10 uM progesterone 7 hours


title: 8156-MH-7

value: Cortex


title: frog_embryo_mRNA_stage_2_PAL_seq_v4

value: embryo


title: 8156-MH-6

description: CDS outlier

value: WT


title: D685_TI2

tag: gender

value: female


title: DFAM-15354_Quizartinib_Bonemarrow XQBN02

value: Bone marrow


title: Pooled SHARE-seq sublibraries from human kidney tissues (ATAC-seq Novaseq S4 1st run)

description: snATAC-seq, paired with Novaseq_S4_1_RNA

value: kidney


title: DFAM-15354_Quizartinib_Bonemarrow XQBN01


title: frog_oocyte_mRNA_progesterone_5hr_PAL_seq_v3

value: 10 uM progesterone 5 hours


title: D683_TI1

value: China

value: CD


title: 8156-MH-5

description: CDS outlier

tag: cell line

value: Primary Microglia


title: DFAM-15354_VenetoclaxResistant_vehicletreated_Spleen XCSV04


title: 8156-MH-4

value: Microglia

tag: genotype


title: frog_oocyte_mRNA_progesterone_3hr_PAL_seq_v3

tag: treatment

value: 10 uM progesterone 3 hours


title: D686_TI1

value: male


title: frog_oocyte_mRNA_progesterone_1hr_PAL_seq_v3

tag: tissue

value: 10 uM progesterone 1 hour


title: D681_TI1

tag: patient id


title: DFAM-15354_VenetoclaxResistant_vehicletreated_Spleen XCSV03

value: Spleen


title: D680_TI2

tag: country


title: D648_TI2

tag: diagosis


title: DFAM-15354_VenetoclaxResistant_vehicletreated_Bonemarrow XCBV04


title: D633_TI1

tag: patient group


title: D678_TI1

tag: age


title: frog_oocyte_mRNA_no_progesterone_PAL_seq_v3

value: oocyte


title: DFAM-15354_VenetoclaxResistant_vehicletreated_Bonemarrow XCBV03


title: 8156-MH-3

value: JKO


title: 8156-MH-1

value: Cortex


title: D632_TI1

tag: bmi


title: 7814-MH-4

description: CDS outlier

value: V-SVZ


title: DFAM-15354_VenetoclaxResistant_drugtreated_Spleen XASR07


title: D629_TI1

tag: crp mg_l


title: 7814-MH-3


title: DFAM-15354_VenetoclaxResistant_drugtreated_Spleen XASR03


title: CPEmos_N60_library_frog_oocyte_progesterone_7hr_Tail_seq

description: Libraries enriched by antisese oligos before ligaion, followed by reverse transcription and PCR amplification with library-specific primers

library_source: other

data_processing:
For gene-specific tail-seq of mRNA reporters, data were processed with a custom script available at https://github.com/coffeebond/MPRA_tail_seq.
For PAL-seq v3 or v4, reads were trimmed with cutadapt v3.7 with the parameters “-m 15 --quality-base=64 -q 20,20 --match-read-wildcards -e 0.05 -a NNNNATCTCGTATGCCGTCTTCTGCTTG -O 7”. The trimmed reads were mapped using STAR (v2.7.1a) to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained, the genomic sequences of humans or fish, depending on which spike-in RNAs were used, and the sequences of the poly(A) standards generated previously, with the parameters “--runThreadN 16 --runMode alignReads --outFilterMultimapNmax 1 --outReadsUnmapped Fastx --outFilterType BySJout --outSAMattributes All --outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL-seq.
Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4, primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly.
Supplementary files format and content: For gene-specific tail-seq of mRNA reporters, tab-delimited text files with columns indicating 1) sequence of the variable region; 2) median tail length; 3) number of reads; 4–7) tail lengths at quantile 10, 25, 75, and 90.
Supplementary files format and content: For PAL-seq v3 or v4, tab-delimited files with columns indicating 1) gene ID or poly(A) site ID; 2) read cluster ID; 3) tail length
Library strategy: gene-specific tail-seq

molecule: other

organism: synthetic construct

source_name: Sythetic mRNA reporter library

value: Sythetic mRNA reporter library

value: 10 uM progesterone 7 hours


title: CPEmos_N60_library_frog_oocyte_progesterone_5hr_Tail_seq


title: DFAM-15354_VenetoclaxResistant_drugtreated_Spleen XASR02


title: 7814-MH-2

value: WT


title: 8156-MH-2


title: CPEmos_N60_library_frog_oocyte_progesterone_3hr_Tail_seq


title: DFAM-15354_Venetoclax_Spleen XASN01


title: DFAM-15354_VenetoclaxResistant_drugtreated_Spleen XASR04


title: 7814-MH-1


title: D627_TI1

value: female


title: D621_TI2


title: CPEmos_N60_library_frog_oocyte_progesterone_1hr_Tail_seq


title: DFAM-15354_VenetoclaxResistant_drugtreated_Bonemarrow XABR05-re

value: Bone marrow


title: DFAM-15354_VenetoclaxResistant_drugtreated_Bonemarrow XABR06


title: D618_TI2

value: China


title: 23-CD11b

city: Perth

first: Alexander

last: Tang

address: 35 Stirling Highway, University of Western Australia

department: School of Biomedical Science

institute: University of Western Australia

description:
microglia_rawreadcounts_allsamples.csv
microglia_normalisedcounts_allsamples.csv

data_processing:
First 15 base pairs of sequenced reads were trimmed to remove low-quality sequences using fastp
Trimmed sequence reads were mapped to GrCM39/mm10 using the STAR RNA-seq aligner. STAR-aligned SAM files were converted into BAM files.
Read counts were generated using GenomicAlignments
Normalisation of counts was performed using DESeq2
Assembly: mm10
Supplementary files format and content: comma-separated values files containing the raw readcounts for each sample
Supplementary files format and content: comma-separated values files containing counts that have been normalised using the variance stabilising transformation (VST) for each sample

source_name: CD11b, burn

extract_protocol:
RNA was extracted using the RNeasy Micro Kit (Qiagen). A minimum of 1ng total RNA was sent to the Australian Genome Research Facility (Melbourne, Australia) to generate libraries for sequencing.
RNA libraries for whole transcriptome RNA-seq were prepared using the Illumina Stranded Total RNA Library Preparation Kit with Ribo-Zero Plus, by the technical staff at the Autralian Genome Research Facility (Melbourne, Australia)

value: Brain

value: microglia

value: Thermal burn (7-8% total body surface area)


title: N60_and_N37_PAS_N17_libraries_fish_embryo_shield_7hr_post_injection_Tail_seq

value: None, shield stage


title: D614_TI1

value: CD


title: D612_TI1

value: male


title: DFAM-61786_JQ1_Spleen YJSN02

value: Spleen


title: 22-CD11b


title: N60_and_N37_PAS_N17_libraries_fish_embryo_sphere_4hr_post_injection_Tail_seq

value: None, sphere stage


title: N60_and_N37_PAS_N17_libraries_fish_embryo_30percent_epiboly_5hr_post_injection_Tail_seq

value: None, 30%-epiboly


title: DFAM-15354_VenetoclaxResistant_drugtreated_Bonemarrow XABR02


title: 20-CD11b


title: DFAM-61786_JQ1_Spleen YJSN01


title: D607_TI1


title: D611_TI1


title: N60_and_N37_PAS_N17_libraries_fish_embryo_high_3hr_post_injection_Tail_seq

value: None, high stage


title: 19-CD11b

source_name: CD11b, sham

value: Control (no burn injury)


