title: A12.mEx5.siPuf60.Dox.B

description:
A12.mEx5.siPuf60.Dox.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, knockdown of endogenous Puf60 and dox induction, replicate B
A12.mEx5.siPuf60.Dox.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, knockdown of endogenous Puf60 and dox induction, 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 assessed based on the vast-tools diff module. For Puf60 knockdown/rescue experiments, we defined changing AS events as those with a significant (as defined above) PSI differential and changing in the same direction upon knockdown of endogenous Puf60 in both cell lines (Puf60+Ex5 and Puf60-Ex5). We further defined events rescued by either isoform as those that were knocked down and changed by > 5 PSI and with a likelihood of 0.95 for a change > 0 in the opposite direction upon dox induction in at least one of the cell lines. Finally, isoform-differentially rescued events were those that were rescued by either isoform, and where there was a dPSI of > 10 and a 0.95 likelihood that there was a difference > 0 between rescue with Puf60+Ex5 and Puf60-Ex5, while requiring that no such difference was detected between the uninduced cell lines, with or without Puf60 knockdown.
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.

source_name: Mouse neural cells

growth_protocol: Mouse Neuro-2A (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.    Puf60+Ex5 and Puf60-Ex5 cDNAs were synthesized from N2A cells, cloned into a Gateway pDONR vector (Thermo Fisher Scientific) and transferred to a rtTA3-compatible N-terminal-3xFlag pcDNA5/FRT destination vector (modified from Thermo Fisher Scientific, V6010-20) using the Gateway system. The primers used for cloning are:     AttB Puf60 For: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCGACGGCGACTATAGCTCTCC-3′  AttB Puf60 Rev: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACGCGGAGAGGTCGCTGT-3′     Site-directed mutagenesis was performed on the pcDNA5-Puf60+Ex5 and pcDNA5-Puf60-Ex5 expression vectors to generate siRNA-resistant cDNA sequences using the Q5 Site-Directed Mutagenesis Kit (NEB), following manufacturer’s instructions. The primers used for mutagenesis are:     Q5 Puf60 M10 For: 5′-GAACGGTTCGATAACAGCGACCTCTCC-3′  Q5 Puf60 M10 Rev: 5′-TTGATCGTATACTTCAGCCACCACCTTG-3′    Doxycycline inducible N2A Flp-In rtTA3 stable cell lines were generated as previously described (Gonatopoulos-Pournatzis et al., 2018). Briefly, N2A Flp-In rtTA3 stable cell lines capable of inducible cDNA expression were created through co-transfection of 200 ng of a rtTA3 compatible pcDNA5/FRT plasmid (modified from Thermo Fisher Scientific, V6010-20), with 2 μg of pOG44 Flp-recombinase expression vector (Thermo Fisher Scientific, V600520). Cell lines with successful cDNA integration were selected and maintained using 200 mg/mL Hygromycin.

value: Mouse neural cells

value: Flp-In rtTA3 allele of siRNA-resistant Puf60 cDNA lacking exon 5

value: Flp-In rtTA N2A cell lines stably expressing doxycycline-inducible, siRNA-resistant Puf60-Ex5 constructs were transfected with 15nM (final concentration) of siRNA (Dharmacon) for silencing Puf60 (J-051526-10). 24 hours post-transfection, fresh media was added with doxycycline (2μg/ml final dox concentration) to induce protein expression. Cells were harvested 48 hours post-transfection (24 hours post-induction).


title: JL019 Allo 2 CD4+ d6

status: Public on Jul 15 2022

type: SRA

city: Stanford

first: Juliane

last: Lohmeyer

address: 269 W. Campus Drive

country: USA

institute: Stanford University

library_source: transcriptomic

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

molecule: total RNA

organism: Mus musculus

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.)

tag: tissue

value: spleen + lymph nodes

tag: cell line

value: primary

tag: cell type

value: T cell

tag: treatment

value: GVHD, untreated


title: Monte047ME

city: New York

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

molecule: polyA RNA

organism: Homo sapiens

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: age

tag: phase

tag: gender

value: F


title: D04.ESC.Puf60.pEx5.HET.A

city: Toronto

first: Ulrich

last: Braunschweig

state: Ontario

address: 160 College Street

department: Donnelly Centre

country: Canada

institute: University of Toronto

description: D04.ESC.Puf60.pEx5.HET.A: RNA-seq of CGR8 mouse ESCs engineered to heterozygously express Puf60 with constitutive exon5, replicate A

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.

source_name: Mouse CGR8 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).

