title: RNA-seq Lin- WT Rep2

data_processing:
RNA-seq reads were trimmed using TrimGalore (v0.6.0, -q 15) and then aligned to mm10 reference genome by TopHat (v2.1.1, --b2-very-sensitive --no-coverage-search).
Reads with low alignment quality were filtered out using Samtools (v1.10, -q 30).
Aligned reads for each gene in UCSC mm10 GTF file were counted by featureCounts (v1.6.3) and normalized using DEseq2 (v1.30.1).
Genome_build: mm10
Supplementary_files_format_and_content: txt, gene-level read counts obtained using featureCounts.

source_name: RNA-seq Lin- WT

growth_protocol: Lin- cells were collected by depleting lineage-positive cells from the bone marrow. LSK+ (Lin-SCA-1+c-Kit+) and LSK- (Lin-SCA-1-c-KIT+) were sorted by FACS in 1xPBS.

extract_protocol:
Total RNA from WT and XistΔ/Δ mouse bone marrow Lin- cells (~200,000) was extracted using TRIzol reagent (Invitrogen), and RNA from LSK+ or LSK- cells (~30,000-50,000) was extracted using RNeasy Plus Micro Kit (Qiagen, 74004). For Lin- samples, total RNA from 3 mice were combined per sample. For LSK+ or LSK- cells, total RNA from 2 mice were combined per sample.
RNA-seq libraries of Lin- cells were prepared using Ovation RNA-Seq System Kit (NuGen, 0403-32 and 0348-32), and LSK+ or LSK- RNA-seq libraries were prepared using SMART-Seq v4 Ultra Low Input RNA Kit (Takara, 634888) and KAPA HyperPrep kit (KAPA, 07962347001).

treatment_protocol: Bones (tibias and femurs) were dissected, and bone marrow (BM) was flushed with 1xPBS supplemented with 2% FBS (2% F/PBS) into a 50-ml tube (Falcon) using a 22.5 gauge needle. Cell suspension was pipetted up and down for several times and filtered through a 70μm cell strainer (Falcon, 352350) to obtain a single-cell suspension. Red blood cells in the cell suspension were lysed by ACK Lysing buffer (Gibco, A10492-01) according to the manufacturer's instructions. All antibodies used for FACS staining were from BD Pharmingen unless noted. To label lineage-positive cells, the following antibody cocktail (1:200) containing biotinylated antibodies against CD11b (M1/70), Gr-1 (RB6-8C5), CD3ε (145-2C11), B220 (RA3-6B2), CD8a (53-6.7), CD4 (GK1.5), and TER-119 (TER-119) in combination with BV421-conjugated Streptavidin (BD, 5832259; 1:200) was used. To distinguish between LSK+ and LSK- cells, biotinylated lineage-specific antibody cocktail was used in combination with FITC-conjugated SCA-1 (D7; 1:100) and PE-conjugated c-KIT (2B8; 1:100) antibodies. Labelled cells were filtered through a 70 μm cell strainer before sorting. 7-Amino-Actinomycin D (7-AAD, BD559925; 1:200) solution was used to label the nonviable cells. For LSK+ and LSK- cells, sorting was conducted on MoFlo Astrios Cell Sorter (BD) or MA900 Multi-Application Cell Sorter (Sony).

value: Bone marrow Lin- cells

value: Wild type

value: Female

tag: age

value: 129Sv4/Jae x B6


title: RNA-seq Lin- XistΔ/Δ Rep2

status: Public on Jul 15 2022

type: SRA

city: Durham

first: Eda

last: Yildirim

state: NC

address: DUMC Box 3709

department: Cell Biology

country: USA

institute: Duke University School of Medicine

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

source_name: RNA-seq Lin- XistΔ/Δ

tag: cell type

tag: genotype

value: Xist knockout

tag: Sex

tag: strain


title: day05.H3K27me3.Rep1

city: La Jolla

first: Christopher

last: Benner

state: California

address: 9500 Gilman Dr. MC 0640

department: Medicine

institute: University of California, San Diego (UCSD)

description:
H3K27me3 ChIP-seq at Day 5 (cardiac mesoderm) rep 1
peaks-H3K27me3-D05.bed.gz

library_source: genomic

data_processing:
base-calling use bcl2fastq version 2.17
Reads were aligned with bowtie2 (ATAC/ChIPseq) or STAR (RNAseq) to the human hg19 genome using default parameters. Only reads that mapped uniquely to the genome (MAPQ >=10) were considered for downstream analyses. ATAC/ChIPseq peaks were found using HOMER's getDifferentialPeaksReplicates.pl and gene expression FPKM values were calculated using HOMER's analyzeRepeats.pl program using GENCODE transcript annotations.
Genome_build: hg19
Supplementary_files_format_and_content: BED files with peak positions (hg19), FPKM transcript levels in a tab-delimited text file

molecule: genomic DNA

organism: Homo sapiens

source_name: ES derived cardiac mesoderm

growth_protocol: Human pluripotent stem cells were grown in E8 on Geltrex-coated plates and differentiated into cardiomyocytes and intervening staged using modulation of WNT signalling

extract_protocol:
ATAC-seq was performed as previously described (PMID: 29434377). ChIP-seq experiments for all histone marks at all time points were performed as previously described (PMID: 22763441). Detailed protocols for ChIP-seq including antibodies used and antibodies validations performed are available at www.encodedcc.org associated with each experiment described here. They can also be found at the links below. Standard ChIP-seq, Tissue fixation & sonication: https://www.encodeproject.org/documents/3125496b-c833-4414-bf5f84dd633eb30d/@@download/attachment/Ren_Tissue_Fixation_and_Sonication_v060614.pdf, Immunoprecipitation: https://www.encodeproject.org/documents/89795b31-e65a-42ca-9d7bd75196f6f4b3/@@download/attachment/Ren%20Lab%20ENCODE%20Chromatin%20Immunoprecipitation%20Protocol_V2.pdf. For RNA-seq, total RNA was extracted with Trizol according to protocol (Thermo Scientific, 15596-026).
ATAC-seq libraries were created using Nextera Tn5 transposase with Illumina adapters and PCR-amplified as previously described (PMID: 29434377). ChIP-seq libraries were created as described here: https://www.encodeproject.org/documents/4f73fbc3-956e-47ae-aa2d41a7df552c81/@@download/attachment/Ren_ChIP_Library_Preparation_v060614.pdf. RNA-seq libraries were constructed using standard Illumina TruSeq LT library prep.

treatment_protocol: ZIC3 knock out lines were generated through CRISPR/Cas9-mediated targeting of exon1 that is common to ZIC3 isoforms

tag: tissue

value: ES derived cardiac mesoderm

tag: differentiation time

value: H9 MLC2v:H2B

tag: chip antibody

value: H3K27me3 AM61017 Active Motif

tag: perturbation

value: wild type


title: SH-SY5Y_H2O2- NuMA (TDIF) KD OGAP-seq rep2

city: Sheffield

first: Sherif

last: El-Khamisy

state: South Yorkshire

address: Firth Court, University of Sheffield, Western Bank

department: Molecular Biology and Biotechnology

country: United Kingdom

institute: University of Sheffield

description: MH = Untreated/H2O2-, R90 = H2O2 treated 90 minute recovery, WT = Wild type with respect to NuMA (TDIF), KD = NuMA (TDIF) Knockdown, IN = Input, IP = OGAP-seq

data_processing:
Data mapped using bwa mem version 0.7.17 (parameters: -M -k 25)
Data was filtered prior to downstream analyses. Reads with a quality score of less than 30 were also removed using samtools view (parameters: -q 30)
bigwigs were generated using MACS2 version 2.1.1.2. Fragment pileup and control (IgG) lambda were output to BEDgraph files using MACS2 peak calling (parameters: -B –SPMR). The sample and control lambda BEDgraph signal tracks were then compared using MACS2 bdgcomp (parameters: -m FE), and the resulting bedgraph files converted to bigwigs
Metagene profiles were generated using bam2geneprofile from CGAT scripts using gene annotations derived from Ensembl 85, with reporter set to genes and no normalisation
Assembly: hg38
Supplementary files format and content: Fold-change relative to IgG bigwigs

source_name: Neuroblastoma cell line

growth_protocol: SH-SY5Y cells were cultured in DMEM/F12 (1:1) (1X) + GlutaMAX supplemented with 1X non-essential amino acids, 100 U/ml penicillin and 100 μg/ml streptomycin and 10% fetal bovine serum. Differentiation was performed by culturing in 2.5% fetal bovine serum supplemented with 20μM retinoic acid for 6 days. Cells were grown at 37°C in a humidified atmosphere containing 5% carbon dioxide (CO2).

extract_protocol:
Genomic DNA was extracted using the Qiagen Blood and Tissue Kit. The DNA was digested with OGG1 or Fpg and then the labelled with 5mM biotin labelled aldehyde-reactive probe (ARP). The DNA was subsequenetly sheared by sonication and the labelled DNA was enriched using MyOne Dynabeads, then eluted in TE Buffer. The DNA was then repaired using the PreCR Repair Mix and purified usin the MinElute Clean Up Kit.
Libraries were prepared according to NEB's instructions accompanying the NEBNext Ultra II DNA Library Prep Kit for Illumina with Sample Purification Beads (Cat. No.: E7103L) and NEBNext Multiplex Oligos for Illumina (Index Primer Set 1) (Cat. No.: E7335L). Briefly, the DNA ends were end repaired, 5'phosphorylated and dA-tailing was done using the NEBNext Ultra II End Prep Enzyme Mix, then adaptors were ligated and Uracils were excised using the USER enzyme. The DNA was then cleaned up and enriched by PCR for 12 cycles and then cleaned up with the sample purification beads, before being sequenced on the Illumina NovaSeq 6000.

treatment_protocol: Cells were either left untreated or treated with 10 µM hydrogen peroxide (H2O2) in cold PBS for 10 minutes in the dark, on ice and then washed with PBS. Cells were then allowed to recover in H2O2-free media for 90 minutes at 37°C.

tag: cell line

value: Neuroblastoma cell line

value: NuMA (TDIF) Knockdown


title: Tet2/3 DKO CD4 SP IgG control

city: New York

first: Tarmo

last: Äijö

address: 162 5th Avenue

department: Center for Computational Biology

institute: Flatiron Institute

data_processing:
Library strategy: CUT&RUN
bcl2fastq ver 2.20.0
Adapter trimming and quality filtering of the sequencing libraries was done using fastp (0.21.0) with the default parameters.
The sequencing libraries were mapped against mm10 using Bowtie 2 (2.4.1) (--very-sensitive -X 2000). Mitochondrial reads were removed after alignment. Additional filtering was done using samtools (1.12) using the following parameter values: -q 30 -h -b -F 1804 -f 2.
Reads with identical sequences were filtered and only one was retained for subsequent analysis.
The peaks were identified from the pooled samples against controls using HOMER (4.10) (findPeaks -style factor).
The coverage tracks were generated from the samples obtained by pooling the biological replicates using HOMER.
Genome_build: mm10
Supplementary_files_format_and_content: Coverage tracks (bigWig) produced by HOMER.

source_name: Thymus

growth_protocol: All mice were bred and maintained under specific pathogen-free conditions at University of North Carolina (UNC) Genetic Medicine building, in a facility managed by the Division of Comparative Medicine at UNC Chapel Hill. The experimental procedures were approved by the UNC Institutional Animal Care and Use Committee.