title: N60_and_N37_PAS_N17_libraries_fish_embryo_512cell_2hr_post_injection_Tail_seq

value: Sythetic mRNA reporter library

value: None, 512-cell stage


title: 24-CD11b


title: CPEmos_N60_library_frog_oocyte_uninjected_Tail_seq


title: DFAM-61786_JQ1_Bonemarrow YJBN02-re


title: D606_TI1


title: DFAM-15354_VenetoclaxResistant_drugtreated_Bonemarrow XABR04


title: N60_and_N37_PAS_N17_libraries_fish_embryo_32cell_1hr_post_injection_Tail_seq

value: None, 32-cell stage


title: D605_TI1


title: 18-CD11b


title: 21-CD11b


title: N60_and_N37_PAS_N17_libraries_fish_embryo_uninjected_Tail_seq


title: 17-CD11b


title: DFAM-61786_JQ1_Bonemarrow YJBN01


title: C487_TI2

value: Control

value: Rural

value: female


title: DFAM-61786_Birinapant_Bonemarrow YTBN09

value: Bone marrow


title: B323_TI2

value: China

value: Urban


title: DFAM-61786_Birinapant_Bonemarrow YTBN04


title: 15-CD11b

value: Brain

value: microglia


title: 16-CD11b


title: N60_and_N37_PAS_N17_libraries_frog_embryo_stage_10_8hr_post_injection_Tail_seq

description: Direct ligaion to total RNA, followed by reverse transcription and PCR amplification with library-specific primers.

value: None, stage 10


title: 13-CD11b


title: N60_and_N37_PAS_N17_libraries_frog_embryo_stage_12_20hr_post_injection_Tail_seq

value: None, stage 12


title: 10-CD11b

value: Thermal burn (7-8% total body surface area)


title: N60_and_N37_PAS_N17_libraries_frog_embryo_stage_9_6hr_post_injection_Tail_seq

value: None, stage 9


title: B321_TI2

value: male


title: DFAM-61786_Birinapant_Bonemarrow YTBN02


title: B320_TI2


title: DFAM-61786_Birinapant_Spleen YTSN07

value: Spleen


title: 8-CD11b

value: Control (no burn injury)


title: N60_and_N37_PAS_N17_libraries_frog_embryo_stage_7_3hr_post_injection_Tail_seq

value: Sythetic mRNA reporter library

value: None, stage 7


title: 7-CD11b


title: B319_TI2

value: Control


title: DFAM-61786_Birinapant_Spleen YTSN05


title: B238_TI2


title: 3-CD11b


title: N60_and_N37_PAS_N17_libraries_frog_embryo_stage_6_2hr_post_injection_Tail_seq

value: None, stage 6


title: DFAM-61786_Birinapant_Bonemarrow YTBN03


title: DFAM-61786_Birinapant_Spleen YTSN04


title: N60_and_N37_PAS_N17_libraries_frog_embryo_stage_3_1hr_post_injection_Tail_seq

value: None, stage 3


title: B236_TI2

value: female


title: B322_TI2

value: China


title: N60_and_N37_PAS_N17_libraries_frog_embryo_stage_8_4hr_post_injection_Tail_seq

value: None, stage 8


title: 2-CD11b

value: Brain


title: DFAM-61786_Birinapant_Spleen YTSN03


title: N60_and_N37_PAS_N17_libraries_frog_embryo_stage_1_5min_post_injection_Tail_seq

value: None, stage 1


title: B232_TI2

value: Urban


title: 24-ACSA

description:
astrocytes_rawreadcounts_allsamples.csv
astrocytes_normalisedcounts_allsamples.csv

source_name: ACSA, burn

value: astrocytes


title: B235_TI2


title: DFAM-61786_Birinapant_Spleen YTSN01


title: N60_and_N37_PAS_N17_libraries_frog_embryo_uninjected_Tail_seq


title: 23-ACSA

value: Thermal burn (7-8% total body surface area)


title: N60_and_N37_PAS_N17_libraries_frog_oocyte_progesterone_7hr_Tail_seq

value: 10 uM progesterone 7 hours


title: 22-ACSA


title: B230_TI2


title: N60_and_N37_PAS_N17_libraries_frog_oocyte_progesterone_3hr_Tail_seq

value: 10 uM progesterone 3 hours


title: DFAM-61786_Birinapant_Spleen YTSN02

value: Spleen


title: N60_and_N37_PAS_N17_libraries_frog_oocyte_progesterone_5hr_Tail_seq

value: 10 uM progesterone 5 hours


title: 20-ACSA


title: 21-ACSA


title: DFAM-61786_QuizartinibResistant_drugtreated_ Spleen YQSR06


title: DFAM-61786_QuizartinibResistant_drugtreated_ Spleen YQSR07


title: B231_TI2


title: B228_TI2


title: N60_and_N37_PAS_N17_libraries_frog_oocyte_uninjected_Tail_seq

value: Sythetic mRNA reporter library


title: DFAM-61786_QuizartinibResistant_drugtreated_ Spleen YQSR05


title: DFAM-61786_Quizartinib_Spleen YQSN03


title: B212_TI2


title: DFAM-61786_Quizartinib_Spleen YQSN02


title: 18-ACSA

source_name: ACSA, sham

value: Control (no burn injury)


title: N60_and_N37_PAS_N17_libraries_frog_oocyte_progesterone_1hr_Tail_seq

value: 10 uM progesterone 1 hour


title: 17-ACSA


title: DFAM-61786_Quizartinib_Spleen YQSN04


title: 19-ACSA


title: B211_TI2

value: Control

value: male


title: B226_TI2

value: female


title: B223_TI2

value: China


title: 16-ACSA


title: 15-ACSA


title: B207_TI2


title: DFAM-61786_QuizartinibResistant_drugtreated_ Bonemarrow YQBR07

value: Bone marrow


title: 13-ACSA

value: Brain


title: B206_TI2


title: DFAM-61786_QuizartinibResistant_drugtreated_ Bonemarrow YQBR05


title: DFAM-61786_Quizartinib_Bonemarrow YQBN04


title: TMA4, Visium

city: Rome

first: Antonio

last: Agostini

address: Largo Francesco Vito, 1

country: Italy

institute: Università Cattolica del Sacro Cuore

description: Visium

data_processing:
The demultiplexing, barcoded processing, gene counting were made using the  Space Ranger software v1.3.1 following standard workflow
Assembly: GRCh38
Supplementary files format and content: matrix files, json files, csv

source_name: Pancreatic Tissue

extract_protocol:
5μm section for each TMA were positioned in each  indivdual capture area, and processed following manufacturer reccomendations.
Libraries were obtained according to the manufacter's instructions: For Visium the tissue were processed following the Visim Spatial Gene Expression for FFPE.