value: Mouse CGR8 embryonic stem cells

tag: differentiation stage

value: Embryonic stem cell

tag: mutation

value: Constitutive inclusion of Puf60 exon 5 (heterozygous)

tag: cloning

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: COVIDT30

value: Acute

value: M


title: D03.ESC.Puf60.pEx5.HOM.A

description: D03.ESC.Puf60.pEx5.HOM.A: RNA-seq of CGR8 mouse ESCs engineered to homozygously express Puf60 with constitutive exon5, replicate A

value: Constitutive inclusion of Puf60 exon 5 (homozygous)


title: Covid-Tx-35-RNA


title: JL027 Treg 1 CD8+ d6

value: GVHD, Treg treatment


title: 15_1uM_H2O2_Empty_6DT1

city: Bethesda

first: Howard

last: Yang

state: MD

address: 37 CONVENT DR, Room 5120

department: CCR

institute: NCI

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

tag: strain

value: mammary cancer cell line

value: treated with 1uM H2O2


title: A09.mEx5.siPuf60.Dox.A

description:
A09.mEx5.siPuf60.Dox.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, knockdown of endogenous Puf60 and dox induction, replicate A
A09.mEx5.siPuf60.Dox.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, knockdown of endogenous Puf60 and dox induction, replicate A

value: Mouse neural cells

value: Flp-In rtTA3 allele of siRNA-resistant Puf60 cDNA lacking exon 5

value: Flp-In rtTA N2A cell lines stably expressing doxycycline-inducible, siRNA-resistant Puf60-Ex5 constructs were transfected with 15nM (final concentration) of siRNA (Dharmacon) for silencing Puf60 (J-051526-10). 24 hours post-transfection, fresh media was added with doxycycline (2μg/ml final dox concentration) to induce protein expression. Cells were harvested 48 hours post-transfection (24 hours post-induction).


title: JL019 Allo 1 CD4+ d6

value: spleen + lymph nodes

value: primary

value: T cell


title: 4_NT_Lenti_6DT1

description: wildtype cells

value: untreated


title: D02.ESC.Puf60.mEx5.HOM.A

description: D02.ESC.Puf60.mEx5.HOM.A: RNA-seq of CGR8 mouse ESCs engineered to homozygously express Puf60 lacking exon5, replicate A

value: Mouse CGR8 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: 3_NT_Lenti_6DT1

description: wildtype cells


title: JL019 Allo 2 CD8+ d6

value: GVHD, untreated


title: COVIDT16

value: whole blood

value: Acute


title: JL027 Allo 2 CD8+ d6


title: A11.mEx5.siPuf60.B

description:
A11.mEx5.siPuf60.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, knockdown of endogenous Puf60 without induction, replicate B
A11.mEx5.siPuf60.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, knockdown of endogenous Puf60 without induction, replicate B

value: Flp-In rtTA N2A cell lines stably expressing doxycycline-inducible, siRNA-resistant Puf60-Ex5 constructs were transfected with 15nM (final concentration) of siRNA (Dharmacon) for silencing Puf60 (J-051526-10). 24 hours post-transfection, fresh media was added without doxycycline. Cells were harvested 48 hours post-transfection.


title: 2_NT_Lenti_6DT1

description: wildtype cells


title: JL027 Allo 2 CD4+ d6


title: JL019 Allo 1 CD8+ d6


title: 13_1uM_H2O2_Empty_6DT1


title: A10.mEx5.siNTC.B

description:
A10.mEx5.siNTC.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, control non-targeting knockdown without induction, replicate B
A10.mEx5.siNTC.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, control non-targeting knockdown without induction, replicate B

value: Flp-In rtTA N2A cell lines stably expressing doxycycline-inducible, siRNA-resistant Puf60-Ex5 constructs were transfected with 15nM (final concentration) of non-targeting control (NTC) (D-001810-10) siRNA. 24 hours post-transfection, fresh media was added without doxycycline. Cells were harvested 48 hours post-transfection.


title: 1_NT_Lenti_6DT1

description: wildtype cells

value: mammary cancer cell line


title: 16_1uM_H2O2_Empty_6DT1

value: treated with 1uM H2O2


title: A08.mEx5.siPuf60.A

description:
A08.mEx5.siPuf60.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, knockdown of endogenous Puf60 without induction, replicate A
A08.mEx5.siPuf60.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, knockdown of endogenous Puf60 without induction, replicate A

value: Mouse neural cells


title: A07.mEx5.siNTC.A

description:
A07.mEx5.siNTC.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, control non-targeting knockdown without induction, replicate A
A07.mEx5.siNTC.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively skipped exon 5 without induction, control non-targeting knockdown without induction, replicate A

value: Flp-In rtTA3 allele of siRNA-resistant Puf60 cDNA lacking exon 5


title: Monte012RN

value: F


title: 9_NT_Empty_6DT1


title: COVIDT13


title: JL027 Allo 1 CD8+ d6

value: spleen + lymph nodes


title: COVIDT29

value: M


title: A06.pEx5.siPuf60.Dox.B

description:
A06.pEx5.siPuf60.Dox.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, knockdown of endogenous Puf60 and dox induction, replicate B
A06.pEx5.siPuf60.Dox.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, knockdown of endogenous Puf60 and dox induction, replicate B

value: Flp-In rtTA3 allele of siRNA-resistant Puf60 cDNA including exon 5

value: Flp-In rtTA N2A cell lines stably expressing doxycycline-inducible, siRNA-resistant Puf60+Ex5 constructs were transfected with 15nM (final concentration) of siRNA (Dharmacon) for silencing Puf60 (J-051526-10). 24 hours post-transfection, fresh media was added with doxycycline (2μg/ml final dox concentration) to induce protein expression. Cells were harvested 48 hours post-transfection (24 hours post-induction).