extract_protocol:
500.000 CD4 single positive (SP) cells, sorted by Flow cytometry,  were used as starting material and were processed using the CUT&RUN kit (Epicypher).
. The antibodies used for these experiments are: GATA3 (clone: D13C9) XP (Cell Signalling, cat no: 5852), IgG (Epicypher, cat no: 13-0042). 3ng DNA (corresponding to small fractions of DNA) were used for library preparation utilizing NEBNext Ultra II DNA Library Prep Kit (NEB). The adaptor was diluted to 1:12.5 for adaptor ligation. The adaptor-ligated fragments were amplified for 12 PCR cycles using a 10 sec 60 degrees Celsius annealing/extension step. After amplification, libraries were purified with 1.0x volume of AMPure XP beads (Beckman). The concentration of DNA was measured using the double stranded (ds) DNA High Sensitivity (HS) assay in a Qubit 4 Fluorometer (Invitrogen).  The quality control and the size of the library was determined using High Sensitivity (HS) D1000 Screen Tape assay (Agilent, cat no: 5067-5584), including HS D1000 screen tape and HS D1000 reagents (such as HS D1000 sample buffer and HS D1000 ladder, Agilent, cat no: 5067-5585), in a Tapestation 4150 (Agilent).

treatment_protocol: Mice were euthanized by CO2 asphyxiation and thymi were isolated and dissociated to acquire single-cell suspensions.  Total thymocytes were depleted of CD24hi cells by staining with biotinylated mouse anti-CD24 (clone:M1/69) purchased from Biolegend. Unwanted cells were depleted by binding to mouse streptavidin magnetic beads (anti-mouse Rapidspheres, cat no 19860, Stemcell Technologies) according to the manufacturer’s instructions. Enriched cells were stained with viability dye (eBioscience) to distinguish live cells from. Cells were stained with aGalactosyl-Ceramide loaded tetramer (conjugated with PE, obtained from NIH tetramer core), TCRb (clone:H57-597) conjugated with PERCP/Cy5.5 (Biolegend), anti-mouse CD4 AF488 (clone:RM4-5), CD8 (clone:53-6.7) conjugated with Brilliant Violet 650 or APC.  For the thymic differentiation related studies, live CD4+, CD8-, TCRb+, tetramer - cells were sorted and usedfor CUT&RUN. The purity of the samples after sorting was >98%.

tag: genetic background

tag: cells

tag: treatment

value: untreated

tag: antibody

value: IgG (Epicypher, cat no:13-0042)


title: SH-SY5Y_H2O2+ 90 min recovery WT OGAP-seq rep1


title: Tet2/3 DKO CD4 SP Gata3 BR1

value: GATA3 (clone:D13C9) XP (Cell Signalling, cat no:5852)


title: SH-SY5Y_H2O2+ 90 min recovery WT Input rep2


title: day05.H3K27me3.Rep2

description:
H3K27me3 ChIP-seq at Day 5 (cardiac mesoderm) rep 2
peaks-H3K27me3-D05.bed.gz


title: SH-SY5Y_H2O2- WT OGAP-seq rep2


title: WT CD4 SP Gata3 BR2


title: day02.H3K27me3.Rep1

description:
H3K27me3 ChIP-seq at Day 2 (mesoderm) rep 1
peaks-H3K27me3-D02.bed.gz

source_name: ES derived mesoderm

value: ES derived mesoderm


title: day00.H3K27me3.Rep2

description:
H3K27me3 ChIP-seq at Day 0 (ESC) rep 2
peaks-H3K27me3-D00.bed.gz

source_name: Embryonic stem cell

value: Embryonic stem cell


title: SH-SY5Y_H2O2+ 90 min recovery WT Input rep1


title: WT CD4 SP IgG control 2


title: Tet2/3 DKO CD4 SP Gata3 BR2


title: RNA-seq Lin- WT Rep1


title: day02.H3K27me3.Rep2

description:
H3K27me3 ChIP-seq at Day 2 (mesoderm) rep 2
peaks-H3K27me3-D02.bed.gz


title: 4_D_imsc_blasts

city: Newcastle upon Tyne

first: Graham

last: Smith

address: Framlington Place

department: Bioinformatics Support Unit

institute: Newcastle University

data_processing:
Quality assessment with FastQC (version 0.11.5)
transcript abundance was quantified from raw reads with Salmon (version 0.8.2) using the reference human transcriptome (hg38) defined by GENCODE release 27
R package tximport (version 1.6.0) was used to estimate raw gene-level counts and abundances from Salmon’s transcript-level counts
R package DESeq2 (version 1.18.1) was used to generate gene-level normalized counts
Assembly: hg38
Supplementary files format and content: raw_counts.txt: tab-delimited text containing unnormalized gene-level counts
Supplementary files format and content: normalized_counts.txt: tab-delimited text containing normalized gene-level counts

source_name: patient derived xenograft sample

growth_protocol: L707 cells were maintained in SFEMII media on co-culture with either iMSC or iANG. 7_G_Before_Feeder_L707D is patient derived xenograft sample harvested from the mice and RNA taken on thawing

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.0 ug of total RNA was used for the construction of sequencing libraries.
Sequencing libraries were prepared using the TruSeq Stranded mRNA Sample Preparation Kit [Illumina] following manufacturer's instructions.

treatment_protocol: L707 cells were maintained in SFEMII media on co-culture with either iMSC or iANG. Cells were co-cultured over a seven day period

value: patient derived xenograft sample

value: leukaemia cell, acute lymphoblastic leukaemia

value: following 7 days co-culture with iMSC cells

tag: time

value: Day 7


title: 3_C_iangio_blasts

value: following 7 days co-culture with iANG cells


title: day80.ATAC.Rep1

description:
ATAC-seq at Day 80 (ventricular cardiomyocyte) rep 1
peaks-ATAC-D80.bed.gz

source_name: ES derived ventricular cardiomyocyte

value: ES derived ventricular cardiomyocyte


title: day00.H3K27me3.Rep1

description:
H3K27me3 ChIP-seq at Day 0 (ESC) rep 1
peaks-H3K27me3-D00.bed.gz


title: SH-SY5Y_H2O2- WT OGAP-seq rep1


title: 7_G_Before_Feeder_L707D

value: Day 0


city: Toronto

first: Ulrich

last: Braunschweig

state: Ontario

address: 160 College Street

department: Donnelly Centre

country: Canada

institute: University of Toronto

description: L1_W001: SPAR-seq of 108 multi-exon gene regions in N2A cells, control non-targeting knockdown (T0001), replicate 1

data_processing:
CASAVA 1.8 was used for base calling.
Fastq files contain demultiplexed reads which were assigned to the appropriate treatment by matching to expected forward and reverse barcodes (sequenced in dedicated reads; not supplied).
Forward and reverse event reads were mapped to custom junction libraries representing all expected splice variants (supplied as processed files), using bowtie with settings --best -v 3 -k 1 --trim3 26 --trim5 10. Trimming was done to remove lower-quality, uninformative ends and because forward reads had a missing base call at position 11 due to a power outage. Junction libraries were first constructed from NCBIm37/mm9 gene annotations, and then refined based on the results of de novo mapping of the reads from one full Illumina lane from each cell line using tophat (default settings with -i 40 and providing Ensembl transcript annotations for NCBIm37), after quality trimming (minimum MAPQ of 33) from the 3'-end with the fastx toolkit. Mapped reads were counted per junction.
Alternative splicing analysis: Percent-spliced-in (PSI) values were calculated for each alternative exon, or part thereof in the case of alternative 5'-/3'-splice sites ('event'), from read tallies as follows: PSI was calculated independently from forward and reverse reads as the percentage of reads supporting inclusion divided by the total number of reads for the event. For alternative 5'/3'-ss events that were part of an alternative exon (Foxm1.A2, Mta1.A1, Uspl1.A3), the PSI was instead calculated with reference to the total reads supporting inclusion of that exon in order to assess independent regulation of alternative splice site usage. Only the read direction in which all reads unambiguously supported either inclusion or exclusion of an event was used, and the average if that applied for both. Exploratory analysis revealed that PSI values showed low-level batch effects per 96-well plate which we moderated by subtracting a weighted plate median in which negative controls (siNT and mock treatment) were given 20x more weight than other samples. To derive variances, and because PSI values are not nearly normally distributed but roughly follow a beta distribution, we elected to fit a beta distribution to each pair of replicate treatments, as well as to all negative controls from both replicates, using maximum-likelihood fitting as implemented in the fitdistr() function from the R package MASS {Venables & Ripley, Modern Applied Statistics with S (2002)}. Iterative optimization of shape parameters was initiated with settings x=PSI, shape1=1, shape2=1, method=”L-BFGS-B”, lower=0.01, and upper=mean number of reads supporting each PSI value. A modified Strictly Standardized Mean Difference (SSMD {Zhang, Genomics (2007)}) was then calculated based on the formula: SSMD = (μt – μc)/SQRT(vart + varc) where μt and μc are the means (corresponding to the PSI), and vart and varc the variances of the beta distributions fitted to the treatment replicates and negative controls, respectively. Events for which not all reads from at least one direction were informative (Mff.A2, Tead1.A2) were excluded from further analysis, as were events in individual treatments with less than 20 reads in one or both replicates (~9% of all event x treatment combinations). Additionally, the following events were removed from further splicing analysis: expression only events or constitutive exons, Fgf4, Gapdh, Srpk2, Srrm4; events with consistently low read counts: Atg13.A1, H2afy.A2, Tcf7l1 (N2A only), Dnmt3b (N2A only); or because measured inclusion was biased by differential length of isoforms:, Fgfr1, Sall4.
Expression analysis: Read counts from all splice variants of each gene were used to estimate relative mRNA expression levels, reads per million reads (RPMs). Differential expression analysis based on raw read counts was performed using the generalized linear models workflow from the R package edgeR. Plate, position (edge/interior) and treatment were used as design factors (where all treatments of each type of control were treated as replicates.) Models fitted using estimateDisp() to estimate the common, trended, and tag-wise dispersion for the CGR8 and N2A data separately. Subsequently, differences attributable to treatment, plate, or position contrasts were extracted with glmLRT() and represented as log2-fold changes with associated FDR. Fold-changes for treatments are relative to the siNT and mock-treatment controls.
Genome_build: mm10
Supplementary_files_format_and_content: Processed files are either tab-delimited  tables (.tab) or FASTA files (.fa).   JunctionLibrary_design.csv contains information about sequence and 1-based coordinates of all elements that make up each event, along with vast-tools ID (vastEVENT) and information about how elements are connected. See https://github.com/UBrau/SPARpipe for information about library design.  Junctions108_11-101_fwd.fa and Junctions108_11-101_rev.fa contain all possible junctions given the event elements and read length for forward and reverse reads, respectively.  Events108_11-101.tab indicates which junctions were used to calculate PSI for each element.  Treatments_fullScreen.csv contains information about all 1536 control and experimental knockdowns.  ExprContrasts_fullScreen.csv contains the design factors for differential expression analysis.  ReadsPerEvents.tab.gz contains raw read counts per treatment and event element.  PSI.raw.tab.gz contains raw PSI for each treatment and event element.  dPSI.norm.tab.gz contains normalized and filtered dPSI.  SSMD.norm.tab.gz contains normalized and filtered SSMD.  Expression.Treatment.tab.gz contains results from differential expression analysis (differential expression due to treatment).

source_name: Mouse neuroblastoma cells

growth_protocol: Mouse neuroblastoma (N2A) cells were grown in DMEM supplemented with 10% FBS, sodium pyruvate, MEM non-essential amino acids, and penicillin/streptomycin and maintained at 37°C with 5% CO2.