value: Pancreatic Tissue


title: DFAM-61786_Quizartinib_Bonemarrow YQBN02


title: 11-ACSA


title: 12-ACSA


title: DFAM-61786_Quizartinib_Bonemarrow YQBN01


title: IPMN61, Geomx

description: Nanostring GeoMx

data_processing:
Sequencing data was uploaded to the Illumina BaseSpace hub and processed with DRAGEN to obtain .dcc files
Assembly: GRCh38
Supplementary files format and content: tabular .dcc files
Library strategy: GEOmx Spatial Transcriptomics

source_name: High-grade IPMN

extract_protocol:
5μm sections from to TMA were used for library construction for spatial transcriptomics
Two IPMN TMA obtained from APGI were analyzed for spatial transcriptomics using GeoMX Human Whole Transcriptome Atlas (Nanostring, USA) following the provided protocol. TMAs were stained with GeoMX morphology kit to mark neoplastic cells (PanCK), and immune cells (CD45). After staining all TMA cores that showed signs of detachment and degradation, or contained few IPMN cells (< 100 nuclei) were discarded from the analysis. From the original set of 101 tissue cores from 61 patients, a total of 80 ROI from 46 patients were selected (multiple ROI from the same patient) for segmentation to isolate only the PanCK positive areas and exclude CD45 areas to obtain the specific transcriptome of IPMN cells

value: High-grade IPMN


title: 8-ACSA

value: astrocytes


title: DFAM-61786_Quizartinib_Spleen YQSN01-re


title: DFAM-61786_VenetoclaxResistant_vehicletreated_Spleen YCSV07


title: IPMN60, Geomx

description: Nanostring GeoMx


title: B209_TI2

value: Urban


title: 7-ACSA

value: Thermal burn (7-8% total body surface area)


title: IPMN78, Geomx

description: Nanostring GeoMx

source_name: Borderline IPMN

value: Borderline IPMN


title: DFAM-61786_QuizartinibResistant_drugtreated_ Bonemarrow YQBR06


title: DFAM-61786_VenetoclaxResistant_vehicletreated_Spleen YCSV06

value: Spleen


title: 3-ACSA


title: IPMN58, Geomx

description: Nanostring GeoMx


title: IPMN59, Geomx

description: Nanostring GeoMx


title: 2-ACSA


title: DFAM-61786_VenetoclaxResistant_vehicletreated_Spleen YCSV05-re


title: 24-mRuby

description:
inhibitory_rawreadcounts_allsamples.csv
inhibitory_normalisedcounts_allsamples.csv

source_name: mRuby, burn

value: inhibitory neurons


title: DFAM-61786_VenetoclaxResistant_vehicletreated_Bonemarrow YCBV06


title: IPMN77, Geomx

description: Nanostring GeoMx


title: 23-mRuby


title: DFAM-61786_VenetoclaxResistant_vehicletreated_Bonemarrow YCBV05


title: 10-ACSA


title: IPMN10, Geomx

description: Nanostring GeoMx

source_name: Low-grade IPMN

value: Low-grade IPMN


title: TMA3, Visium

description: Visium


title: 22-mRuby


title: DFAM-61786_VenetoclaxResistant_vehicletreated_Bonemarrow YCBV04


title: 21-mRuby


title: DFAM-61786_VenetoclaxResistant_drugtreated_Spleen YASR03

source_name: Spleen


title: IPMN57, Geomx

description: Nanostring GeoMx


title: IPMN56, Geomx

description: Nanostring GeoMx


title: DFAM-61786_VenetoclaxResistant_vehicletreated_Spleen YCSV04


title: 17-mRuby

source_name: mRuby, sham

value: Control (no burn injury)


title: IPMN55, Geomx

description: Nanostring GeoMx


title: DFAM-61786_Venetoclax_Bonemarrow YABN01

source_name: Bone marrow


title: IPMN76, Geomx

description: Nanostring GeoMx


title: 19-mRuby


title: DFAM-61786_Venetoclax_Spleen YASN01


title: 18-mRuby


title: DFAM-61786_VenetoclaxResistant_drugtreated_Bonemarrow YABR02


title: DFAM-61786_VenetoclaxResistant_drugtreated_Spleen YASR02


title: 16-mRuby


title: IPMN53, Geomx

description: Nanostring GeoMx


title: 12-mRuby


title: DFAM-61345_Control_Bonemarrow ZCBN02


title: IPMN54, Geomx

description: Nanostring GeoMx


title: DFAM-61345_Control_Spleen ZCSN01


title: 13-mRuby


title: IPMN52, Geomx

description: Nanostring GeoMx


title: DFAM-61345_Control_Bonemarrow ZCBN01


title: DFAM-15354_Control_Spleen XCSN02


title: IPMN9, Geomx

description: Nanostring GeoMx


title: IPMN74, Geomx

description: Nanostring GeoMx


title: IPMN8, Geomx

description: Nanostring GeoMx


title: 10-mRuby


title: 11-mRuby


title: DFAM-61345_Control_Spleen ZCSN02


title: 15-mRuby


title: IPMN75, Geomx

description: Nanostring GeoMx


title: IPMN2, Geomx

description: Nanostring GeoMx


title: 20-mRuby


title: DFAM-15354_Control_Spleen XCSN01-re


title: 8-mRuby


title: 7-mRuby


title: DFAM-15354_Control_Bonemarrow XCBN02


title: IPMN50, Geomx

description: Nanostring GeoMx


title: IPMN51, Geomx

description: Nanostring GeoMx


title: IPMN49, Geomx

description: Nanostring GeoMx


title: DFAM-15354_Control_Bonemarrow XCBN01-re


title: 2-mRuby


title: DFAM-15354_Control_Spleen XCSN01


title: 24-GFP

description:
excitatory_rawreadcounts_allsamples.csv
excitatory_normalisedcounts_allsamples.csv

source_name: GPF, burn

value: excitatory neurons


title: DFAM-61786_Control_Spleen YCSN03-re


title: IPMN48, Geomx

description: Nanostring GeoMx


title: 3-mRuby


title: 22-GFP


title: Bleo_lin-pos_Mouse_4

city: Biberach

first: Meshal

last: Ansari

address: Birkendorfer Straße 65

institute: Boehringer Ingelheim

description:
10x
muc17859

data_processing:
After sequencing, the processing of next-generation sequencing reads of the scRNA-seq data was performed using CellRanger version 3.1.0 (10x Genomics) with a modified genome, by using the mus musculus reference genome (mm10) and manually adding the sequence of the dt-Tomtato gene. We performed unsupervised analysis for multiple subsets of the data with scanpy (v1.6.0). We filtered genes that were expressed in at least 10 cells. Barcodes with less than 250 genes detected and a mitochondrial fraction of less than 10% were excluded, and those with a number of transcripts between 500 to maximal 50 000 were retained.
Assembly: mm10 + tdTomato sequence
Supplementary files format and content: Raw count matrices, seperated by treatment (Tcf21Labeled_BLEO_raw_counts.mtx, Tcf21Labeled_PBS_raw_counts.mtx) from 10x experiments with 19559 genes and 24322 cells in market matrix format and corresponding column (Tcf21Labeled_genes.txt) and row (Tcf21Labeled_BLEO_barcodes.txt, Tcf21Labeled_PBS_barcodes.txt) labels. Cell-level metadata (Tcf21Labeled_metadata.txt).