title: A05.pEx5.siPuf60.B

description:
A05.pEx5.siPuf60.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, knockdown of endogenous Puf60 without induction, replicate B
A05.pEx5.siPuf60.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, knockdown of endogenous Puf60 without induction, replicate B

value: Flp-In rtTA N2A cell lines stably expressing doxycycline-inducible, siRNA-resistant Puf60+Ex5 constructs were transfected with 15nM (final concentration) of siRNA (Dharmacon) for silencing Puf60 (J-051526-10). 24 hours post-transfection, fresh media was added without doxycycline. Cells were harvested 48 hours post-transfection.


title: COVID-T1


title: 14_1uM_H2O2_Empty_6DT1


title: 12_NT_Empty_6DT1

value: untreated


title: JL027 CD8+ d0

value: primary

value: T cell

value: pretransplant


title: 10_NT_Empty_6DT1


title: COVIDT17

value: whole blood

value: Acute


title: A04.pEx5.siNTC.B

description:
A04.pEx5.siNTC.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, control non-targeting knockdown without induction, replicate B
A04.pEx5.siNTC.B: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, control non-targeting knockdown without induction, replicate B

value: Flp-In rtTA N2A cell lines stably expressing doxycycline-inducible, siRNA-resistant Puf60+Ex5 constructs were transfected with 15nM (final concentration) of non-targeting control (NTC) (D-001810-10) siRNA. 24 hours post-transfection, fresh media was added without doxycycline. Cells were harvested 48 hours post-transfection.


title: JL019 CD8+ d0


title: A03.pEx5.siPuf60.Dox.A

description:
A03.pEx5.siPuf60.Dox.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, knockdown of endogenous Puf60 and dox induction, replicate A
A03.pEx5.siPuf60.Dox.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, knockdown of endogenous Puf60 and dox induction, replicate A


title: COVIDT8


title: JL027 CD4+ d0


title: A02.pEx5.siPuf60.A

description:
A02.pEx5.siPuf60.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, knockdown of endogenous Puf60 without induction, replicate A
A02.pEx5.siPuf60.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, knockdown of endogenous Puf60 without induction, replicate A


title: COVIDT22


title: E12_Input_Rbm38

description:
E12_Input_Rbm38: Size-matched input for iCLIP-seq of Rbm38 in N2A cells
Extracted RNA was reverse transcribed using primer Rt14clip: /5Phos/NNTGCCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGCANN, where N are random bases.

data_processing:
Library strategy: iCLIP-Seq
RTA 2.0
91-nt raw reads that consisted of 3 random positions, a 4-nt multiplexing barcode, and another 2 random positions, followed by the cDNA sequence, were initially trimmed to 49 nt from the 3'-end, and duplicates were discarded. Reads were de-multiplexed, and the random positions, barcodes, and any 3'-bases matching Illumina adaptors were removed. Remaining reads longer than 25 nt were mapped to the mouse genome/transcriptome (Ensembl annotation of NCBIm37) using TopHat with default settings. To prevent false assignment of reads from repetitive regions, any reads with a mapping quality < 3 were removed from further analysis. Note that this record contains demultiplexed but un-trimmed reads.
Peaks were first derived on individual replicates of CLIP and input using Piranha (https://github.com/smithlabcode/piranha). Regions which were called as a peak in 2/3 replicates or in size-matche input were kept and merged. Enrichment of CLIP over input reads was then assessed in individual peaks using the exact test mode in edgeR, and peaks with an enrichment of at least two-fold and FDR < 0.05 were deemed significant.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text with 1-based coordinates and enrichment (log2FC) of CLIP reads over input reads with associated raw (pval) and adjusted (FDR) p-values derived using edgeR.

source_name: 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:
Lysates were treated with Turbo DNase (Thermo Fisher Scientiﬁc) and RNase I (Thermo Fisher Scientiﬁc) diluted 1/250 in PBS for 5 minutes at 37°C to digest genomic DNA and partially digest RNA to short fragments of an optimal size range. RNA-protein complexes were puriﬁed using 100 mL of protein G Dynabeads (Thermo Fisher Scientiﬁc) and 5μg of anti-Flag M2 (Sigma-Aldrich) antibody, with 2% (20ul) of the total lysate retained for use as a size-matched input (SMI) control. SMI samples were processed as previously described (Van Nostrand et al., 2016). Following stringent high salt washes, the immunoprecipitated RNA was 3′ end dephosphorylated and ligated to a pre-adenylated adaptor at the 3′ end, as described previously. After extensive additional washes and buffer exchange, the immunoprecipitated RNA was 5′ end-labeled using ATP [γ-32P]. The puriﬁed protein-RNA complexes and SMI control samples were separated with SDS-PAGE and transferred to nitrocellulose membrane. Protein was digested using proteinase K, the RNA was puriﬁed using phenol/chloroform.
Purified RNA was reverse transcribed into cDNA. The cDNA was size selected by resolving samples on a 6% TBE-Urea gel, circularized to add the adaptor to the 5′ end, digested at the internal BamHI site, and then PCR ampliﬁed using AccuPrime SuperMix I (Thermo Fisher Scientiﬁc). The ﬁnal PCR libraries were puriﬁed by agarose gel electrophoresis using gel extraction columns (QIAGEN). The eluted DNA from the low, middle, and high fractions was pooled together and submitted for sequencing.