extract_protocol:
Forty-eight hours post-transfection, total RNA were purified from cultured cells using RNeasy Plus 96 Kit (Qiagen), as per the manufacturer’s instructions.
For each sample, a multiplex RT-PCR assay was first applied to simultaneously amplify 108 AS and/or expression events in a single reaction. The multiplex RT-PCR reaction was carried out in 96-well plates using the OneStep RT-PCR kit (Qiagen) as recommended by the manufacturer, with the following changes: reactions were performed in a volume of 20 uL with 2 uL of the purified total RNA as input, and a mixture of 50 pairs of primers was added to each reaction with a final concentration of 0.25 uM for each individual forward and reverse primer. Four identical Veriti 96-well Thermal Cyclers (Applied Biosystems) were used with the following conditions: 50°C for 30 minutes, 95°C for 15 minute, 30 cycles of 94°C for 40 seconds, 58°C for 1 minute (slow ramp rate), 72°C for 3 minutes, and a final extension step at 72°C for 10 minutes. Two sets of barcodes were incorporated into forward and reverse primers, respectively, after the universal adaptors and added to the amplicons in the second PCR reaction to allow demultiplexing all reads that were derived from a particular treatment. Primers sequences were: forward primer, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT (N denotes i5 barcode); reverse primer, CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (N denotes i7 primer). The second PCR was performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific), as per the manufacturer’s instructions. For each 20 uL of reaction, 1 uL of the multiplex RT-PCR reaction products was used as templates. The thermal cycling conditions were as follows: 98°C for 30 seconds, 15 cycles of 98°C for 10 seconds, 65°C for 30 seconds, 72°C for 30 seconds, and a final extension step at 72°C for 5 minutes. The resulting libraries were pooled and sequenced.

treatment_protocol: Twenty-four hours prior to transfection, N2A cells were seeded in 96-well plates at 5000 cells per well. Cells were transfected with the SMARTpool siRNAs at 50 nM final concentration using DharmaFECT1 reagent (Dharmacon), as recommended by the manufacturer.

value: Mouse neuroblastoma cells


title: day80.ATAC.Rep2

description:
ATAC-seq at Day 80 (ventricular cardiomyocyte) rep 2
peaks-ATAC-D80.bed.gz

value: H9 MLC2v:H2B


title: day15.ATAC.Rep2

description:
ATAC-seq at Day 15 (primitive cardiomyocite) rep 2
peaks-ATAC-D15.bed.gz

source_name: ES derived primitive cardiomyocite

value: ES derived primitive cardiomyocite

value: wild type


title: D35.DIV7.Rbm38KO.B

description: D35.DIV7.Rbm38KO.B: RNA-seq of in vitro differentiated neurons with deletion of Rbm38, replicate B

data_processing:
RTA 3.4.4
For AS analysis, RNA-Seq data were processed using the vast-tools pipeline, version 2.2 https://github.com/vastgroup/vast-tools (Braunschweig et al., 2014; Irimia et al., 2014; Tapial et al., 2017). From primary output, events with poor coverage or junction balance were filtered out (vast-tools quality column score 3 other than SOK/OK/LOW/VLOW for cassette exon [CE], microexon [MIC], and alternative 5' or 3' splice site [Alt5/3] events or coverage less than 10 reads for intron retention [IR] events; score 4 other than OK/B1/B2/Bl/Bn for CE and MIC events and score 5 of less than 0.05 for IR events). Differential AS was assessed through the vast-tools diff module available with the main pipeline. Changes were considered significant if they were greater than 10 dPSI/dPIR and the expected minimum change was different from zero at p > 0.95 according to vast-tools’ diff module.
Gene expression changes were analyzed by pseudo-aligning pre-trimmed reads to GENCODE vM21 transcripts using Salmon v0.14.1 (Patro et al., 2017) and aggregated per gene using the R package tximport (Soneson et al., 2015). Differential expression was then assessed using the classic mode (exactTest) edgeR (McCarthy et al., 2012), and genes with an FDR < 0.05 and a greater than 2-fold change were considered differential. For each comparison, only genes expressed at a minimum of 5 RPKM in one or both conditions were considered.
Genome_build: mm10
Supplementary_files_format_and_content: All files are in tab-delimited format. vast-tools.AltSplicing_differentiation.tab contains primary vast-tools output. AltSplicing_differentiation.tab contains filtered PSI values and annotations of differential AS events. Expression_differentiation.tab contains edgeR-estimated RPM, fold changes and associated FDR.

molecule: polyA RNA

source_name: Glutamatergic neurons derived in vitro from mouse embryonic stem cells

growth_protocol: CGR8 mouse embryonic stem cells (ESCs) were cultured as described previously on gelatin-coated plates (Gabut et al., 2011; Han et al., 2013) and maintained at 37°C with 5% CO2.  In vitro differentiation of CRISPR-Cas9-edited mouse CGR8 ESCs into glutamatergic neurons was performed as described previously (Gueroussov et al., 2015; Hubbard et al., 2013). The CGR8 lines were maintained for a minimum of two passages prior to differentiation. Initially, 5x106 cells were transferred to a 15 cm non-adhesive (not coated) plate containing 30mL of differentiation medium (GMEM containing 5% KnockOut Serum replacement [Thermo Fisher Scientiﬁc], 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and without LIF). This point was designated as ‘Days In Vitro’ (DIV)-7. Half of the growth media on each plate was replaced with fresh media every 48 hours by collecting cell aggregates to the center of the plates by swirling. The media was supplemented with 6μM retinoic acid (RA) (Sigma-Aldrich) at DIV-4 and DIV-2. On DIV0 (also referred to as EB Day8), cellular aggregates were dissociated with TrypLE Express for 5 minutes at 37°C. Trypsinization was halted with soybean trypsin inhibitor (Thermo Fisher Scientiﬁc) and after gentle dissociation by trituration, cells were pelleted for 5 minutes at 300xg. Neural progenitors were washed in N2 medium (Neurobasal-A medium with 1x N2 vitamins, 2 mM glutamine and antibiotics [Thermo Fisher Scientiﬁc]), counted using a hemocytometer, and plated at a density of 1.5x105cells/cm2 onto poly-D-Lysine and Laminin coated dishes. The plated neural progenitors were washed with N2 medium after 24 hours to remove residual serum and non-adherent cells. At DIV2, the N2 medium was replaced with B27 medium (Neurobasal-A supplemented with antibiotics, 2mM glutamine and 1xB27 vitamins [Thermo Fisher Scientiﬁc]). Subsequently, differentiating neurons were provided with full medium changes with B27 on DIV4 and DIV7.

extract_protocol:
Total RNA was extracted using the RNeasy Mini Kit (QIAGEN). RT-PCR assays were performed using the OneStep RT-PCR kit (QIAGEN), as per the manufacturer’s instructions.
1000 ng per sample was used in the library preparation. To obtain larger insert size (540 nt on average after size selection), Illumina’s TruSeq Stranded mRNA sample preparation protocol was modified as follows: 1) fragmentation time and temperature was reduced from 8 minutes at 98 C to 2 minutes at 80 C. Average size distribution prior to library preparation was 232 nt versus 740 nt, respectively; 2) after the final library was generated, the samples were loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1 hour at 100 V. The libraries were size selected between 400 and 600 bp. Excised gel fragments were purified using the Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen). The rest of the protocol was adapted as described in Illumina’s TruSeq Stranded mRNA sample preparation guide (Part# 15031047 Rev. E, Illumina).

tag: differentiation stage

value: Glutamatergic neurons at day 7 after neuronal progenitor stage

tag: mutation

value: Deletion of Rbm38

tag: cloning

value: CGR8 clonal cell lines harboring a CRISPR-Cas9 mediated ~1300bp deletion within the Rbm38 locus (Rbm38 KO) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting within Rbm38 (Rbm38_gRNA2:5′-GCTCCCCGAGTGTGTTTCCG-3′) and (Rbm38_gRNA4:5′-CCTTGATCCAGCGCACCTAC-3′) were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). For the generation of Rbm38 knockout cells, CGR8 cells were simultaneously transiently transfected with PX459-Rbm38_gRNA2 and PX459-Rbm38_gRNA4 plasmids, using Lipofectamine 2000 (Thermo Fisher Scientiﬁc) following the manufacturer’s instructions. 24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Rbm38 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Rbm38 was detected by PCR ampliﬁcation of gDNA using the following primers:    Rbm38 KO screen For:5′-GGACAGCGTGGGGAACTC-3′  Rbm38 KO screen Rev:5′-CCACACCCCCAATACACAAT-3′  Rbm38 KO screen Internal:5′- GCTGTCCAGGCCATCAGTA-3′     Positive clones were further analyzed by Sanger sequencing. Rbm38 protein depletion was confirmed by Western Blot.


title: human_patients_1_2_3_4_5_tcr

city: Minneapolis

first: Todd

middle: P

last: Knutson

state: MN

address: 117 Pleasant St SE

department: Minnesota Supercomputing Institute

institute: University of Minnesota

description: Mixed samples: T cells derived from the bone marrow of leukemia patients

data_processing:
10X Genomics Cell Ranger (scRNAseq) software was used to demultiplex samples and generate fastq files.
Each gel bead in emulsion (GEM) was identified and all gene specific reads were mapped via cellranger "count" (ver. 5.0.1) using the mouse reference genome (mm10) or human reference genome (GRCh38), or antibody specific tags (HTOs and ADTs). T-cell receptor contigs were recovered by running Cellranger "vdj" software with a reference provided by 10X genomics.
HTO/ADT counts were generated by cellranger count using the specific antibody-oligo tags sequences used in this experiment. Experimental arms or patient samples were classified using HTO counts respectively via GMM-Demux software (ver. 0.2.1.3).
Raw counts data was imported into R (ver 4.0.3) for downstream processing, including normalization, principal components analysis, differential gene expression testing, single cell clustering, etc. primarily using the Seurat R package (ver. 4.0.1).
Genome_build: refdata-gex-mm10-2020-A, refdata-gex-GRCh38-2020-A, refdata-cellranger-vdj-GRCm38-alts-ensembl-5.0.0, refdata-cellranger-vdj-GRCh38-alts-ensembl-5.0.0 (all provided by 10X Genomics)
Supplementary_files_format_and_content: tab-delimited text files include raw or normalized read count values for each sample (GEX, HTO, ADT) or tab-delimited text and json files of T-cell receptor contigs (TCR)

source_name: mixed sample, patients 1-5, bone marrow T-cells

extract_protocol:
For the mouse studies, CD44+CD4+ T cells from the indicated arms of mice were sorted using a BD FACSAria sorter. Cells from different treatment arms were labeled with hashtag antibodies, as well as CITE-Seq antibodies to CD25, OX40, TIGIT, PD1, Tim3, LAG3 (Biolegend). Sorted CD44+CD4+ T cells were resuspended at 106 ml in 50% FBS in 1×PBS and captured using 10x Genomics Single Cell 5’ Solution. Single cell TCR sequencing was performed using the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 (10x Genomics) according to the manufacturer’s instructions. For the human studies, cells were sorted using a BD FACSAria sorter. Ten-thousand cells from each of the three sorted fractions were pooled together and resuspended at 10^6 ml in 50% FBS in 1×PBS before capture using 10x Genomics Single Cell 5’ Solution. Single cell TCR sequencing was performed using the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 (10x Genomics).
For 10X Genomics scRNA-seq, we generated three libraries that measure: (1) mRNA transcript expression (GEX), (2) sample/tissue-specific hashtag oligos (HTO) and protein marker levels using antibody derived tags (ADT), and (3) T cell receptor specific mRNA reperitorires. Cells were harvested and stained as described above. The sample was split into three libraries (GEX, HTO/ADT, and TCR). Reverse transcription PCR and library preparation were carried out under the Chromium Single Cell 5’ v1.1 protocol (10X Genomics) per manufacturer’s recommendations. The libraries were sequenced on the NovaSeq 6000 with 2x150 bp paired-end reads (Illumina).