source_name: Lung tissue

extract_protocol:
Cells were counted and stained with anti-mouse Epcam CD326-AlexaFluor647 antibody (1:100, Biolegend, 118212), CD31-APC (1:250, Invitrogen, 17-0311-82), CD45-APC (1:250, Biolegend, 103112), Lyve1-AF647 (Invitrogen, 50-0443-82), Ter119-APC (1:100, Biolegend, 116218), and Zombie Violet for viability (BioLegend, 423113). All stainings were performed per 10 MIo cells in 1ml. Cells were stained with Zombie violet according to manufacturer's instructions for 15 min at RT, washed once with PBS and  blocked with anti-mouse FC block for 10min on ice. Staining occurred for 20min at 4°C in the dark in the FACS buffer (PBS + 0.1% BSA), and cells were washed once and filtered with a cell strainer (70µM)  before subjecting them to FACS sorting. Mesenchymal cells were sorted as ZombieViolet-/APC- and TCF21 lineage was distinguished by the dt-Tomato signal in the PE channel. Data acquisition was performed in a BD Fortessa flow cytometer (Becton Dickinson, Heidelberg, Germany). 30.000 cells per lineage and mouse were sorted into cooled RPMI media.
The sorted cells were were diluted to 1000 cells/µl, and loaded on the 10x Chromium Next GEM Chip G with a targeted cell recovery of 10,000. The following steps were completed according to the manufacturer’s protocol (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1). Libraries have been pooled according to their minimal required read counts (35,000 or 50,000 reads/cell for 3′ gene expression libraries, 20,000 reads/cell for 5′ gene expression libraries, and 5000 reads/cell for TCR libraries). Illumina paired-end sequencing was performed with 150 or 200 (3′ gene expression) on a NovaSeq 6000.

treatment_protocol: Tcf21iCre(+/-);R26tdTomato(+/-)-lineage labeled mice, both female and male,  were  treated with 100mg/kg Tamoxfien 3 days before the start and randomly divided into two groups: saline-only (NaCl), or bleomycin (Bleo). Lung injury and pulmonary fibrosis were induced by single-dose administration of bleomycin hydrochloride (Sigma Aldrich, Germany), which was dissolved in sterile NaCl and given at 3.5U/kg bodyweight by oropharyngeal instillation. The control group was treated with sterile NaCl only. Mice were  sacrificed at day 14  after instillation.

value: Bleo

value: positive

tag: number of cells


title: IPMN47, Geomx

description: Nanostring GeoMx


title: DFAM-15354_Control_Bonemarrow XCBN01


title: IPMN73, Geomx

description: Nanostring GeoMx


title: Bleo_lin-neg_Mouse_4

description:
10x
muc17860

value: negative


title: IPMN1, Geomx

description: Nanostring GeoMx


title: PBS_lin-neg_Mouse_4

description:
10x
muc17858

value: PBS


title: DFAM-61786_Control_Bonemarrow YCBN03-re

value: Bone marrow


title: DFAM-61786_Control_Spleen YCSN02


title: 20-GFP

source_name: GFP, burn

value: Brain

tag: strain


title: DFAM-61786_Control_Spleen YCSN03


title: 19-GFP

source_name: GFP, sham


title: 18-GFP


title: PBS_lin-neg_Mouse_3

description:
10x
muc17854


title: IPMN46, Geomx

description: Nanostring GeoMx


title: DFAM-61786_Control_Bonemarrow YCBN03


title: PBS_lin-pos_Mouse_3

description:
10x
muc17853


title: 17-GFP


title: 16-GFP


title: Bleo_lin-neg_Mouse_2

description:
10x
muc17852


title: IPMN45, Geomx

description: Nanostring GeoMx


title: IPMN44, Geomx

description: Nanostring GeoMx


title: DFAM-61786_Control_Bonemarrow YCBN02


title: PBS_lin-neg_Mouse_2

description:
10x
muc17850


title: 15-GFP


title: Bleo_lin-pos_Mouse_2

description:
10x
muc17851


title: control_TCR

city: HOUSTON

first: Navid

last: Sobhani

state: TEXAS

address: 1 BAYLOR PLAZA

department: Medicine, Epidemiology and Population Sciences

institute: Baylor College of Medicine

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v7.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: h5 or comma separated values

source_name: tumor

extract_protocol:
Tumors from CTLA-4-humanized MC38 syngeneic mice (n = 3 - 5 mice) were extracted for single cell suspension using a gentleMACSTM Octo Dissociator. The cells were stained with anti-mouse CD45 antibody (PE-Cy7, Clone 30-F11, Cat. 103114, Lot: B354212) and sorted with a BD FACSAriaII Cell Sorting Flow Cytometer
Library preparation of CD45+ sorted cells for single-cell- RNA sequencing (scRNA-seq) was performed using the 10X Genomics Chromium Single Cell 5’ Library & Gel Bead reagent kit. For TCR sequencing, the Chromium Single Cell V(D)J Enrichment kit was used.
scRNA-seq and TCR-seq

value: TILS

value: tumor

value: 7-8 weeks old


title: IPMN42, Geomx

description: Nanostring GeoMx

value: High-grade IPMN


title: control

description: 10x Genomics


title: PBS_lin-pos_Mouse_2

description:
10x
muc17849


title: 8-GFP


title: IPMN41, Geomx

description: Nanostring GeoMx


title: D11_mTCR

description: 10x Genomics


title: IPMN43, Geomx

description: Nanostring GeoMx


title: PBS_lin-neg_Mouse_1

description:
10x
muc17846


title: IPMN72, Geomx

description: Nanostring GeoMx


title: 3-GPF

value: Thermal burn (7-8% total body surface area)


title: WT.M3

city: Columbus

first: Amy

last: Hite

state: OH - Ohio

address: 1800 Cannon Drive 250 Lincoln Tower

department: Biomedical Informatics

institute: The Ohio State University

data_processing:
Raw reads were aligned to mouse reference genome GRCm38 with HISAT2 v2.1.0. Gene wise counts were generated with featureCounts from the subread package v1.5.1 for genes annotated by ensembl Mus_musculus.GRCm38.102, counting the primary alignment in the case of multimapped reads. Raw counts were normalized by voom.
Assembly: GRCm38
Supplementary files format and content: voom normalized counts

source_name: hippocampal tissue

extract_protocol:
Total RNA was extracted from cryopulverized hippocampal tissue by PureLink™ RNA Mini Kit (Invitrogen, 012183025) according to the manufacturer’s instructions following lysis with the provided lysis buffer.
RNA cleaning and concentration was done using Zymo Research, RNA Clean & Concentrator™-5 kit (cat# R1015) following the manufacturer’s protocol. Fluorometric quantification of RNA and RNA integrity analysis was carried out using RNA Biochip and Qubit RNA Fluorescence Dye (Invitrogen). cDNA libraries were generated using NEBNext® Ultra™ II Directional (stranded) RNA Library Prep Kit for Illumina (NEB #E7760L). Ribosomal RNA was removed using NEBNext rRNA Depletion Kit (human, mouse, rat) (E #E6310X). Libraries were indexed using NEBNext Multiplex Oligos for Illumina Unique Dual Index Primer Pairs (NEB #644OS/L). Library prep generated cDNA was quantified and analyzed using Agilent DNA chip and Qubit DNA dye.