treatment_protocol: N2A cells were crosslinked (150mJ/cm2) at 254 nm with a Stratalinker 1800.

value: Neuroblastoma cells

value: Rbm38 was expressed in an N2A cell line engineered with the Flp-in system under the control of doxycyclin, and expression was induced with 1μg/ml doxycycline for 24 hours prior to crosslinking.

tag: antibody


title: A01.pEx5.siNTC.A

description:
A01.pEx5.siNTC.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, control non-targeting knockdown without induction, replicate A
A01.pEx5.siNTC.A: RNA-seq of N2A cells carrying an inducible Puf60 allele with constitutively included exon 5 without induction, control non-targeting knockdown without induction, replicate A

value: Mouse neural cells


title: JL027 Treg d0


title: JL019 CD4+ d0


title: JL027 Allo 1 CD4+ d6

value: GVHD, untreated


title: E11_Rbm38_BR3

description:
E11_Rbm38_BR3: iCLIP-seq of Rbm38 in N2A cells, replicate BR3
Extracted RNA was reverse transcribed using primer Rt16clip: /5Phos/NNTTAANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNTTAANN, where N are random bases.

value: Flag monoclonal antibody M2 (Sigma-Aldrich)


title: E10_Input_Puf60mEx5

description:
E10_Input_Puf60mEx5: Size-matched input for iCLIP-seq of Puf60 lacking exon 5 in N2A cells
Extracted RNA was reverse transcribed using primer Rt6clip: /5Phos/NNCCGGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNCCGGNN, where N are random bases.

value: Puf60 lacking exon 5 was expressed in an N2A cell line engineered with the Flp-in system under the control of doxycyclin, and expression was induced with 1μg/ml doxycycline for 24 hours prior to crosslinking.


title: B23.Rbm38.B

description: B23.Rbm38.B: RNA-seq of N2A cells upon siRNA-mediated knockdown 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_Rbm38etc.tab contains primary vast-tools output. AltSplicing_Rbm38etc.tab contains filtered PSI values and annotations of differential AS events. Expression_Rbm38etc.tab contains edgeR-estimated RPM, fold changes and associated FDR.

growth_protocol: Mouse Neuro-2A (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.

treatment_protocol: N2A cells were transfected with siGENOME siRNA SMARTpools (Dharmacon), or non-targeting control SMARTpools, using Lipofectamine RNAiMAX (Invitrogen), as recommended by the manufacturer. Cells were harvested 48 hours post-transfection.


title: B27.siNT.C

description: B27.siNT.C: RNA-seq of N2A cells upon control non-targeting siRNA-mediated knockdown, replicate C


title: JL019 Treg d0

value: spleen + lymph nodes


title: E09_Puf60mEx5_BR3

description:
E09_Puf60mEx5_BR3: iCLIP-seq of Puf60 lacking exon 5 in N2A cells, replicate BR3
Extracted RNA was reverse transcribed using primer Rt13clip: /5Phos/NNTCCGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNCGGANN, where N are random bases.


title: COVIDT5

value: M


title: COVIDT7


title: 11_NT_Empty_6DT1

value: mammary cancer cell line


title: E08_Puf60mEx5_BR2

description:
E08_Puf60mEx5_BR2: iCLIP-seq of Puf60 lacking exon 5 in N2A cells, replicate BR2
Extracted RNA was reverse transcribed using primer Rt10clip: /5Phos/NNGACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGTCNN, where N are random bases.


title: B21.Khsrp.B

description: B21.Khsrp.B: RNA-seq of N2A cells upon siRNA-mediated knockdown of Khsrp (Fubp2), replicate B


title: COVID-T2


title: B20.Khsrp_Fubp1.B

description: B20.Khsrp_Fubp1.B: RNA-seq of N2A cells upon siRNA-mediated knockdown of Fubp1 and Khsrp (Fubp2), replicate B


title: COVIDT28

value: F


title: E07_Puf60mEx5_BR1

description:
E07_Puf60mEx5_BR1: iCLIP-seq of Puf60 lacking exon 5 in N2A cells, replicate BR1
Extracted RNA was reverse transcribed using primer Rt9clip: /5Phos/NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNTGGCNN, where N are random bases.


title: B11.Rbm38.A

description: B11.Rbm38.A: RNA-seq of N2A cells upon siRNA-mediated knockdown of Rbm38, replicate A


title: COVIDT21


title: spinal cord, control, EAE day 17, biol rep 3

city: Shanghai

first: Yue

last: Wu

address: 320 Yueyang Road

department: Institute of Neuroscience

country: China

institute: Chinese Academy of Sciences

data_processing:
The library construction and sequencing was performed by Illumina Hiseq-PE250 at Novogene Corporation. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina following manufacture's recommendations.
Clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data.
Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5.
Quantification of gene expression level featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Assembly: mm10
Supplementary files format and content: raw_count_table

source_name: spinal cord

extract_protocol: The spinal cord of 2~3-month-old Drd2 cKO mice and littermate control were harvested using Trizol reagent.

value: spinal cord

value: 2-3 months

tag: genotype

value: wt

tag: condition

value: EAE day 17


title: E05_Rbm38_BR1

description:
E05_Rbm38_BR1: iCLIP-seq of Rbm38 in N2A cells, replicate BR1
Extracted RNA was reverse transcribed using primer Rt16clip: /5Phos/NNTTAANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNTTAANN, where N are random bases.