treatment_protocol: For the mouse studies, C57BL/6 mice were injected with 2500 LM138 cells via tail vein. Mice were bled at 14 days post-leukemia challenge, then treated with 75mg/kg nilotinib via oral gavage 5 days/week for 2 weeks. Some mice were treated with 10mg/kg InVivoPlus anti-mouse PDL1 (BioXCell, clone: 10F.9G2), or an isotype control antibody (Rat IgG2b, kappa) via intraperitoneal injection on days 14, 16 and 18 post-leukemia establishment. For the 1-week treatment schedule, mice were treated with nilotinib 5 days/week for 1 week starting at day 14 post leukemia establishment, and with anti-PDL1 or isotype control antibodies. For the CD4 and CD8 T cell depletion studies, mice were treated with 22mg/kg InVivoPlus anti-CD4 (BioXCell, clone: GK1.5) or InVivoPlus anti-CD8 (BioXCell, clone: YTS 169.4) depleting antibodies on days 7, 9 and 11 post-leukemia establishment. For the human studies, leukemia blasts, T cells (CD45+CD3+) and other immune cells (CD45+CD3-CD10-) were isolated from bone marrow biopsy samples from five leukemia patients.

value: leukemia blasts, T cells , and other immune cells

value: bone marrow

tag: sample type

value: single cell T cell receptor mRNA


title: SH-SY5Y_H2O2- WT Input rep2

tag: cell type

value: Neuroblastoma cell line

tag: genotype


title: human_patients_1_2_3_4_5_hto

tag: strain

value: single cell HTO feature barcodes


title: WT CD4 SP Gata3 BR1

value: untreated

value: GATA3 (clone:D13C9) XP (Cell Signalling, cat no:5852)


title: RNA-seq Lin- XistΔ/Δ Rep1

value: Bone marrow Lin- cells

value: Xist knockout

tag: Sex

value: Female

tag: age

value: 129Sv4/Jae x B6


title: human_patients_1_2_3_4_5_gex

tag: tissue

value: single cell mRNA


title: D34.DIV7.Puf60.pEx5.HET.B

description: D34.DIV7.Puf60.pEx5.HET.B: RNA-seq of in vitro differentiated neurons engineered to heterozygously express Puf60 with constitutive exon5, replicate B

value: Glutamatergic neurons derived in vitro from mouse embryonic stem cells

value: Constitutive inclusion of Puf60 exon 5 (heterozygous)

value: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated constitutive inclusion of Puf60 exon 5 (Puf60+Ex5) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting the upstream (Puf60_gRNA_Up1:5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1:5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). In order to engineer constitutive inclusion of Puf60 exon 5, CGR8 cells were co-transfected with either PX459-Puf60_gRNA_Up1 or PX459-Puf60_gRNA_Dn1, and a HDR template containing the following sequence:   agggtgctgcttggttaatggaagtaggctgtagccctttatagggctcatgaactgaggagtaacacccaccctctagtaggtatgttttgaagaatgtcttcaggcctgaaatgggcattgttggagggaaggtcccttgcctccttacttgttctggggtggtctgaggccctggcaaggcctgttgaggaaactaagaggctagggtgcctggtagcctatgagtaatgagtctttggtcctggttctgttttctgggctctggaggacaataaaggttggctgttctccaagactctgggatcaaattgtcaacagaggcaggcagactgctctcctgttcatgaagaggccattgtgttgtcctacagggcacctgcctggtttaatgtgtcccatccacaagtcatgtctgacgaaggcctgagtgggtatagcaatgtctgaagggaaggcctgcattctaaccaggcaggagatagtttgtattggggcttctcttttgctttaattttagagtctagggataaatggcttatggggtttctgggaccacatgagaaatgttgagtttgctgctgaacaactcccctgtttcctacttcagGGCACAGAATCCATCAAGATGGAAAATGGACAAAGCACAGGCACCAAGCTGGGGCTGCCTCCCCTGACGCCCGAGCAGCAGGAGGCCCTCCAGAAGgtgagagtgcatctcctgggcacagctacttaaccagggttggaaaggcaggccatccccctgcaggctgggccccccagtgctcctcactgccatctcttgcagGCCAAGAAATATGCAATGGAGCAGAGCATCAAGAGTGTGCTGGTGAAACAGACCATCGCGCACCAGCAGCAGCAGCTCACCAACCTGCAGATGGCAGCAGTGACAATGGGCTTTGGAGATCCTCTCTCACCTTTGCAATCGATGGCAGCTCAACGGCAGCGGGCACTGGCTATCATGTGCCGGGTCTATGTGGGCTCCATCTACTATGAGCTGGGAGAAGACACTATTCGCCAGGCCTTTGCTCCCTTTGGCCCCATCAAGAGCATTGATATGTCCTGGGACTCCGTTACCATGAAGCATAAGgtaagcagatgagagctaccctggggctctagcttgccctctgtctgctttccttgccttctaacaccttcctgtattcagGGCTTTGCCTTCGTGGAGTACGAGGTCCCAGAAGCTGCGCAGCTCGCTTTGGAGCAGATGAACTCTGTGATGCTTGGGGGCAGGAACATCAAGgtaaggacaagaaagtttggtcttagccgggcagtggtggcacacgcctttaatctcagcacttgggaggcagaggcaggcggatttctgagtttgaggccagcctggtctacaagtgagttccaggacagccagggctagacagagaaaccctgtcttgaaaaaccaaaaaaagaaaaaaagtttggtcttggacctagaataattgttccgtgaagttacatacatacaagcagtcaccacaggcctaggctgatgtttcttcttgaccttcttcagGTGGGAAGACCCAGCAACATTGGACAAGCCCAACCCATCATAGATCAGCTGGCTGAGGAAGCTAGGGCTTTCAACCGAATATATGTGGCCTCTGTACATCAAGACCTGTCTGATGATGACATCAAGAGTGTATTTGAAGCCTTTGGCAAGATCAAGTCTTGTACGCTGGCCCGGGACCCCACAACTGGCAAGCACAAGGGCTATGGTTTTATCGgtgagtttgggaagg     24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Puf60 exon 5 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Puf60 detected by PCR ampliﬁcation of gDNA using the following primers:    Puf60 HDR screen For:5′-TTGGAAATGGCTTTGGTAGG-3′  Puf60 HDR screen Rev:5′-CAAGTACTGGCCACCCAGAT-3′     Positive clones were further analyzed by Sanger sequencing. Constitutive splicing inclusion of Puf60 exon 5 was confirmed by RT-PCR.


title: day15.ATAC.Rep1

description:
ATAC-seq at Day 15 (primitive cardiomyocite) rep 1
peaks-ATAC-D15.bed.gz

value: ES derived primitive cardiomyocite

tag: differentiation time

value: H9 MLC2v:H2B

tag: chip antibody

tag: perturbation

value: wild type


title: WT CD4 SP Gata3 BR3

tag: genetic background

tag: cells


title: Peer_SCM

city: Atlanta

first: Chris

last: Scharer

state: GA

address: 1510 Clifton Rd, Suite 3086A

department: Microbiology and Immunology

institute: Emory University

data_processing:
Fastq reads were mapped to the hg38 reference genome using STAR with the default parameters.
Reads mapping to exons for unique ENTREZ genes were annotated and normalized using the GenomicRanges package in R/Bioconductor.
Assembly: hg38
Supplementary files format and content: RPKM normalized gene expression counts for all detected genes.

source_name: T-ALL cell line

growth_protocol: Human T-ALL cell lines Loucy and Peer were grown in RPMI with 20% FBS and Jurkat were grown in RPMI with 10% FBS.

extract_protocol:
After the treatment conditions, RNA was extracted using the RNAeasy mini kit (cat. no. 74104) from Qiagen.
200-1000 ng of total RNA was used with the KAPA mRNA HyperPrep kit with polyA selection beads and 10 rounds of PCR to generate RNA-seq libraries.

treatment_protocol: Human T-ALL cells were treated with vehicle or methotrexate (50 nM) for 24 hours prior to extracting RNA for sequencing.  Note:  We performed sequencing on 1 replicate per conditon.

tag: disease

value: T-cell acute lymphoblastic leukemia

tag: media

value: bone marrow stromal cell

tag: cell line

value: Peer


title: day07.ATAC.Rep2

description:
ATAC-seq at Day 7 (cardiac progenitor cells) rep 2
peaks-ATAC-D07.bed.gz

source_name: ES derived cardiac progenitor cells

value: ES derived cardiac progenitor cells


title: day05.ATAC.Rep1

description:
ATAC-seq at Day 5 (cardiac mesoderm) rep 1
peaks-ATAC-D05.bed.gz

value: ES derived cardiac mesoderm


title: D33.DIV7.Puf60.pEx5.HOM.B

description: D33.DIV7.Puf60.pEx5.HOM.B: RNA-seq of in vitro differentiated neurons engineered to homozygously express Puf60 with constitutive exon5, replicate B

tag: differentiation stage

value: Glutamatergic neurons at day 7 after neuronal progenitor stage

tag: mutation

value: Constitutive inclusion of Puf60 exon 5 (homozygous)


title: day07.ATAC.Rep1

description:
ATAC-seq at Day 7 (cardiac progenitor cells) rep 1
peaks-ATAC-D07.bed.gz


title: organoid 2

city: Santa Barbara

first: Stella

middle: M. K.

last: Glasauer

state: CA

address: Biological Sciences II

department: Neuroscience Research Institute

institute: University of California Santa Barbara

description: drop-seq

library_source: transcriptomic single cell

data_processing:
Gene expression matrices were generated using the drop-seq tools package version 1.13 (https://github.com/broadinstitute/Drop-seq/releases)
Assembly: hg19
Supplementary files format and content: matrix files

source_name: iPSC-derived cerebral organoid

growth_protocol: organoids were generated using the method described in Lancaster et al., 2013 ( DOI: 10.1038/nature12517 )

extract_protocol:
Individual cerebral organoids were dissociated using the Worthington Papain Dissociation System (Worthington). Organoids  were cut into pieces with a sterile scalpel blade and washed twice in PBS prior to dissociation. Incubation in papain/DNaseI solution was performed at 37 ºC and 5% CO2 with occasional agitation. After the first 30 minutes of incubation, organoids were triturated 10 times with a fire-polished glass Pasteur pipette, followed by further incubation and trituration every 15 minutes until dissociation was complete. The total incubation time in papain/DNaseI solution was 90 minutes. After adding Inhibitor Solution and DNaseI in EBSS, the dissociated cells were pelleted by centrifugation for 3 minutes at 300 g. The cells were resuspended in 500 µl ice-cold PBS/0.01% BSA using a wide-bore plastic pipette tip and passed through a 35 µm cell strainer. An aliquot of the single-cell suspension was mixed with Trypan blue and analyzed for viability and cell concentration on a Countess II FL Automated Cell Counter (ThermoFisher). cells were diluted to 110 cells/µl in PBS/0.01% BSA.
Drop-seq was performed as described in Macosko et al. (Macosko et al., 2015; DOI: 10.1016/j.cell.2015.05.002). Briefly cells and barcoded beads (Chemgenes, 132 beads/ul in lysis buffer) were run on an aquapel-treated microfluidic drop-seq device (FlowJEM) for co-encapsulation in nanoliter-sized droplets. After droplet-breakage, reverse transcription and Exonuclease I treatment, beads were counted, and 2,500 beads were apportioned per PCR tube for cDNA amplification. The amplified cDNA libraries were purified using SPRI beads (Beckman Coulter) and quantified on a Fragment Analyzer (Agilent). Tagmentations were performed using the Illumina Nextera XT kit (Illumina), and the resulting libraries were purified in two consecutive rounds of SPRI beads-based size selection (0.6x beads to sample ratio followed by 1x beads to sample ratio). The size and concentration of the final libraries were measured on a Fragment Analyzer and a Qubit Fluorometer (ThermoFisher), respectively.