value: hippocampal tissue

value: WT

tag: Sex

value: M


title: WT.M2


title: D11+anti-PD-1_mTCR

description: 10x Genomics

tag: cell type


title: D11+anti-PD-1

description: 10x Genomics


title: 2-GFP


title: IPMN40, Geomx

description: Nanostring GeoMx


title: Ipilimumab_mTCR

description: 10x Genomics


title: Bleo_lin-pos_Mouse_1

description:
10x
muc17847


title: IPMN39, Geomx

description: Nanostring GeoMx


title: Human iPS, ChIA-PET

city: Seattle

first: Chia-Lin

last: Wei

state: WA

address: 3720 15th Ave NE

department: The Northwest Genomics Center (NWGC)

institute: University of Washington

description: PolII ChIA-PET

library_source: genomic

data_processing:
ChIA-PET data were processed using ChIA-PET Utilities  (https://github.com/cheehongsg/CPU) on hg19 reference genome. Interaction data or loops with significant (called with Chiasig method by Paulsen 2014; PMID: 25114054) PETs >= 3 and PET distance > 8 kb are used for downstream analyses.
ChIP-seq fastq files were trimmed with trim_galore (v0.4.0)  with error rate 0.2 and min quality 30, then mapped with bwa aln. ChIP vs Input peaks were called using MACS version 2.1.0.20151222  (options: --nomodel --extsize 250 -B --SPM)
Assembly: hg19
Supplementary files format and content: ChIA-PET: non-redundant paired BAM files and Chiasig output interaction files (coordinates of 2 anchors at the first six columns, interaction frequency at the 8th).
Supplementary files format and content: ChIP-seq: narrowPeak and BAM alignment files

source_name: hiPSC

extract_protocol:
10 million for formaldehyde crosslinked cells were suspended with 100  μl 0.55% SDS and incubated at RT for 10 min, 62°C for 10 min and 37°C for 10 min to permeabilize nuclei, which was followed by adding 25 μl 20% TritonX-100 with an incubation of 30 min at 37°C to quench SDS, and then 50 μl of blunt-end four-cutter AluI (cat# R0137L, NEB), 50 μl 10 × CutSmart buffer and 275 μl H2O incubating at 37°C overnight for in situ digestion. After pelleted and washed once with 1 ml 1× CutSmart buffer, the nuclei were suspended in 500 μl A tailing solution composed of 50 μl 10 × CutSmart buffer, 10 μl BSA (cat#B9000S, NEB), 10 μl 10 mM dATP (cat#N0440S, NEB), 10 μl Klenow (3’-5’ exo-) (cat#M0202L, NEB), and 420 μl H2O and incubated at RT for 1 hr.  The in situ proximity ligation with biotinylated bridge linker was performed by adding 200 μl 5 × ligation buffer (cat#B6058S, NEB), 6 μl Bridge linker (200 ng/ul), 10 μl T4 DNA ligase (cat#M0202L, NEB) and 284 μl H2O and incubating at 16°C overnight.
The nuclei with in situ proximity ligation were then subjected to sonication and chromatin immunoprecipitation with anti-RNApolII antibody (8WG16, cat# 920102, Biolegend, San Diego, CA), Tn5 tagmentation, biotin selection, PETs were amplified by PCR and sequenced.
in-situ ChIA-PET nuclear proximity ligation

tag: developmental stage

value: PGP1 line Passage 45

value: hiPSC

tag: chip antibody

value: RNAPII monoclonal antibody (8WG16, cat# 920102, Biolegend, San Diego, CA)


title: IPMN71, Geomx

description: Nanostring GeoMx

value: Borderline IPMN


title: PBS_lin-pos_Mouse_1

description:
10x
muc17845

value: positive


title: IPMN37, Geomx

description: Nanostring GeoMx


title: WT.F3

value: F


title: IPMN70, Geomx

description: Nanostring GeoMx


title: IPMN38, Geomx

description: Nanostring GeoMx


title: Bleo_lin-neg_Mouse_1

description:
10x
muc17848

value: Bleo

value: negative


title: WT.F1


title: DAY15_CHIR

city: Xiamen

first: Jie

last: Ren

state: Fujian Province

address: 2999 Jinshan Road

department: Institute of Cardiovascular Diseases

country: China

institute: Xiamen Cardiovascular Hospital of Xiamen University

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v6.1.1.
Assembly: GRCh38
Supplementary files format and content: *.tsv: Transcript counts in tab-separated values files.

source_name: H1 human embryonic stem cell line

extract_protocol:
hPSC-derived cardiac cells at day15 were dissociated to single cells by 4 mg/ml collagenase type 4 treatment in PBS for 20 minutes at 37°C and then Accutase treatment for 15 minutes at 37°C. Dissociated cells were stained with DAPI and then live cells were sorted using MoFlo Astrios EQS.
Libraries were performed according to the manufacturer’s instructions (Single Cell 3’ Reagent Kit v3 protocol, 10x Genomics). Briefly, cardiac cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

tag: cell line

value: H1 human embryonic stem cell line

value: hPSC-derived cardiac cells

value: CHIR

tag: time point

value: Day 15


title: Human Cardiomyocyte, ChIA-PET

description: PolII ChIA-PET

source_name: CM

value: Differentiation day 35 cells

value: CM

value: human CM


title: Ipilimumab

description: 10x Genomics

value: TILS

value: tumor

value: 7-8 weeks old


title: PD-1_mTCR

description: 10x Genomics


title: WT.F2

value: hippocampal tissue

tag: genotype

value: WT


title: DAY11_scRNA-seq

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v6.1.1.  Further processing of the scRNA-seq data was performed in R software v4.1.0 with Seurat v4.3.0.  Cells were retained if they had greater than 1800 and fewer than 6000 genes and their mitochondrial content was below 25%.  The transcript counts.tsv file was generated by exporting the "counts" data from the Seurat object as a data frame.
Assembly: GRCh38
Supplementary files format and content: Transcript counts as .tsv files

extract_protocol:
hPSC-derived cardiac cells at day15 were dissociated to single cells by 4 mg/ml collagenase type 4 treatment in PBS for 20 minutes at 37°C and then Accutase treatment for 15 minutes at 37°C. Dissociated cells were stained with DAPI and then live cells were sorted using MoFlo Astrios EQS.
libraries were performed according to the manufacter’s instructions (Single Cell 3’ Reagent Kit v3 protocol, 10x Genomics).  Briefly, cardiac cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM).  The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode.  The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction.  During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: Cardiac cells


title: D11

description: 10x Genomics

tag: tissue


title: C11.M1

value: M


title: IPMN36, Geomx

description: Nanostring GeoMx

value: High-grade IPMN


title: DAY15_DMSO

description: 10x Genomics

tag: treatment

value: DMSO


title: PD-1

description: 10x Genomics


title: C11.F3

value: F


title: C11.M3


title: M1 nuclei, train, mouse2, shallow snRNAseq

city: Kingston

first: Ying

last: Zhang

state: RI

address: 120 Flagg Rd

department: Cell and Molecular Biology

institute: University of Rhode Island

description:
10x Genomics
Mouse 2 shallow

data_processing:
Sequencing results were processed using the Cell Ranger v4.0.0 pipeline in order to demultiplex, perform alignment, UMI counting, and aggregate samples.
Assembly: mm10
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz

molecule: nuclear RNA

source_name: caudal forelimb area of primary motor cortex

extract_protocol:
All brains were serially sectioned in ice-cold, carbogen-bubbled ACSF on a VT1000S vibratome (Leica) with polytetrafluoroethane-coated razor blades (Ted Pella) at 0.15 mm/sec and 100 Hz, dividing the whole cerebrum into 400 µm coronal slices. Each slice between 0 and 1.2 mm anterior to bregma (2-4 slices per animal) was dissected under a stereomicroscope using a microsurgical stab knife to cut out the caudal forelimb area (CFA) in the motor cortex of the right hemisphere (~1 to 2 mm from the midline). All extracted tissue was recovered in ice-cold, nuclease-free, 0.22 µm sterile-filtered cryoprotective nuclear storage buffer, composed of 0.32 M sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 10 mM Trizma hydrochloride buffer (pH 8.0), 1 mM dithiothreitol, 0.02 U/µl SUPERase•In RNAse Inhibitor (Invitrogen), and 1X cOmplete Protease Inhibitor Cocktail with EDTA (Roche). Tissue was then flash frozen using a metal CoolRack M90 (Biocision) pre-chilled to -80˚C and stored at -80˚C until nuclear isolation. Single nucleus RNA-seq was provided by the Center for Epigenomics, UC San Diego using the Droplet-based Chromium Single-Cell 3’ solution (10x Genomics, v3.1 chemistry) [5]. Nuclei preparation was adapted from Lacar et al. 2016 (PMID: 27090946). The flash frozen mouse motor cortex tissues were thawed on ice for ~30-50 minutes before centrifugation for 3 minutes at 100 rcf in a 4 °C, swinging bucket centrifuge with setting run 3/3 (5920, Eppendorf). The supernatant was removed and the tissue was resuspended in 1 ml of  douncing buffer consisting of 0.25M sucrose (S1888, Sigma), 25mM KCl (AM9610G, Invitrogen), 5mM MgCl2 (194698, Mp Biomedicals Inc.), 10 mM Tris-HCl pH 7.5 (15567027, Thermo Fischer Scientific), 1mM DTT (D9779, Sigma), 1X protease inhibitor (05056489001, Roche), 0.1% Triton X-100 (T8787-100ML, Sigma), and 0.2 U/ul RNasin RNase inhibitor (PAN21110, Promega) in molecular biology grade water (46000-CM, Corning). The tissue was then transferred to a dounce homogenizer on ice and dounced 25 times with a loose plunger and 25 times with a tight plunger. Suspension was then passed through a Celltrix 30 μM filter (04-004-2326, Sysmex) and washed with 300 ul of douncing buffer before being centrifuged for 10 minutes at 1000 rcf. Pellet was then washed with an additional 1 ml of douncing buffer without Triton X-100 and centrifuged again for 10 minutes at 1000 rcf. Supernatant was then discarded, and the pellet was resuspended in 500 ul of sort buffer consisting of 1 mM EDTA (15575020, Invitrogen), 0.2 U/ul RNasin, and 1% BSA in PBS before staining with 3 μM DRAQ7 (#7406S, Cell Signaling Technology). Nuclei were then incubated on ice for 10 minutes and ~20-30,000 nuclei were sorted using a 100 μm chip in an SH800 sorter (Sony) into 50 ul of collection buffer consisting of 1 U/ul RNasin and 5% BSA in PBS. Samples were then centrifuged for 15 minutes at 1000 rcf; the supernatant was removed, leaving behind ~20 ul, and an additional 25 ul of reaction buffer consisting of 0.2 U/ul RNasin and 1% BSA in PBS was added for a total volume of ~45-50 ul.
The prepared nuclei were visually inspected and manually counted using a hemocytometer before loading 15,000 onto a Chromium Controller for 10x GEM generation in the Single Cell 3’ v3.1 kit (1000268, 10x Genomics).  Libraries were generated using the Chromium Single Cell 3′ Library Construction Kit v3.1 (1000190, 10x Genomics) according to manufacturer specifications. CDNA was amplified for 12 PCR cycles. SPRISelect reagent (Beckman Coulter) was used for size selection and clean-up steps. Final library concentration was assessed by Qubit dsDNA HS Assay Kit (Thermo-Fischer Scientific) and fragment size was checked using Tapestation High Sensitivity D1000 (Agilent) to ensure that fragment sizes were distributed normally about 500 bp.

treatment_protocol:
All mice received a controlled amount of water (1mL/day) on a petri dish in their home cages on the first 10 days. On days 11-13, the control group mice (n=3) continued to receive 1 mL/day in their home cage, whereas the training group mice (n=3) received 1 mL/day, in part as reward during a lever-press task (0.3 – 0.8 mL) and the rest in their home cage immediately after completing the task.
Mice in the training group performed a lever-press task as previously described. Briefly, a lever was positioned for head-fixed mice to press it with their left paw. A trial began with a 6-kHz tone marking a cue period (10 s) during which a successful lever-press was rewarded with water (∼15 μl/trial), followed by an intertrial interval (12 s). A success was defined as lever-press crossing two thresholds (∼1.5 mm and ∼2.6 mm below the resting position) within 200 ms. With these criteria, a distinct and brisk downward press was considered as successful lever-press. No successful lever-press during the cue period triggered a loud white noise and an intertrial interval. Lever-press during intertrial intervals was neither rewarded nor punished. Each daily session ended after mice completed 100 trials.

value: 13 weeks old

value: water restriction, motor training


title: M1 nuclei, control, mouse5, shallow snRNAseq

description:
10x Genomics
Mouse 5 shallow

value: caudal forelimb area of primary motor cortex

value: water restriction, no motor training


title: C11.M2


title: MAA4788, Alzheimer's

data_processing:
Assess raw read QC with FastQC v0.11.5 before trimming and after trimming.  Trimming performed with TrimGalore v0.4.5 specifying RRBS and Illumina with a quality cutoff of 20.  Quality and adapter trimmed reads aligned to bismark generated indexes for GRCm38 with bismark v0.22.1 using bowtie2 v2.3.5.1 with options " --N 1 --L 15 -D 50 --score_min L,-0.6,-0.6 -p 10 --X 600"  Methylations extracted using bismark_methylation_extractor.
Assembly: GRCh38
Supplementary files format and content: cpg methylation calls

source_name: temporal lobe brain

extract_protocol:
NEXTflex Bisulfite Library Prep Kit for Illumina Sequencing (Bioo Scientific, PerkinElmer, MA) was used to convert the study sample genomes to bisulfite-converted genomes.
Illumina sequencing libraries were prepared using Zymo EZ-DNA methylation kit.

tag: disease state

value: Alzheimer's

value: temporal lobe brain


title: M1 nuclei, train, mouse6, shallow snRNAseq

description:
10x Genomics
Mouse 6 shallow


title: IPMN35, Geomx

description: Nanostring GeoMx

source_name: High-grade IPMN


title: WT.M1


title: MAA5190, Normal

value: Normal


title: MAA4660, Normal


title: C11.F1


title: M1 nuclei, train, mouse4, shallow snRNAseq

description:
10x Genomics
Mouse 4 shallow


title: IPMN34, Geomx

description: Nanostring GeoMx


title: MAA4807, Alzheimer's


title: M1 nuclei, control, mouse3, shallow snRNAseq

description:
10x Genomics
Mouse 3 shallow


title: IPMN33, Geomx

description: Nanostring GeoMx


title: IPMN32, Geomx

description: Nanostring GeoMx


title: C11.F2


title: IPMN31, Geomx

description: Nanostring GeoMx


title: 958, Heart, isoproterenol, 21 days

city: Columbia

first: Jacob

middle: Jeffrey

last: Russell

state: MO

address: 1401 Ashland Rd, Apartment E

department: Biomedical Sciences

institute: University of Missouri

data_processing:
Subsequently, unique sequences with length in 18~26 nucleotide were mapped to specific species precursors in miRBase 22.0 by BLAST search to identify known miRNAs and novel 3p- and 5p- derived miRNAs. Length variation at both 3’ and 5’ ends and one mismatch inside of the sequence were allowed in the alignment.
Assembly: The remaining sequences were mapped to other selected species precursors (with the exclusion of specific species) in miRBase 22.0 by BLAST search, and the mapped pre-miRNAs were further BLASTed against the specific species genomes to determine their genomic locations.
Supplementary files format and content: Differential expression of miRNAs based on normalized deep-sequencing counts was analyzed by selectively using Fisher exact test, Chi-squared 2X2 test, Chi-squared nXn test, Student t test, or ANOVA based on the experiments design. The significance threshold was set to be 0.01 and 0.05 in each test.