title: B09.Khsrp.A

description: B09.Khsrp.A: RNA-seq of N2A cells upon siRNA-mediated knockdown of Khsrp (Fubp2), replicate A


title: B08.Khsrp_Fubp1.A

description: B08.Khsrp_Fubp1.A: RNA-seq of N2A cells upon siRNA-mediated knockdown of Fubp1 and Khsrp (Fubp2), replicate A


title: Monte006SA

value: whole blood


title: spinal cord, control, EAE day 17, biol rep 2


title: Monte-004-AF-Clinic

value: Acute


title: E06_Rbm38_BR2

description:
E06_Rbm38_BR2: iCLIP-seq of Rbm38 in N2A cells, replicate BR2
Extracted RNA was reverse transcribed using primer Rt14clip: /5Phos/NNTGCCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGCANN, where N are random bases.


title: Monte018MS


title: E04_Input_Puf60pEx5

description:
E04_Input_Puf60pEx5: Size-matched input for iCLIP-seq of Puf60 including exon 5 in N2A cells
Extracted RNA was reverse transcribed using primer Rt6clip: /5Phos/NNCCGGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNCCGGNN, where N are random bases.

value: Puf60 including exon 5 was expressed in an N2A cell line engineered with the Flp-in system under the control of doxycyclin, and expression was induced with 1μg/ml doxycycline for 24 hours prior to crosslinking.


title: Monte007DS


title: B04.Fubp1.A

description: B04.Fubp1.A: RNA-seq of N2A cells upon siRNA-mediated knockdown of Fubp1, replicate B


title: E03_Puf60pEx5_BR3

description:
E03_Puf60pEx5_BR3: iCLIP-seq of Puf60 including exon 5 in N2A cells, replicate BR3
Extracted RNA was reverse transcribed using primer Rt13clip: /5Phos/NNTCCGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNCGGANN, where N are random bases.

value: Neuroblastoma cells

value: Puf60 including exon 5 was expressed in an N2A cell line engineered with the Flp-in system under the control of doxycyclin, and expression was induced with 1μg/ml doxycycline for 48 hours prior to crosslinking.


title: spinal cord, Drd2mGFAP cKO, EAE day 17, biol rep 2

value: Drd2mGFAP conditional knockout


title: spinal cord, Drd2mGFAP cKO, EAE day 17, biol rep 3


title: spinal cord, Drd2mGFAP cKO, basal, biol rep 3

value: Basal


title: B03.siNT.B

description: B03.siNT.B: RNA-seq of N2A cells upon control non-targeting siRNA-mediated knockdown, replicate B

value: Mouse neural cells


title: spinal cord, control, EAE day 17, biol rep 1


title: E02_Puf60pEx5_BR2

description:
E02_Puf60pEx5_BR2: iCLIP-seq of Puf60 including exon 5 in N2A cells, replicate BR2
Extracted RNA was reverse transcribed using primer Rt10clip: /5Phos/NNGACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGTCNN, where N are random bases.

value: Flag monoclonal antibody M2 (Sigma-Aldrich)


title: spinal cord, Drd2mGFAP cKO, EAE day 17, biol rep 1


title: Monte016MG


title: Monte005HL


title: E01_Puf60pEx5_BR1

description:
E01_Puf60pEx5_BR1: iCLIP-seq of Puf60 including exon 5 in N2A cells, replicate BR1
Extracted RNA was reverse transcribed using primer Rt9clip: /5Phos/NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNTGGCNN, where N are random bases.


title: spinal cord, Drd2mGFAP cKO, basal, biol rep 1


title: B02.Fubp1.A

description: B02.Fubp1.A: RNA-seq of N2A cells upon siRNA-mediated knockdown of Fubp1, replicate A


title: spinal cord, Drd2mGFAP cKO, basal, biol rep 2


title: Monte011LA


title: spinal cord, control, basal, biol rep 3


title: siPOOL_neg_2

city: Zurich

first: Constance

last: Ciaudo

email: cciaudo@ethz.ch, ciaudobeyer@gmail.com

address: Otto-Stern-Weg 7

department: D-BIOL

country: Switzerland

institute: ETH

data_processing:
fastq files were obtained, as provided by sequencing facility (functional genomics center zurich)
Adapters were initially trimmed using TrimGalore with arguments “--stringency 3 –illumina”
Next, trimmed reads were polyA-tail-trimmed using TrimGalore with argument “--polyA”
Finally, processed files were mapped and analyzed using snakePipes' RNA-seq pipeline (v2.3.1) with arguments “--libraryType 1 –featureCountOptions ‘--primary’ –mode alignment,alignment-free,deepTolls_qc –sampleSheet mir290_vs_wt.conf”
Genome_build: ENSEMBL GRCm38 v98
Supplementary_files_format_and_content: counts

source_name: Embryonic stem cells

growth_protocol: mESCs were cultured in DMEM, 15% serum 100 U/ml LIF, 0.1 mM 2-beta-mercaptoethanol, 1% Pen/Strep on gelatin coated plates.