value: iPSC-derived cerebral organoid

value: 7 months


title: organoid 3

description: drop-seq


title: organoid 1

description: drop-seq


title: Peer_ACM

value: adipocyte-conditioned medium


title: WT CD4 SP IgG control 1

tag: treatment

value: untreated

tag: antibody

value: IgG (Epicypher, cat no:13-0042)


title: Monte049AC

first: Zeguo

last: Sun

address: 1468 Madison Avenue

institute: Ichan School of Medicine at Mount Sinai

data_processing:
Quality of raw sequencing reads were summarized with fastqc v0.11.9(38).  After trimming the adapter sequences using cutadapt v2.9 (39), cleaned reads were mapped to the human genome (GRCh38) using STAR 2.5.3a (40) with default parameters. Reads mapped to each gene locus was counted by HTseq 0.11.2 with “-m union --nonunique none”(41).
Genome_build: GRCh38
Supplementary_files_format_and_content: tab delimited xls file, raw count of each gene in each sample

source_name: Whole Blood

extract_protocol:
A single sample of blood (2.5-5ml) was collected for peripheral blood transcriptome analysis after consent in a PAXgene tube with RNA stabilization reagents. The date of sample collection was considered enrollment date. PAXgene samples from all patients were transported to the central laboratory at Mount Sinai and stored at -80C. RNA extraction and processing were done in batches of ten by 2 experienced operators. Total RNA was stored at -80C till sequencing.
QIAamp RNA Blood Kit was used to extract high quality RNA from total blood (<1% DNA contamination; Qiagen, CA, USA). Total RNA (>500 ng per sample; 260/280 ratio = 2.07  0.08) was poly-A selected and amplified for library preparation and subsequent sequencing (Genewiz Inc, NJ, USA). 150bp paired-end sequencing was performed on the prepared libraries (Illumina, CA, USA) to generate 20-30 million reads per sample, and raw reads data was stored in FASTQ files for analysis.

treatment_protocol: Inclusion criteria: Adult KTRs (>18 years), who provided informed consent for mechanistic studies and sample collection, at both hospital systems (Montefiore Medical Center and Icahn School of Medicine at Mount Sinai) were enrolled in the study cohort during the study period (May 2020 to May 2021). Consented KTRs from inpatient facilities or outpatient transplant clinics were included based on: (a) COVID-19 PCR positivity. Nasopharyngeal and/or oropharyngeal swabs were collected in 3ml viral transport media. RNA extraction and real-time PCR was performed using one of three commercial methods at both institutions; (b) diagnosis of antecedent COVID-19 infection by positive COVID-19 Spike and Nucleocapsid IgG serology. IgG antibody testing was performed using the Abbott SARS-CoV-2 IgG antibody test on the Abbott Architect Immunoassay Analyzer. Testing was performed on serum samples following manufacturer’s instructions. The assay is a chemiluminescent microparticle immunoassay intended for the qualitative detection of IgG antibodies to SARS-CoV-2. COVID-19 infected KTRs were classified as acute (or early) cases (enrollment date <4 weeks from PCR positivity and post-acute (or late) cases (enrollment date >4 weeks from initial COVID-19 diagnosis), with the latter no matter enrolled inpatient or outpatient. Exclusion criteria: pregnant women, inability to give consent, active malignancy, or prior COVID vaccination.

value: whole blood

tag: phase

value: Acute

tag: gender

value: M


title: day02.ATAC.Rep1

description:
ATAC-seq at Day 2 (mesoderm) rep 1
peaks-ATAC-D02.bed.gz

value: ES derived mesoderm

tag: genotype

value: H9 MLC2v:H2B


title: D32.DIV7.Puf60.mEx5.HOM.B

description: D32.DIV7.Puf60.mEx5.HOM.B: RNA-seq of in vitro differentiated neurons engineered to homozygously express Puf60 lacking exon5, replicate B

value: Glutamatergic neurons derived in vitro from mouse embryonic stem cells

value: Deletion of Puf60 exon 5 (homozygous)

tag: cloning

value: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated deletion of Puf60 exon 5 (Puf60-Ex5) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting the upstream (Puf60_gRNA_Up1:5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1:5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). For deletion of Puf60 exon 5, CGR8 cells were simultaneously transiently transfected with PX459-Puf60_gRNA_Up1 and PX459-Puf60_gRNA_Dn1 plasmids, using Lipofectamine 2000 (Thermo Fisher Scientiﬁc) following the manufacturer’s instructions. 24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Puf60 exon 5 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Puf60 was detected by PCR ampliﬁcation of gDNA using the following primers:    Puf60 Ex5 deletion screen For:5′- TCCACTGGGCCCTACTCTAA-3′  Puf60 Ex5 deletion screen Rev:5′- CTTGACCCCAACCCAAGAG-3′     Positive clones were further analyzed by Sanger sequencing. Deletion of Puf60 exon 5 was confirmed by RT-PCR.


title: Monte064VR

tag: tissue

tag: age


title: Monte067

value: F


title: day05.ATAC.Rep2

description:
ATAC-seq at Day 5 (cardiac mesoderm) rep 2
peaks-ATAC-D05.bed.gz

value: ES derived cardiac mesoderm

tag: differentiation time

tag: chip antibody

value: wild type


title: Monte069BD


title: D28.DIV7.Puf60.pEx5.HET.A

description: D28.DIV7.Puf60.pEx5.HET.A: RNA-seq of in vitro differentiated neurons engineered to heterozygously express Puf60 with constitutive exon5, replicate A

value: Glutamatergic neurons at day 7 after neuronal progenitor stage

value: Constitutive inclusion of Puf60 exon 5 (heterozygous)

value: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated constitutive inclusion of Puf60 exon 5 (Puf60+Ex5) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting the upstream (Puf60_gRNA_Up1:5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1:5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). In order to engineer constitutive inclusion of Puf60 exon 5, CGR8 cells were co-transfected with either PX459-Puf60_gRNA_Up1 or PX459-Puf60_gRNA_Dn1, and a HDR template containing the following sequence:   agggtgctgcttggttaatggaagtaggctgtagccctttatagggctcatgaactgaggagtaacacccaccctctagtaggtatgttttgaagaatgtcttcaggcctgaaatgggcattgttggagggaaggtcccttgcctccttacttgttctggggtggtctgaggccctggcaaggcctgttgaggaaactaagaggctagggtgcctggtagcctatgagtaatgagtctttggtcctggttctgttttctgggctctggaggacaataaaggttggctgttctccaagactctgggatcaaattgtcaacagaggcaggcagactgctctcctgttcatgaagaggccattgtgttgtcctacagggcacctgcctggtttaatgtgtcccatccacaagtcatgtctgacgaaggcctgagtgggtatagcaatgtctgaagggaaggcctgcattctaaccaggcaggagatagtttgtattggggcttctcttttgctttaattttagagtctagggataaatggcttatggggtttctgggaccacatgagaaatgttgagtttgctgctgaacaactcccctgtttcctacttcagGGCACAGAATCCATCAAGATGGAAAATGGACAAAGCACAGGCACCAAGCTGGGGCTGCCTCCCCTGACGCCCGAGCAGCAGGAGGCCCTCCAGAAGgtgagagtgcatctcctgggcacagctacttaaccagggttggaaaggcaggccatccccctgcaggctgggccccccagtgctcctcactgccatctcttgcagGCCAAGAAATATGCAATGGAGCAGAGCATCAAGAGTGTGCTGGTGAAACAGACCATCGCGCACCAGCAGCAGCAGCTCACCAACCTGCAGATGGCAGCAGTGACAATGGGCTTTGGAGATCCTCTCTCACCTTTGCAATCGATGGCAGCTCAACGGCAGCGGGCACTGGCTATCATGTGCCGGGTCTATGTGGGCTCCATCTACTATGAGCTGGGAGAAGACACTATTCGCCAGGCCTTTGCTCCCTTTGGCCCCATCAAGAGCATTGATATGTCCTGGGACTCCGTTACCATGAAGCATAAGgtaagcagatgagagctaccctggggctctagcttgccctctgtctgctttccttgccttctaacaccttcctgtattcagGGCTTTGCCTTCGTGGAGTACGAGGTCCCAGAAGCTGCGCAGCTCGCTTTGGAGCAGATGAACTCTGTGATGCTTGGGGGCAGGAACATCAAGgtaaggacaagaaagtttggtcttagccgggcagtggtggcacacgcctttaatctcagcacttgggaggcagaggcaggcggatttctgagtttgaggccagcctggtctacaagtgagttccaggacagccagggctagacagagaaaccctgtcttgaaaaaccaaaaaaagaaaaaaagtttggtcttggacctagaataattgttccgtgaagttacatacatacaagcagtcaccacaggcctaggctgatgtttcttcttgaccttcttcagGTGGGAAGACCCAGCAACATTGGACAAGCCCAACCCATCATAGATCAGCTGGCTGAGGAAGCTAGGGCTTTCAACCGAATATATGTGGCCTCTGTACATCAAGACCTGTCTGATGATGACATCAAGAGTGTATTTGAAGCCTTTGGCAAGATCAAGTCTTGTACGCTGGCCCGGGACCCCACAACTGGCAAGCACAAGGGCTATGGTTTTATCGgtgagtttgggaagg     24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Puf60 exon 5 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Puf60 detected by PCR ampliﬁcation of gDNA using the following primers:    Puf60 HDR screen For:5′-TTGGAAATGGCTTTGGTAGG-3′  Puf60 HDR screen Rev:5′-CAAGTACTGGCCACCCAGAT-3′     Positive clones were further analyzed by Sanger sequencing. Constitutive splicing inclusion of Puf60 exon 5 was confirmed by RT-PCR.


title: D27.DIV7.Puf60.pEx5.HOM.A

description: D27.DIV7.Puf60.pEx5.HOM.A: RNA-seq of in vitro differentiated neurons engineered to homozygously express Puf60 with constitutive exon5, replicate A

value: Constitutive inclusion of Puf60 exon 5 (homozygous)


title: Loucy_ACM

value: T-cell acute lymphoblastic leukemia

value: adipocyte-conditioned medium

value: Loucy


title: Peer_RPMI

value: RPMI

value: Peer


title: SH-SY5Y_H2O2- WT Input rep1

description: MH = Untreated/H2O2-, R90 = H2O2 treated 90 minute recovery, WT = Wild type with respect to NuMA (TDIF), KD = NuMA (TDIF) Knockdown, IN = Input, IP = OGAP-seq

tag: cell type

value: Neuroblastoma cell line


title: day00.ATAC.Rep1

description:
ATAC-seq at Day 0 (ESC) rep 1
peaks-ATAC-D00.bed.gz

value: Embryonic stem cell

value: H9 MLC2v:H2B

tag: perturbation


title: Monte071GS

value: whole blood

value: M


title: day02.ATAC.Rep2

description:
ATAC-seq at Day 2 (mesoderm) rep 2
peaks-ATAC-D02.bed.gz

value: ES derived mesoderm


title: D26.DIV7.Puf60.mEx5.HOM.A

description: D26.DIV7.Puf60.mEx5.HOM.A: RNA-seq of in vitro differentiated neurons engineered to homozygously express Puf60 lacking exon5, replicate A

value: Glutamatergic neurons derived in vitro from mouse embryonic stem cells

value: Deletion of Puf60 exon 5 (homozygous)

value: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated deletion of Puf60 exon 5 (Puf60-Ex5) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting the upstream (Puf60_gRNA_Up1:5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1:5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). For deletion of Puf60 exon 5, CGR8 cells were simultaneously transiently transfected with PX459-Puf60_gRNA_Up1 and PX459-Puf60_gRNA_Dn1 plasmids, using Lipofectamine 2000 (Thermo Fisher Scientiﬁc) following the manufacturer’s instructions. 24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Puf60 exon 5 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Puf60 was detected by PCR ampliﬁcation of gDNA using the following primers:    Puf60 Ex5 deletion screen For:5′- TCCACTGGGCCCTACTCTAA-3′  Puf60 Ex5 deletion screen Rev:5′- CTTGACCCCAACCCAAGAG-3′     Positive clones were further analyzed by Sanger sequencing. Deletion of Puf60 exon 5 was confirmed by RT-PCR.