source_name: Whole Heart

growth_protocol: Mice were housed on a

extract_protocol:
Qiagen miRNEasy Mini Kit (217004)
Quality control analysis and quantification of the DNA library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip.
Raw reads were subjected to an in-house program, ACGT101-miR (LC Sciences, Houston, Texas, USA) to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA) and repeats.

treatment_protocol: Mice received miniosmotic pumps that delivered either sterile saline (control) or isoproterenol at a dose of 30 mg/kg a day. Another group of mice received coadministration of minocycline at 50 mg/kg via daily IP injections

value: Whole Heart

value: 12 weeks of age

value: Male

value: Isoproterenol


title: IPMN30, Geomx

description: Nanostring GeoMx


title: 957, Heart, minocycline + isoproterenol, 21 days

value: WT

value: Minocycline + Isoproterenol


title: IPMN69, Geomx

description: Nanostring GeoMx

source_name: Borderline IPMN

value: Borderline IPMN


title: MAA4382, Alzheimer's


title: AD.M2

value: hippocampal tissue

value: M


title: MAA4617, Alzheimer's

value: F


title: M1 nuclei, control, mouse1, shallow snRNAseq

description:
10x Genomics
Mouse 1 shallow

value: 13 weeks old


title: AD.M1


title: MAA4308, Normal


title: MAA4414, Alzheimer's


title: 954, Heart, isoproterenol, 21 days


title: 955, Heart, minocycline + isoproterenol, 21 days


title: 956, Heart, control, 21 days

value: Vehicle (sterile saline)


title: MAA4494, Normal


title: IPMN29, Geomx

description: Nanostring GeoMx

value: High-grade IPMN


title: IPMN68, Geomx

description: Nanostring GeoMx


title: AD.F3


title: IPMN7, Geomx

description: Nanostring GeoMx

source_name: Low-grade IPMN

value: Low-grade IPMN


title: MAA4130, Normal


title: AD.F2


title: AD.M3


title: AD.F1


title: IPMN28, Geomx

description: Nanostring GeoMx


title: 953, Heart, minocycline + isoproterenol, 21 days


title: B083

data_processing:
Raw reads were aligned to the GRCh37 genome reference using STAR v2.5.3a.
CircRNA expression counts were obtained using CIRCexplorer3.
Assembly: grch37
Supplementary files format and content: circrna expression counts

source_name: Pediatric acute lymphoblastic leukemia

extract_protocol:
Mini AllPrep DNA/RNA kits from Qiagen
Ribo-Zero Gold Kit according to Illumina's protocol

tag: diagnosis

value: bone marrow

tag: gender

tag: cohort

value: discovery


title: IPMN80, Geomx

description: Nanostring GeoMx


title: B082


title: AD-C11.M3


title: 951, Heart, isoproterenol, 21 days

tag: age


title: AD-C11.M2


title: IPMN66, Geomx

description: Nanostring GeoMx


title: B081


title: 950, Heart, control, 21 days


title: IPMN67, Geomx

description: Nanostring GeoMx


title: AD-C11.M1


title: AD-C11.F3


title: B078

value: HHD


title: 949, Heart, control, 21 days

value: Whole Heart


title: IPMN27, Geomx

description: Nanostring GeoMx


title: 948, Heart, isoproterenol, 21 days

value: 12 weeks of age


title: 952, Heart, minocycline + isoproterenol, 21 days

value: Male


title: IPMN26, Geomx

description: Nanostring GeoMx


title: AD-C11.F1


title: AD-C11.F2


title: IPMN25, Geomx

description: Nanostring GeoMx


title: B077


title: 947, Heart, control, 21 days


title: B074


title: B080

value: MLL


title: 944, Heart, control, 21 days

value: WT


title: M1 nuclei, train, mouse6, deep snRNAseq

description:
10x Genomics
Mouse 6 deep

value: caudal forelimb area of primary motor cortex

value: water restriction, motor training


title: IPMN24, Geomx

description: Nanostring GeoMx


title: 946, Heart, minocycline + isoproterenol, 21 days

value: Minocycline + Isoproterenol


title: M1 nuclei, control, mouse5, deep snRNAseq

description:
10x Genomics
Mouse 5 deep

value: water restriction, no motor training


title: IPMN23, Geomx

description: Nanostring GeoMx


title: IPMN65, Geomx

description: Nanostring GeoMx

value: Borderline IPMN


title: B073

value: F


title: B071


title: 943, Heart, isoproterenol, 21 days

value: Isoproterenol


title: IPMN22, Geomx

description: Nanostring GeoMx

value: High-grade IPMN


title: M1 nuclei, train, mouse4, deep snRNAseq

description:
10x Genomics
Mouse 4 deep

value: 13 weeks old


title: B070

value: bone marrow


title: 942, Heart, minocycline + isoproterenol, 21 days


title: M1 nuclei, control, mouse3, deep snRNAseq

description:
10x Genomics
Mouse 3 deep


title: B075

value: discovery


title: 941, Heart, isoproterenol, 21 days


title: B069


title: M1 nuclei, train, mouse2, deep snRNAseq

description:
10x Genomics
Mouse 2 deep


title: 940, Heart, control, 21 days

value: Vehicle (sterile saline)


title: IPMN6, Geomx

description: Nanostring GeoMx

value: Low-grade IPMN


title: B065


title: IPMN20, Geomx

description: Nanostring GeoMx


title: M1 nuclei, control, mouse1, deep snRNAseq

description:
10x Genomics
Mouse 1 deep


title: 939, Heart, minocycline + isoproterenol, 21 days

value: Whole Heart

value: 12 weeks of age


title: KO-IR-4

city: Yuzhong District

first: Lili

last: Huang

state: Chongqing

address: Chongqing

institute: The Second Affiliated Hospital, Chongqing Medical University

data_processing:
We applied the reads filtering towards the raw reads after sequencing to achieve the clean data following the criteria: a)10% base quality <15 b) 13% base quality <20.
Hisat2 2.0.4 mapping to reference genome
count counting
Supplementary files format and content: mm10
Supplementary files format and content: TXT file contain counts for each sample

source_name: kidney cortex

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
Total RNA was extracted from cell or animal tissue by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Bioanalyzer 2200 (Aligent) and kept at -80℃.The RNA with RIN >8.0 is right for cDNA library construction.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Bilateral flank incisions were made to expose the kidneys, and renal ischemia was induced for 22 min using micro-clamps and micro-clips (RS-5420/RS-5410, Roboz, UK). The micro-clamps were removed.

value: kidney cortex

value: ALR knockdownWT+Ischemia/reperfusion

value: Ischemia/reperfusion


title: Chronic screen

city: Amsterdam

first: Chun-Pu

last: Lin

address: Plesmanlaan 121

department: Molecular Oncology and Immunology

country: Netherlands

institute: NKI-AvL

data_processing:
Align to Brie mouse library
MAGeCK (v0.5.7) was used to perform the analysis of the screen.
Assembly: Other, align to Brie mouse sgRNA library
Supplementary files format and content: MAGeCK analysis output, txt file
Library strategy: Custom Library DNA seq, "Brie Library"

source_name: splenic

growth_protocol: CD8+ T cells were maintained in CD8 T cell medium (RPMI, 10% FBS, 100U/ml penicillin, 100μg/ml streptomycin, 10ng/ml IL-2 (ImmunoTools), and 50μM 2-mercaptoethanol (Merck)) at a concentration of 1x10^6 cells/ml

extract_protocol:
Genomic DNA was isolated using the Blood and Cell culture MAXI Kit, according to manufacturer’s instructions.
sgRNAs were amplified using a one-step barcoding PCR with NEBNext High Fidelity 2X PCR Master Mix and the forward and reverse primers. The hexa-N nucleotide stretch contains a unique barcode identifying each sample following deep sequencing.