extract_protocol:
RNA was harvested using Trizol reagent.
RNA libraries were prepared for sequencing using QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina on 500ng of RNA

treatment_protocol: mESCs were transfected with one of the siPOOLs (obtained from siTOOLs Biotech GmbH) at a final concentration of 5nM using the Lipofectamine RNAiMax transfection reagent (Invitrogen). Cell were harvested after 36 hours of transfection.

value: 129/Ola background

value: Blastocyst

value: MiR-290-295 knockout cells


title: spinal cord, control, basal, biol rep 2


title: siPOOL_tfap4_2


title: B01.siNT.A

description: B01.siNT.A: RNA-seq of N2A cells upon control non-targeting siRNA-mediated knockdown, replicate A


title: siPOOL_tfap4_1


title: spinal cord, control, basal, biol rep 1


title: YY1 ChIP-seq Lin- XistΔ/Δ Rep1

city: Durham

first: Eda

last: Yildirim

state: NC

address: DUMC Box 3709

department: Cell Biology

institute: Duke University School of Medicine

library_source: genomic

data_processing:
ChIP-seq reads were trimmed using TrimGalore and then aligned to mm10 reference genome by Bowtie2 (v2.3.5.1, --qc-filter --very-sensitive).
Reads with alignment quality lower than 30 or reads aligned to mitochondrial DNA were removed using Samtools (v1.10).
For YY1 ChIP-seq, duplicated reads were removed by MarkDuplicates tool of Picard (v2.0.1; http://broadinstitute.github.io/picard/).
ChIP-seq results of two biological replicates were merged to make bigwig files, metagene profiles and heatmaps using deepTools (v3.5.0) (Ramirez et al., 2016) with CPM normalization method.
For YY1 ChIP-seq, peaks were called using findPeaks (-region -size 500 -minDist 500 -F 0 -L 4 -C 2) program in HOMER (v4.9.1) (Heinz et al., 2010) and consensus YY1 peak sites from all samples were calculated by DiffBind R package (v3.0.13, minOverlap = 2; https://bioconductor.org/packages/DiffBind/).
Genome_build: mm10
Supplementary_files_format_and_content: bed (YY1 peaks called by HOMER) and bigwig (H3K27me3 and H3K4me3 distribution).

molecule: genomic DNA

source_name: YY1 ChIP-seq Lin- XistΔ/Δ

growth_protocol: Lin- cells were collected by depleting lineage-positive cells from the bone marrow.

extract_protocol:
ChIP experiments were performed as previously described with minor modifications (Kadota and Nagata, 2014). Briefly, to perform H3K27me3 and H3K4me3 ChIP, mouse bone marrow Lin- cells (~3x106) were crosslinked with 1% formaldehyde for 10 min at RT. The reaction was stopped by adding glycine (final concentration, 125 mM) for 5 min at RT. Crosslinked cells were treated with hypotonic buffer (10 mM HEPES-NaOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 % NP-40, 1 mM DTT, 1 mM PMSF, 1x protease inhibitor cocktail (Sigma, P8340)) at 4ºC for 10 min and collected in a tube. The cells were lysed in 150 ml of ChIP lysis buffer (50 mM Tris-HCl, pH 7.9, 10 mM EDTA, 1% SDS, 1 mM PMSF, protease inhibitor cocktail (Sigma, P8340)) on ice for 10 min, and sonicated for 35 cycles (30’’ ON + 30’’ OFF) using Diagenode Bioruptor to shear the chromatin to 200-1,000 bp fragments. The lysate was diluted with 9x volumes of ChIP dilution buffer (16.7 mM Tris-HCl, pH 7.9, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 1 mM PMSF). The fragmented chromatin was pre-cleared using 15 ml of pre-washed Protein A/G agarose beads (Thermo Fisher, 20423) for 1 hour at 4ºC. The pre-cleared chromatin fragments were equally separated into two aliquots. Each aliquot was incubated with either 2 ml of anti-H3K27me3 (mAbcam6002; Abcam) or 2 ml of anti-H3K4me3 (ab8580; Abcam) antibodies at 4ºC overnight with rotation, followed by incubation with pre-washed Protein A/G agarose beads (Thermo Scientific, 20423) for 1-2 hours at 4ºC. DNA-protein complexes attached to agarose beads were washed at 4ºC with the following buffers: low salt wash buffer (20 mM Tris-HCl, pH 7.9, 100 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) for 10 min, high salt wash buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) for 10 min, the LiCl buffer (10 mM Tris-HCl, pH 7.9, 250 mM LiCl, 1% NP-40, 1% deoxycholate acid, 1 mM EDTA) for 10 min, and twice with TE buffer for 5 min. DNA-protein complexes were eluted by incubating the beads in elution buffer (1% SDS, 0.1 M NaHCO3) for 15 min at RT. Reverse crosslinking was performed by incubating samples in 200 mM of NaCl at 65ºC overnight followed by Proteinase K (20 mg/ml) treatment for 2 hrs at 55ºC. DNA was purified using MiniElute PCR purification kit (QIAGEN, 28006). To perform YY1 ChIP, bone marrow Lin- cells (~1.5x106) were crosslinked with 1% formaldehyde for 15 min at RT and the reaction was stopped by adding glycine (final concentration, 125 mM) for 5 min at RT. Crosslinked cells were treated with cell lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 1 mM DTT, 1 mM PMSF, 1x protease inhibitor cocktail (Sigma, P8340)) for 10 min at 4ºC. To digest chromatin into <1,000 bp fragments, the pellet was washed twice with MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 0.2% NP-40, 1 mM DTT, 1 mM PMSF, 1x protease inhibitor cocktail), resuspended in 200 ml of MNase reaction buffer containing 2.5U of MNase (NEB, M0247) and incubated for 10 min at 37ºC. MNase digestion was stopped by adding 20 ml of 0.5M EDTA into the reaction. Nuclear membrane was broken up by sonicating the lysate for 3 cycles (output level 6, 10’’ ON + 20’’ OFF) using Microson ultrasonic cell disruptor (Misonix). Lysate was spun at 21,000xg for 10 min at 4ºC, and the supernatant containing fragmented chromatin was incubated with 2 ml of anti-YY1 antibody (D5D92Z, Cell Signaling, 46395) overnight at 4ºC. DNA-protein complexes were pulled down with addition of 10 ml of pre-washed Protein G Magnetic Beads (Invitrogen, 10003D) and incubation for 1-2 hours at 4ºC. DNA-protein complexes were eluted in elution buffer (1% SDS, 0.1 M NaHCO3) by incubating at 65ºC for 40 min, and reverse crosslinked as described above. All ChIP DNA samples were purified using MinElute PCR purification kit (Qiagen,28006) and quantified using the fluorometric quantitation Qubit 2.0 system (Thermo Fisher Scientific).
ChIP-seq libraries were prepared using KAPA HyperPrep kit (KAPA, 07962347001). Libraries were sequenced on Illumina HiSeq 4000 (single-end; H3K27me3, and H3K4me3 ChIP-seq) and on Illumina NovaSeq 6000 (paired-end; YY1 ChIP-seq).