title: Monte039


title: Monte043

value: Acute

value: F


title: D31.DIV7.WTprtl.B

description: D31.DIV7.WTprtl.B: RNA-seq of in vitro differentiated neurons, parental wild type, replicate B

value: Glutamatergic neurons at day 7 after neuronal progenitor stage

value: Parental wild type


title: PBN Reaction 3

city: Seattle

first: Richard

middle: D

last: Palmiter

state: WA

address: 1959 NE Pacific St

institute: UW/HHMI

description:
PBN_M3
Live single-cell suspension generated from PBN tissue from 3 female and 2 male mice pooled prior to library generation.

data_processing:
Demultiplexing was performed with 1 mismatch allowed using Illumina bcl2fastq v2.18.0.12. Initial read processing and generation of digital expression matrices was performed using 10X Genomics Cell Ranger v3.1.0 with 128 GB of memory on 24 cores.
Assembly: mm10 3.0.0
Supplementary files format and content: Data is provided as HDF5 matrices generated by Cell Ranger v3.1.0.

source_name: brain (parabrachial nucleus)

extract_protocol:
C57BL/6J mice were transcardially perfused with a cold artificial CSF solution containing NMDG (NMDG-aCSF), modified from (Ting et al., 2018). All steps were performed under continuous oxygenation and CO2 buffering using 95%/5% O2/CO2. Brains were rapidly harvested, and coronal slices (200 µm) spanning the PBN were prepared using a vibrating microtome (VT1200, Leica Biosystems). Slices were allowed to recover in NMDG-aCSF containing 500 nM TTX, 10 µM AP-V, 10 µM DNQX (NMDG-aCSF-R) for 20 minutes and the PBN was subsequently isolated using tissue punches (500-750 μm). The isolated tissue was then enzymatically digested using 1 mg/mL Pronase (Roche) for 50 minutes, and enzymatic digestion was quenched with 0.05% BSA. Cells were then mechanically dissociated using a fire-polished glass pipet with an internal diameter of 200-300 µm, filtered through a 40 μm strainer, washed, and depleted of dead cellular fragments using a commercial kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The remaining cells were resuspended in PBS containing 0.05% BSA at a concentration of 1,000 cells/µL.
Single-cell RNA libraries were generated using Chromium Single-Cell 3’ v2 chemistry (10X Genomics, Pleasanton, CA) following the standard manufacturer protocol. A pool of ~17,000 cells harvested from 5 mice were loaded per reaction, with the first pool run on a single reaction and the second pool spread across three reactions. For each library, cDNA amplification was performed using 12 cycles and indexing was performed using 11 cycles. Library size distribution and concentration were determined using a Qubit HS DNA Assay (Invitrogen, Waltham, MA) or High Sensitivity D5000 ScreenTape (Agilent Technologies, Santa Clara, CA). Each reaction library was sequenced on two lanes of an Illumina HiSeq 4000 using 2x150 chemistry by Genewiz, Inc (South Plainfield, NJ) following the standard 10X Genomics v2 paired-end configuration. Approximately 875 million reads were generated per lane with a mean sequencing saturation of 82.6%. Sequences were aligned to the mm10-3.0.0 genome, and digital expression matrices were created using 10X Genomics Cell Ranger v3.1.0 with 128 GB of memory on 24 cores.

tag: strain

value: brain (parabrachial nucleus)

tag: developmental stage

value: adult

tag: Sex

value: pooled male and female


title: Monte052PF


title: Jurkat_ACM

tag: disease

value: T-cell acute lymphoblastic leukemia

tag: media

value: adipocyte-conditioned medium

tag: cell line

value: Jurkat


title: Monte068NM


title: D29.DIV7.Rbm38KO.A

description: D29.DIV7.Rbm38KO.A: RNA-seq of in vitro differentiated neurons with deletion of Rbm38, replicate A

tag: differentiation stage

tag: mutation

value: Deletion of Rbm38

value: CGR8 clonal cell lines harboring a CRISPR-Cas9 mediated ~1300bp deletion within the Rbm38 locus (Rbm38 KO) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting within Rbm38 (Rbm38_gRNA2:5′-GCTCCCCGAGTGTGTTTCCG-3′) and (Rbm38_gRNA4:5′-CCTTGATCCAGCGCACCTAC-3′) were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). For the generation of Rbm38 knockout cells, CGR8 cells were simultaneously transiently transfected with PX459-Rbm38_gRNA2 and PX459-Rbm38_gRNA4 plasmids, using Lipofectamine 2000 (Thermo Fisher Scientiﬁc) following the manufacturer’s instructions. 24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Rbm38 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Rbm38 was detected by PCR ampliﬁcation of gDNA using the following primers:    Rbm38 KO screen For:5′-GGACAGCGTGGGGAACTC-3′  Rbm38 KO screen Rev:5′-CCACACCCCCAATACACAAT-3′  Rbm38 KO screen Internal:5′- GCTGTCCAGGCCATCAGTA-3′     Positive clones were further analyzed by Sanger sequencing. Rbm38 protein depletion was confirmed by Western Blot.


title: PBN Reaction 2

description:
PBN_M2
Live single-cell suspension generated from PBN tissue from 3 female and 2 male mice pooled prior to library generation.


title: Loucy_RPMI

value: RPMI

value: Loucy


title: Passage 42 FHL124 cells_rep2

city: Augusta

first: Xingjun

last: Fan

address: 1460 Laney Walker Blvd, Room CB1119

department: Cellular Biology and Anatomy

institute: Augusta University

data_processing:
Data were processed using Partek Ffow
Unaligned reads were trimmed based on PHRED quality score with a minimal read >25nt.
Trimmed reads were aligned to the latest human genome assembly hg19 using STAR aligner.
Aligned reads were quantitatively aligned to Ensembi Transcripts release 100.
Genome_build: hg19
Supplementary_files_format_and_content: TPM

source_name: Human lens epithelium

growth_protocol: FHL124 cells were grown in MEM with 5% FBS, 100 U/ml of penicillin and streptomycin at 35℃ in a humidified 5% CO2 incubator.

extract_protocol:
Total RNA was extracted by TRIzol reagent
NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, Ipswich, MA) combined with SMARTer Apollo NGS library prep system (Takara Bio USA, Mountain View, CA) was used. The library from enriched polyA RNA was created by using the NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs).

treatment_protocol: Non-treatment

value: Lens epithelial cells, FHL124

value: cellular aging model

tag: treatment

value: non-treated

value: Human lens epithelial cell line


title: Loucy_SCM

value: bone marrow stromal cell


title: day00.ATAC.Rep2

description:
ATAC-seq at Day 0 (ESC) rep 2
peaks-ATAC-D00.bed.gz

value: Embryonic stem cell

tag: genotype

value: H9 MLC2v:H2B

value: wild type


title: JL027 Syn CD8+ d6

city: Stanford

first: Juliane

last: Lohmeyer

address: 269 W. Campus Drive

institute: Stanford University

data_processing:
Sequencing reads were checked using FastQC v.0.11.7. Estimated transcript counts and transcripts per million (TPM) for the mouse genome assembly GRCm38 (mm10) were obtained using the pseudo-aligner Kallisto. Transcript-level abundance was quantified and summarized into gene level using the tximport R package. Differential gene expression was performed using the DESeq2 R package version 1.22.221. TCRα and TCRβ sequence analysis was performed with VDJFasta
Assembly: mouse genome assembly GRCm38 (mm10)
Supplementary files format and content: RNAseq: csv file with normalized counts for each sample
Supplementary files format and content: TCRseq: csv file with counts and proportion for each TCR sequence and sample

source_name: spleen + lymph nodes

extract_protocol:
RNA was extracted using the TRizol RNA isolation method combined with the RNeasy MinElute Cleanup (Qiagen).
The Clontech SMARTer v4 Kit (Takara Bio USA, Inc.) was used to generate full-length cDNA prior to library preparation with the Nextera XT DNA Library Prep Kit (Illumina, Inc.). For TCR sequencing, libraries were prepared from the synthesized full-length cDNA using the nested PCR method reported previously (Han et al., Saligrama et al.)

value: spleen + lymph nodes

value: primary

value: T cell

value: syngeneic control, untreated


title: Jurkat_RPMI

value: T-cell acute lymphoblastic leukemia

value: Jurkat


title: D25.DIV7.WTprtl.A

description: D25.DIV7.WTprtl.A: RNA-seq of in vitro differentiated neurons, parental wild type, replicate A

value: Glutamatergic neurons derived in vitro from mouse embryonic stem cells

value: Glutamatergic neurons at day 7 after neuronal progenitor stage

value: Parental wild type

tag: cloning


title: Monte026CD

tag: tissue

value: whole blood

tag: phase

tag: gender

value: M


title: D23.EB8.Rbm38KO.B

description: D23.EB8.Rbm38KO.B: RNA-seq of ESC-derived embryoid bodies with deletion of Rbm38, replicate B

source_name: Embryoid bodies (day 8) derived from mouse embryonic stem cells

value: Embryoid bodies at day 8 of differentiation

value: Deletion of Rbm38

value: CGR8 clonal cell lines harboring a CRISPR-Cas9 mediated ~1300bp deletion within the Rbm38 locus (Rbm38 KO) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting within Rbm38 (Rbm38_gRNA2:5′-GCTCCCCGAGTGTGTTTCCG-3′) and (Rbm38_gRNA4:5′-CCTTGATCCAGCGCACCTAC-3′) were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). For the generation of Rbm38 knockout cells, CGR8 cells were simultaneously transiently transfected with PX459-Rbm38_gRNA2 and PX459-Rbm38_gRNA4 plasmids, using Lipofectamine 2000 (Thermo Fisher Scientiﬁc) following the manufacturer’s instructions. 24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Rbm38 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Rbm38 was detected by PCR ampliﬁcation of gDNA using the following primers:    Rbm38 KO screen For:5′-GGACAGCGTGGGGAACTC-3′  Rbm38 KO screen Rev:5′-CCACACCCCCAATACACAAT-3′  Rbm38 KO screen Internal:5′- GCTGTCCAGGCCATCAGTA-3′     Positive clones were further analyzed by Sanger sequencing. Rbm38 protein depletion was confirmed by Western Blot.