treatment_protocol: sgRNA library containing T-cells were stimulated under three different conditions: (1) Intense: T-cells were stimulated with plate-bound CD3 antibody for two times in two days. (2) Acute: T-cells were stimulated once with CD3 antibody for 24h. Cells were then removed from the plates and cultured for another 3d before harvesting. (3) Chronic: T-cells were co-cultured daily with fresh D4M.OVA tumor cells for 11d (11 times). For the resting condition, cells were refreshed daily without adding tumor cells. Both stimulated and resting cells were harvested for analysis after 11 days (11 times) tumor stimulation.

value: splenic

value: Primary OT-I cell

value: CD8 T cell

value: cells expressing Brie sgRNA library

value: Chronic screen


title: B063

value: F


title: KO-IR-3


title: Intense and Acute screen

value: Intense and Acute screen


title: IPMN79, Geomx

description: Nanostring GeoMx

value: Borderline IPMN


title: KO-IR-2


title: B062

value: bone marrow

value: M


title: Control-IR-4

value: WT+Ischemia/reperfusion


title: IPMN19, Geomx

description: Nanostring GeoMx

value: High-grade IPMN


title: B061

value: discovery


title: IPMN21, Geomx

description: Nanostring GeoMx


title: Mouse_oocyte_mRNA_GV_rep2_PAL_seq_v4

data_processing:
For gene-specific tail-seq of mRNA reporters, data were processed with a custom script available at https://github.com/coffeebond/MPRA_tail_seq.
For PAL-seq v3 or v4, reads were trimmed with cutadapt v3.7 with the parameters “-m 15 --quality-base=64 -q 20,20 --match-read-wildcards -e 0.05 -a NNNNATCTCGTATGCCGTCTTCTGCTTG -O 7”. The trimmed reads were mapped using STAR (v2.7.1a) to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained, the genomic sequences of humans or fish, depending on which spike-in RNAs were used, and the sequences of the poly(A) standards generated previously, with the parameters “--runThreadN 16 --runMode alignReads --outFilterMultimapNmax 1 --outReadsUnmapped Fastx --outFilterType BySJout --outSAMattributes All --outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL-seq.
Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4, primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly.
Supplementary files format and content: For gene-specific tail-seq of mRNA reporters, tab-delimited text files with columns indicating 1) sequence of the variable region; 2) median tail length; 3) number of reads; 4–7) tail lengths at quantile 10, 25, 75, and 90.
Supplementary files format and content: For PAL-seq v3 or v4, tab-delimited files with columns indicating 1) gene ID or poly(A) site ID; 2) read cluster ID; 3) tail length
Library strategy: PAL-seq v4

source_name: oocyte

extract_protocol:
Frog oocytes and embryos were lysed in ice-cold buffer RL (20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1% [v/v] Triton X-100, 100 µg/ml cycloheximide, cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer], and 200 units/ml SUPERase•In) in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri-reagent for RNA isolation. Fish embryos were de-chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. After removing all E3 medium, Tri-Reagent was added to the embryos for RNA isolation. Mouse GV-oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus-oocyte-complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG-induced mice (16 hr), denuded with 3 mg/ml hyaluronidase for 2 min, washed, and then collected in Tri-reagent for RNA isolation.
For gene-specific tail-seq of reporter libraries, total RNA with reporter mRNA libraries was ligated to a pre-adenylated 3ʹ adapter directly in most cases, but for the N60 library injected into fish embryos and frog oocytes, the N37-PAS-N17 library injected into fish embryos and frog oocytes, and the CPEmos-N60 and N60(LC)-PASmos libraries injected into frog oocytes, reporter library mRNAs were enriched by anti-sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009, 8 pmol KXSH010, and 2x SSC (0.3 M NaCl, 30 mM sodium citrate pH 7.0) in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads (Thermo Fisher, 65002) and incubated for 20 min at 23°C on a thermal mixer, shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl) and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined, precipitated with ethanol, and resuspended in 6.5 µl water.
The anti-sense oligo-enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre-adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330, 50 mM HEPES pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol, 1 unit/µl T4 RNA ligase 1 (New England Biolabs, M0204S). The ligation reaction was incubated at 23°C for 150 min. After ligation, RNA was extracted with phenol/chloroform, precipitated with ethanol, and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl, incubated at 65°C for 5 min, and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First-Strand Buffer, 500 µM dNTPs, 5 mM dithiothreitol, 1 unit/µl SUPERase•In, and 200 units SuperScript III (Thermo Fisher, 18080044) at 50°C for 1 hr. After reverse transcription, RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min, followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio-Spin P-30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer (Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR-amplified library was cleaned up twice with AMPure XP beads (Beckman Coulter, A63881) with a beads-to-sample ratio of 1.2.
For sequencing of endogenous mRNA poly(A)-tail length, libraries were prepared with PAL-seq v3 (for frog oocytes) or PAL-seq v4 (for frog embryos, fish embryos, and mouse oocytes) as described previously (PMID: 34213414). When preparing the sequencing libraries of mRNAs from fish embryos, a different 3ʹ adapter (KXS013) was used for 3ʹ-end ligation, and poly(A)-selected mRNA from HeLa cells was used as spike-in, replacing poly(A)-selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer, shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined, precipitated with ethanol, and resuspended in 30 µl water. The oligo-depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample after monitoring consistency.

value: oocyte


title: IPMN4, Geomx

description: Nanostring GeoMx

value: Low-grade IPMN


title: B064


title: Control-IR-1

value: kidney cortex


title: B058


title: IPMN5, Geomx

description: Nanostring GeoMx


title: Control-IR-2

value: Ischemia/reperfusion


title: Mouse_oocyte_mRNA_MII_rep1_PAL_seq_v4


title: Mouse_oocyte_mRNA_MII_rep2_PAL_seq_v4


title: B060


title: KO-4

value: ALR knockdown

value: Sham


title: Mouse_oocyte_mRNA_GV_rep1_PAL_seq_v4


title: KO-2


title: IPMN18, Geomx

description: Nanostring GeoMx


title: B055


title: B054

value: HHD


title: IPMN64, Geomx

description: Nanostring GeoMx


title: Polysome_sgDap5_Act_2

data_processing:
Read mapping was performed using STAR (2.6.0c)
Read counts per gene were quantified using HTSeq (0.12.4)
Read counts were normalised using DEseq2 (1.36.0)
Assembly: mouse GRCm38
Supplementary files format and content: Raw counts

extract_protocol:
For total RNA, cells were lysed with TRIzol (Thermo Fischer) and RNA purified following the manufacturer's instructions). Polysomes were isolated on sucrose gradient as described previously (PMID: 28388414). Fractions 9-13 were collected for RNA isolated using TRIzol.
Libraries were prepared for sequencing using standard Illumina protocols (PolyA-bead enrichment).

treatment_protocol: Cells were stimulated with anti-CD3 (1.25ug per/ 2x10^6 cells) on 24-well stimulation plates for 24h. Cells were then removed from the plates and harvested for sequencing.

value: splenic

value: Primary OT-I cell

value: CD8 T cell

tag: fraction

value: Polysome


title: Polysome_sgDap5_Act_1


title: IPMN3, Geomx

description: Nanostring GeoMx


title: KO-1