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 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.

value: Bone marrow Lin- cells

value: Xist knockout

tag: Sex

value: Female

value: 129Sv4/Jae x B6

tag: chip antibody

value: anti-YY1 (Cell Signaling, 46395)


title: siPOOL_neg_1


title: YY1 ChIP-seq Lin- WT Rep2

source_name: YY1 ChIP-seq Lin- WT

value: Wild type


title: YY1 ChIP-seq Lin- WT Rep1


title: RNA-seq LSK- XistΔ/Δ 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 LSK- XistΔ/Δ

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 LSK- cells


title: Monte021ST

value: whole blood

value: F


title: Monte008LC


title: Monte009OO

value: M


title: Monte001CS


title: Monte002OH


title: WT_1_mESC_CC

value: WT mESCs


title: DKO_CD4_SP_BR1

first: Tarmo

last: Äijö

address: 162 5th Avenue

department: Center for Computational Biology

institute: Flatiron Institute

data_processing:
bcl2fastq ver 2.20.0
Adapter trimming and quality filtering of the sequencing libraries was done using fastp (0.21.0).
The sequencing libraries were mapped against mm10 and the GRCm38.100 transcriptome using STAR (2.7.5a).
The differential expression analysis was done using DESeq2 based on the read counts per gene produced by STAR.
The TCR repertoire extraction was done using MiXCR (3.0.13) from RNA-seq data using the “-p rna-seq” mode
Genome_build: mm10
Supplementary_files_format_and_content: STAR produced gene read counts and MiXCR produced clone count information.

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:
Live, CD24 low enriched cells CD4+, TCRb+, aGalCerCd1d tetramer-, CD8- cells were sorted in a purity higher than 98%. RNA was isolated using the micro RNeasy plus kit (Qiagen
RNA was quantified using HS RNA kit (Invitrogen) in a Qubit 4 Fluorometer (Invitrogen). RNA integrity was assessed in a Bionalyzer (Agilent) using RNA 6000 pico kit (Agilent, cat no: 5067-1513). The RNA that was used for downstream steps had a RIN value of 9 or higher. Libraries were prepared using the SMARTseq kit v4 Ultra Low Input RNA kit for sequencing (Clontech, cat no: 634888) and Nextera  XT (Illumina)
RNA-seq (SMART-seq)

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.  Live, CD24 low enriched cells CD4+, TCRb+, aGalCerCd1d tetramer-, CD8- cells were sorted in a purity higher than 98% and were used for RNA isolation.

tag: cell subset

value: male


title: MiR-290-295_KO2_mESC_CC

tag: tissue


title: DKO_CD4_SP_BR2


title: Monte017TS


title: MiR-290-295_KO1_mESC_HWP8


title: RNA-seq LSK- WT Rep2

source_name: RNA-seq LSK- WT


title: RNA-seq LSK- XistΔ/Δ Rep1


title: WT_CD4_SP_BR2

value: wild type


title: Monte015CL


title: Monte019JC


title: Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato 8 week muscle tamoxifen at 8 weeks

city: Vancouver

first: Tully

middle: Michael

last: Underhill

state: British Columbia

address: 2222 Health Sciences Mall

department: Biomedical Research Centre

institute: University of British Columbia

description: Single cell RNA-seq - adult skeletal muscle resident tdTomato MPs

data_processing:
Samples were demultiplexed using cellranger mkfastq with bclfastq2.
Samples were aligned and output matrices generated using cellranger count for alignment.
cellranger aggregate was used to aggregate multiple samples for combined analysis
Genome_build: mm10 with appended tdTomato and EGFP sequences
Supplementary_files_format_and_content: gene matrices, cell barcode and gene list are included as .mtx, .tsv and .tsv files, respectively

source_name: Mesenchymal Progenitors

growth_protocol: Mice were bred and raised using standard approved animal care committee methodology.