title: Passage 42 FHL124 cells_rep3

value: Lens epithelial cells, FHL124

value: cellular aging model

tag: chip antibody

value: non-treated

value: Human lens epithelial cell line


title: Monte030

tag: age


title: JL019 Syn 2 CD8+ d6

value: spleen + lymph nodes

value: primary

tag: cell type


title: JL019 Syn 2 CD4+ d6

value: T cell

value: syngeneic control, untreated


title: Passage 15 FHL124 cells_rep2

tag: strain


title: D22.EB8.Puf60.pEx5.HET.B

description: D22.EB8.Puf60.pEx5.HET.B: RNA-seq of ESC-derived embryoid bodies engineered to heterozygously express Puf60 with constitutive exon5, replicate B

value: Embryoid bodies (day 8) derived from mouse embryonic stem cells

value: Constitutive inclusion of Puf60 exon 5 (heterozygous)

value: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated constitutive inclusion of Puf60 exon 5 (Puf60+Ex5) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting the upstream (Puf60_gRNA_Up1:5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1:5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). In order to engineer constitutive inclusion of Puf60 exon 5, CGR8 cells were co-transfected with either PX459-Puf60_gRNA_Up1 or PX459-Puf60_gRNA_Dn1, and a HDR template containing the following sequence:   agggtgctgcttggttaatggaagtaggctgtagccctttatagggctcatgaactgaggagtaacacccaccctctagtaggtatgttttgaagaatgtcttcaggcctgaaatgggcattgttggagggaaggtcccttgcctccttacttgttctggggtggtctgaggccctggcaaggcctgttgaggaaactaagaggctagggtgcctggtagcctatgagtaatgagtctttggtcctggttctgttttctgggctctggaggacaataaaggttggctgttctccaagactctgggatcaaattgtcaacagaggcaggcagactgctctcctgttcatgaagaggccattgtgttgtcctacagggcacctgcctggtttaatgtgtcccatccacaagtcatgtctgacgaaggcctgagtgggtatagcaatgtctgaagggaaggcctgcattctaaccaggcaggagatagtttgtattggggcttctcttttgctttaattttagagtctagggataaatggcttatggggtttctgggaccacatgagaaatgttgagtttgctgctgaacaactcccctgtttcctacttcagGGCACAGAATCCATCAAGATGGAAAATGGACAAAGCACAGGCACCAAGCTGGGGCTGCCTCCCCTGACGCCCGAGCAGCAGGAGGCCCTCCAGAAGgtgagagtgcatctcctgggcacagctacttaaccagggttggaaaggcaggccatccccctgcaggctgggccccccagtgctcctcactgccatctcttgcagGCCAAGAAATATGCAATGGAGCAGAGCATCAAGAGTGTGCTGGTGAAACAGACCATCGCGCACCAGCAGCAGCAGCTCACCAACCTGCAGATGGCAGCAGTGACAATGGGCTTTGGAGATCCTCTCTCACCTTTGCAATCGATGGCAGCTCAACGGCAGCGGGCACTGGCTATCATGTGCCGGGTCTATGTGGGCTCCATCTACTATGAGCTGGGAGAAGACACTATTCGCCAGGCCTTTGCTCCCTTTGGCCCCATCAAGAGCATTGATATGTCCTGGGACTCCGTTACCATGAAGCATAAGgtaagcagatgagagctaccctggggctctagcttgccctctgtctgctttccttgccttctaacaccttcctgtattcagGGCTTTGCCTTCGTGGAGTACGAGGTCCCAGAAGCTGCGCAGCTCGCTTTGGAGCAGATGAACTCTGTGATGCTTGGGGGCAGGAACATCAAGgtaaggacaagaaagtttggtcttagccgggcagtggtggcacacgcctttaatctcagcacttgggaggcagaggcaggcggatttctgagtttgaggccagcctggtctacaagtgagttccaggacagccagggctagacagagaaaccctgtcttgaaaaaccaaaaaaagaaaaaaagtttggtcttggacctagaataattgttccgtgaagttacatacatacaagcagtcaccacaggcctaggctgatgtttcttcttgaccttcttcagGTGGGAAGACCCAGCAACATTGGACAAGCCCAACCCATCATAGATCAGCTGGCTGAGGAAGCTAGGGCTTTCAACCGAATATATGTGGCCTCTGTACATCAAGACCTGTCTGATGATGACATCAAGAGTGTATTTGAAGCCTTTGGCAAGATCAAGTCTTGTACGCTGGCCCGGGACCCCACAACTGGCAAGCACAAGGGCTATGGTTTTATCGgtgagtttgggaagg     24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Puf60 exon 5 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Puf60 detected by PCR ampliﬁcation of gDNA using the following primers:    Puf60 HDR screen For:5′-TTGGAAATGGCTTTGGTAGG-3′  Puf60 HDR screen Rev:5′-CAAGTACTGGCCACCCAGAT-3′     Positive clones were further analyzed by Sanger sequencing. Constitutive splicing inclusion of Puf60 exon 5 was confirmed by RT-PCR.


title: PBN Reaction 1

description:
PBN_M1
Live single-cell suspension generated from PBN tissue from 3 female and 2 male mice pooled prior to library generation.

value: brain (parabrachial nucleus)

tag: developmental stage

value: adult

value: pooled male and female


title: D17.EB8.Rbm38KO.A

description: D17.EB8.Rbm38KO.A: RNA-seq of ESC-derived embryoid bodies with deletion of Rbm38, replicate A

value: Embryoid bodies at day 8 of differentiation


title: D16.EB8.Puf60.pEx5.HET.A

description: D16.EB8.Puf60.pEx5.HET.A: RNA-seq of ESC-derived embryoid bodies engineered to heterozygously express Puf60 with constitutive exon5, replicate A


title: D21.EB8.Puf60.pEx5.HOM.B

description: D21.EB8.Puf60.pEx5.HOM.B: RNA-seq of ESC-derived embryoid bodies engineered to homozygously express Puf60 with constitutive exon5, replicate B

value: Constitutive inclusion of Puf60 exon 5 (homozygous)


title: Monte041CD

value: whole blood

value: M


title: Monte066GG


title: Passage 42 FHL124 cells_rep1

value: Lens epithelial cells, FHL124

value: cellular aging model


title: JL019 Syn 1 CD8+ d6


title: Jurkat_SCM

value: T-cell acute lymphoblastic leukemia

value: bone marrow stromal cell

value: Jurkat


title: Monte001CS_2

value: F


title: D20.EB8.Puf60.mEx5.HOM.B

description: D20.EB8.Puf60.mEx5.HOM.B: RNA-seq of ESC-derived embryoid bodies engineered to homozygously express Puf60 lacking exon5, replicate B

value: Deletion of Puf60 exon 5 (homozygous)

value: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated deletion of Puf60 exon 5 (Puf60-Ex5) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting the upstream (Puf60_gRNA_Up1:5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1:5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). For deletion of Puf60 exon 5, CGR8 cells were simultaneously transiently transfected with PX459-Puf60_gRNA_Up1 and PX459-Puf60_gRNA_Dn1 plasmids, using Lipofectamine 2000 (Thermo Fisher Scientiﬁc) following the manufacturer’s instructions. 24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Puf60 exon 5 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Puf60 was detected by PCR ampliﬁcation of gDNA using the following primers:    Puf60 Ex5 deletion screen For:5′- TCCACTGGGCCCTACTCTAA-3′  Puf60 Ex5 deletion screen Rev:5′- CTTGACCCCAACCCAAGAG-3′     Positive clones were further analyzed by Sanger sequencing. Deletion of Puf60 exon 5 was confirmed by RT-PCR.


title: Monte061CM


title: JL027 Syn CD4+ d6

value: spleen + lymph nodes

value: primary

value: T cell

value: syngeneic control, untreated


title: D15.EB8.Puf60.pEx5.HOM.A

description: D15.EB8.Puf60.pEx5.HOM.A: RNA-seq of ESC-derived embryoid bodies engineered to homozygously express Puf60 with constitutive exon5, replicate A

value: Embryoid bodies (day 8) derived from mouse embryonic stem cells

value: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated constitutive inclusion of Puf60 exon 5 (Puf60+Ex5) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting the upstream (Puf60_gRNA_Up1:5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1:5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). In order to engineer constitutive inclusion of Puf60 exon 5, CGR8 cells were co-transfected with either PX459-Puf60_gRNA_Up1 or PX459-Puf60_gRNA_Dn1, and a HDR template containing the following sequence:   agggtgctgcttggttaatggaagtaggctgtagccctttatagggctcatgaactgaggagtaacacccaccctctagtaggtatgttttgaagaatgtcttcaggcctgaaatgggcattgttggagggaaggtcccttgcctccttacttgttctggggtggtctgaggccctggcaaggcctgttgaggaaactaagaggctagggtgcctggtagcctatgagtaatgagtctttggtcctggttctgttttctgggctctggaggacaataaaggttggctgttctccaagactctgggatcaaattgtcaacagaggcaggcagactgctctcctgttcatgaagaggccattgtgttgtcctacagggcacctgcctggtttaatgtgtcccatccacaagtcatgtctgacgaaggcctgagtgggtatagcaatgtctgaagggaaggcctgcattctaaccaggcaggagatagtttgtattggggcttctcttttgctttaattttagagtctagggataaatggcttatggggtttctgggaccacatgagaaatgttgagtttgctgctgaacaactcccctgtttcctacttcagGGCACAGAATCCATCAAGATGGAAAATGGACAAAGCACAGGCACCAAGCTGGGGCTGCCTCCCCTGACGCCCGAGCAGCAGGAGGCCCTCCAGAAGgtgagagtgcatctcctgggcacagctacttaaccagggttggaaaggcaggccatccccctgcaggctgggccccccagtgctcctcactgccatctcttgcagGCCAAGAAATATGCAATGGAGCAGAGCATCAAGAGTGTGCTGGTGAAACAGACCATCGCGCACCAGCAGCAGCAGCTCACCAACCTGCAGATGGCAGCAGTGACAATGGGCTTTGGAGATCCTCTCTCACCTTTGCAATCGATGGCAGCTCAACGGCAGCGGGCACTGGCTATCATGTGCCGGGTCTATGTGGGCTCCATCTACTATGAGCTGGGAGAAGACACTATTCGCCAGGCCTTTGCTCCCTTTGGCCCCATCAAGAGCATTGATATGTCCTGGGACTCCGTTACCATGAAGCATAAGgtaagcagatgagagctaccctggggctctagcttgccctctgtctgctttccttgccttctaacaccttcctgtattcagGGCTTTGCCTTCGTGGAGTACGAGGTCCCAGAAGCTGCGCAGCTCGCTTTGGAGCAGATGAACTCTGTGATGCTTGGGGGCAGGAACATCAAGgtaaggacaagaaagtttggtcttagccgggcagtggtggcacacgcctttaatctcagcacttgggaggcagaggcaggcggatttctgagtttgaggccagcctggtctacaagtgagttccaggacagccagggctagacagagaaaccctgtcttgaaaaaccaaaaaaagaaaaaaagtttggtcttggacctagaataattgttccgtgaagttacatacatacaagcagtcaccacaggcctaggctgatgtttcttcttgaccttcttcagGTGGGAAGACCCAGCAACATTGGACAAGCCCAACCCATCATAGATCAGCTGGCTGAGGAAGCTAGGGCTTTCAACCGAATATATGTGGCCTCTGTACATCAAGACCTGTCTGATGATGACATCAAGAGTGTATTTGAAGCCTTTGGCAAGATCAAGTCTTGTACGCTGGCCCGGGACCCCACAACTGGCAAGCACAAGGGCTATGGTTTTATCGgtgagtttgggaagg     24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Puf60 exon 5 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Puf60 detected by PCR ampliﬁcation of gDNA using the following primers:    Puf60 HDR screen For:5′-TTGGAAATGGCTTTGGTAGG-3′  Puf60 HDR screen Rev:5′-CAAGTACTGGCCACCCAGAT-3′     Positive clones were further analyzed by Sanger sequencing. Constitutive splicing inclusion of Puf60 exon 5 was confirmed by RT-PCR.


title: Monte053BP


title: Thin_EtOH-treated 1

city: Sungnam

first: Danbi

last: Lee

address: sampyeongro 335, Seongnam-Si, Kyounggido, Republic of Korea

department: Department of biological sciences

country: South Korea

institute: CHA university

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence(using fastx_trimmer), then mapped to mm10 (UCSC) whole genome using Bowtie2
Read count extraction using bedtools.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include read count values for each Sample

source_name: Uterus

extract_protocol:
RNA was harvested using Trizol reagent (Invitrogen). 500 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using LEXOGEN Quant-Seq Library Prep Kit (Cat#001.24) standard protocols.

treatment_protocol: 95% EtOH was instilled into one uterus cavity (TS), and saline was treated into the other side cavity as a control (NS).

value: Uterus


title: Monte055DB

value: whole blood


title: Passage 15 FHL124 cells_rep1

value: non-treated

value: Human lens epithelial cell line


title: Thin_EtOH-treated 3


title: Passage 15 FHL124 cells_rep3

value: Lens epithelial cells, FHL124

value: cellular aging model


title: D19.EB8.WTprtl.B

description: D19.EB8.WTprtl.B: RNA-seq of ESC-derived embryoid bodies, parental wild type, replicate B

value: Embryoid bodies at day 8 of differentiation

value: Parental wild type


title: Normal_saline-treated 3


title: Monte013DM

value: Acute

value: M


title: JL019 Treg 2 CD4+ d6

value: GVHD, Treg treatment


title: JL019 Syn 1 CD4+ d6

value: spleen + lymph nodes


title: Thin_EtOH-treated 2


title: COVIDT14


title: PBN Reaction 4

description:
PBN_M4
Live single-cell suspension generated from PBN tissue from 3 female and 2 male mice pooled prior to library generation.

value: brain (parabrachial nucleus)

value: adult

tag: Sex

value: pooled male and female


title: D14.EB8.Puf60.mEx5.HOM.A

description: D14.EB8.Puf60.mEx5.HOM.A: RNA-seq of ESC-derived embryoid bodies engineered to homozygously express Puf60 lacking exon5, replicate A

value: Embryoid bodies (day 8) derived from mouse embryonic stem cells

value: Deletion of Puf60 exon 5 (homozygous)

value: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated deletion of Puf60 exon 5 (Puf60-Ex5) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting the upstream (Puf60_gRNA_Up1:5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1:5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). For deletion of Puf60 exon 5, CGR8 cells were simultaneously transiently transfected with PX459-Puf60_gRNA_Up1 and PX459-Puf60_gRNA_Dn1 plasmids, using Lipofectamine 2000 (Thermo Fisher Scientiﬁc) following the manufacturer’s instructions. 24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Puf60 exon 5 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Puf60 was detected by PCR ampliﬁcation of gDNA using the following primers:    Puf60 Ex5 deletion screen For:5′- TCCACTGGGCCCTACTCTAA-3′  Puf60 Ex5 deletion screen Rev:5′- CTTGACCCCAACCCAAGAG-3′     Positive clones were further analyzed by Sanger sequencing. Deletion of Puf60 exon 5 was confirmed by RT-PCR.


title: D13.EB8.WTprtl.A

description: D13.EB8.WTprtl.A: RNA-seq of ESC-derived embryoid bodies, parental wild type, replicate A


title: 8_1uM_H2O2_Lenti_6DT1

city: Bethesda

first: Howard

last: Yang

state: MD

address: 37 CONVENT DR, Room 5120

department: CCR

institute: NCI

description: wildtype cells

data_processing:
Illumina software RTA 2.4.11 was used for basecalling.
Sequence reads of the samples were trimmed for adapters and low-quality bases using Cutadapt before alignment with the mouse reference genome (mm10) and the  transcripts annotation  using STAR. The gene expression quantification analysis was performed for all samples using   RSEM.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files including raw counts and  FPKM values for each Sample ...

extract_protocol:
Cells with/without 1uM H2O2 treatement were lysed with RLT lysis buffer containing 1% beta mercaptoethanol after 24 hours and RNA was harvased using QIAcube as per Rneasy kit from Qiagen.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: mammary cancer cell line

value: treated with 1uM H2O2


title: Monte048ST

value: whole blood


title: JL019 Treg 1 Treg d6

value: primary

value: T cell


title: JL019 Treg 1 CD4+ d6


title: Normal_saline-treated 2

value: Uterus


title: D11.ESC.Rbm38KO.B

description: D11.ESC.Rbm38KO.B: RNA-seq of CGR8 mouse ESCs with deletion of Rbm38, replicate B

source_name: Mouse CGR8 embryonic stem cells

value: Mouse CGR8 embryonic stem cells

value: Embryonic stem cell

value: Deletion of Rbm38

value: CGR8 clonal cell lines harboring a CRISPR-Cas9 mediated ~1300bp deletion within the Rbm38 locus (Rbm38 KO) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting within Rbm38 (Rbm38_gRNA2:5′-GCTCCCCGAGTGTGTTTCCG-3′) and (Rbm38_gRNA4:5′-CCTTGATCCAGCGCACCTAC-3′) were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). For the generation of Rbm38 knockout cells, CGR8 cells were simultaneously transiently transfected with PX459-Rbm38_gRNA2 and PX459-Rbm38_gRNA4 plasmids, using Lipofectamine 2000 (Thermo Fisher Scientiﬁc) following the manufacturer’s instructions. 24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Rbm38 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Rbm38 was detected by PCR ampliﬁcation of gDNA using the following primers:    Rbm38 KO screen For:5′-GGACAGCGTGGGGAACTC-3′  Rbm38 KO screen Rev:5′-CCACACCCCCAATACACAAT-3′  Rbm38 KO screen Internal:5′- GCTGTCCAGGCCATCAGTA-3′     Positive clones were further analyzed by Sanger sequencing. Rbm38 protein depletion was confirmed by Western Blot.


title: COVIDT15

value: Acute

value: F


title: D05.ESC.Rbm38KO.A

description: D05.ESC.Rbm38KO.A: RNA-seq of CGR8 mouse ESCs with deletion of Rbm38, replicate A


title: 7_1uM_H2O2_Lenti_6DT1

description: wildtype cells


title: JL027 Treg 2 CD8+ d6

value: spleen + lymph nodes

value: GVHD, Treg treatment


title: JL027 Treg Treg d6


title: COVIDT20


title: D10.ESC.Puf60.pEx5.HET.B

description: D10.ESC.Puf60.pEx5.HET.B: RNA-seq of CGR8 mouse ESCs engineered to heterozygously express Puf60 with constitutive exon5, replicate B

value: Constitutive inclusion of Puf60 exon 5 (heterozygous)

value: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated constitutive inclusion of Puf60 exon 5 (Puf60+Ex5) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting the upstream (Puf60_gRNA_Up1:5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1:5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). In order to engineer constitutive inclusion of Puf60 exon 5, CGR8 cells were co-transfected with either PX459-Puf60_gRNA_Up1 or PX459-Puf60_gRNA_Dn1, and a HDR template containing the following sequence:   agggtgctgcttggttaatggaagtaggctgtagccctttatagggctcatgaactgaggagtaacacccaccctctagtaggtatgttttgaagaatgtcttcaggcctgaaatgggcattgttggagggaaggtcccttgcctccttacttgttctggggtggtctgaggccctggcaaggcctgttgaggaaactaagaggctagggtgcctggtagcctatgagtaatgagtctttggtcctggttctgttttctgggctctggaggacaataaaggttggctgttctccaagactctgggatcaaattgtcaacagaggcaggcagactgctctcctgttcatgaagaggccattgtgttgtcctacagggcacctgcctggtttaatgtgtcccatccacaagtcatgtctgacgaaggcctgagtgggtatagcaatgtctgaagggaaggcctgcattctaaccaggcaggagatagtttgtattggggcttctcttttgctttaattttagagtctagggataaatggcttatggggtttctgggaccacatgagaaatgttgagtttgctgctgaacaactcccctgtttcctacttcagGGCACAGAATCCATCAAGATGGAAAATGGACAAAGCACAGGCACCAAGCTGGGGCTGCCTCCCCTGACGCCCGAGCAGCAGGAGGCCCTCCAGAAGgtgagagtgcatctcctgggcacagctacttaaccagggttggaaaggcaggccatccccctgcaggctgggccccccagtgctcctcactgccatctcttgcagGCCAAGAAATATGCAATGGAGCAGAGCATCAAGAGTGTGCTGGTGAAACAGACCATCGCGCACCAGCAGCAGCAGCTCACCAACCTGCAGATGGCAGCAGTGACAATGGGCTTTGGAGATCCTCTCTCACCTTTGCAATCGATGGCAGCTCAACGGCAGCGGGCACTGGCTATCATGTGCCGGGTCTATGTGGGCTCCATCTACTATGAGCTGGGAGAAGACACTATTCGCCAGGCCTTTGCTCCCTTTGGCCCCATCAAGAGCATTGATATGTCCTGGGACTCCGTTACCATGAAGCATAAGgtaagcagatgagagctaccctggggctctagcttgccctctgtctgctttccttgccttctaacaccttcctgtattcagGGCTTTGCCTTCGTGGAGTACGAGGTCCCAGAAGCTGCGCAGCTCGCTTTGGAGCAGATGAACTCTGTGATGCTTGGGGGCAGGAACATCAAGgtaaggacaagaaagtttggtcttagccgggcagtggtggcacacgcctttaatctcagcacttgggaggcagaggcaggcggatttctgagtttgaggccagcctggtctacaagtgagttccaggacagccagggctagacagagaaaccctgtcttgaaaaaccaaaaaaagaaaaaaagtttggtcttggacctagaataattgttccgtgaagttacatacatacaagcagtcaccacaggcctaggctgatgtttcttcttgaccttcttcagGTGGGAAGACCCAGCAACATTGGACAAGCCCAACCCATCATAGATCAGCTGGCTGAGGAAGCTAGGGCTTTCAACCGAATATATGTGGCCTCTGTACATCAAGACCTGTCTGATGATGACATCAAGAGTGTATTTGAAGCCTTTGGCAAGATCAAGTCTTGTACGCTGGCCCGGGACCCCACAACTGGCAAGCACAAGGGCTATGGTTTTATCGgtgagtttgggaagg     24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Puf60 exon 5 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Puf60 detected by PCR ampliﬁcation of gDNA using the following primers:    Puf60 HDR screen For:5′-TTGGAAATGGCTTTGGTAGG-3′  Puf60 HDR screen Rev:5′-CAAGTACTGGCCACCCAGAT-3′     Positive clones were further analyzed by Sanger sequencing. Constitutive splicing inclusion of Puf60 exon 5 was confirmed by RT-PCR.


title: 6_1uM_H2O2_Lenti_6DT1

description: wildtype cells


title: COVIDT10


title: JL027 Treg 2 CD4+ d6


title: JL019 Treg 2 CD8+ d6


title: D08.ESC.Puf60.mEx5.HOM.B

description: D08.ESC.Puf60.mEx5.HOM.B: RNA-seq of CGR8 mouse ESCs engineered to homozygously express Puf60 lacking exon5, replicate B

value: Deletion of Puf60 exon 5 (homozygous)

value: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated deletion of Puf60 exon 5 (Puf60-Ex5) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting the upstream (Puf60_gRNA_Up1:5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1:5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). For deletion of Puf60 exon 5, CGR8 cells were simultaneously transiently transfected with PX459-Puf60_gRNA_Up1 and PX459-Puf60_gRNA_Dn1 plasmids, using Lipofectamine 2000 (Thermo Fisher Scientiﬁc) following the manufacturer’s instructions. 24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Puf60 exon 5 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Puf60 was detected by PCR ampliﬁcation of gDNA using the following primers:    Puf60 Ex5 deletion screen For:5′- TCCACTGGGCCCTACTCTAA-3′  Puf60 Ex5 deletion screen Rev:5′- CTTGACCCCAACCCAAGAG-3′     Positive clones were further analyzed by Sanger sequencing. Deletion of Puf60 exon 5 was confirmed by RT-PCR.


title: SARS-3


title: JL019 Treg 1 CD8+ d6


title: 5_1uM_H2O2_Lenti_6DT1

description: wildtype cells

value: mammary cancer cell line

value: treated with 1uM H2O2


title: Normal_saline-treated 1


title: JL019 Treg 2 Treg d6

value: primary


title: COVIDT6

value: whole blood

value: M


title: COVIDT12


title: D09.ESC.Puf60.pEx5.HOM.B

description: D09.ESC.Puf60.pEx5.HOM.B: RNA-seq of CGR8 mouse ESCs engineered to homozygously express Puf60 with constitutive exon5, replicate B

value: Mouse CGR8 embryonic stem cells

value: Constitutive inclusion of Puf60 exon 5 (homozygous)


title: JL027 Treg 1 CD4+ d6

value: T cell


title: D01.ESC.WTprtl.A

description: D01.ESC.WTprtl.A: RNA-seq of CGR8 mouse ESCs, parental wild type, replicate A

value: Embryonic stem cell

value: Parental wild type


title: COVID-T36

value: Acute

value: F


title: D07.ESC.WTprtl.B

description: D07.ESC.WTprtl.B: RNA-seq of CGR8 mouse ESCs, parental wild type, replicate B