extract_protocol:
FACS purified cells were counted and inspected by hemocytometer before input to the 10X chromium library preparation pipeline and subsequent NGS.
10X Chromium Single Cell 3' v2 or v3

treatment_protocol: To label mesenchymal progenitors of the murine limb, adult dams >7 weeks of age were paired in timed matings and subsequently gavaged with 7.5mg tamoxifen in 300uL sunflower oil at embryonic day 10 or adult animals at 8 weeks of age were injected with five consecutive daily doses of 2.5mg per day in 100uL oil. In both cases adult skeletal muscle was collected at post-natal day 56, dissociated and reporter positive cells isolated for interrogation.

value: adult skeletal muscle


title: WT_CD4_SP_BR3


title: Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato 8 week muscle Tamoxifen at E10


title: H3K4me3 ChIP-seq Lin- XistΔ/Δ Rep2

source_name: H3K4me3 ChIP-seq Lin- XistΔ/Δ

value: anti_H3K4me3 (Abcam, 8580)


title: WT_CD4_SP_BR1

value: female


title: RNA-seq LSK- WT Rep1


title: H3K4me3 ChIP-seq Lin- XistΔ/Δ Rep1


title: ATAC-seq LSK- XistΔ/Δ Rep2

data_processing:
ATAC-seq reads were trimmed using TrimGalore and then aligned to mm10 reference genome by Bowtie2 (v2.3.5.1, --qc-filter --very-sensitive)
Reads with alignment quality lower than 30 or reads aligned to mitochondrial DNA were removed using Samtools (v1.10).
ATAC peaks were called using callpeak program (-q 0.01 -B -f BAMPE) in MACS2 (v2.2.7.1) and consensus ATAC peak sites were calculated by DiffBind.
Genome_build: mm10
Supplementary_files_format_and_content: narrowPeak, ATAC peaks called by MACS2.

source_name: ATAC-seq LSK- XistΔ/Δ

growth_protocol: Bone marrow LSK+ (Lin-SCA-1+c-Kit+) and LSK- (Lin-SCA-1-c-KIT+) were sorted by FACS in 1xPBS.

extract_protocol:
ATAC experiments were performed as previously described with minor modifications (Buenrostro et al., 2015). Briefly, 10,000 bone marrow LSK+ or LSK- cells were lysed in ice-cold lysis buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP-40) for 5 min and then washed twice with 10mM Tris-HCl, pH 7.4. Following the last wash, 25 ml of extracted nuclei was mixed with 25 ml of 2XTD buffer (66 mM Tris-Ac pH 7.8, 132 mM KAc, 20 mM MgAc, 32% Dimethyl Formamide) containing 1 ml of Tn5 with loaded tagmentation adapters. Transposition reaction mixture was incubated using a thermocycler rotating at 500 rpm for 1 hour at 37°C. Tagmented DNA fragments were purified using MinElute PCR Cleanup Kit (Qiagen, 28206) and amplified for 15 PCR cycles using Q5 polymerase (NEB, M0491). PCR products were run on a gel, and fragments of ~200-800 bp size were isolated and libraries were sequenced on MGI MGISEQ-2000 or Illumina NovaSeq 6000 with paired-end reads.
Libraries were constructed by PCR amplificaiton of tagmented DNA fragments.

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). Approximately 10,000 cells were utilized per transposition reaction.


title: COVIDT31


title: ATAC-seq LSK- WT Rep2

source_name: ATAC-seq LSK- WT


title: Gabra3_6KO_Retina_ATAC

city: Seattle

first: Timothy

middle: Joel

last: Cherry

state: WA

department: Pediatrics

institute: University of Washington/Seattle Children's Research Institute

data_processing:
QUANTIFICATION AND STATISTICAL ANALYSIS: Sequence read data processing (ATAC_Seq): Read processing and alignment: Demultiplexed FASTQ files were trimmed with Trimmomatic (version 0.33) using the parameter SLIDINGWINDOW:5:30 (Bolger et al., 2014). Trimmed reads were indexed and aligned to the mouse genome (Mouse GRCm38/mm10 assembly, December 2011) using the Burrows_Wheeler Aligner (bwa) tool (Li and Durbin, 2010) with the parameters: bwa aln  _q 0  _t 4  _n 2  _k 2  _l 32  _e _1  _o 0 and bwa samse  _n 5. Bed files from processed and aligned sequence reads were extended to 200bp and normalized to 10M reads using BEDtools (version 2.23.0) (19) genomeCoverageBED using the -scale parameter before being converted into bigWig format for display on the UCSC genome browser (https://genome.ucsc.edu/).
Genome_build: mm10
Supplementary_files_format_and_content: Bed flies of normalized ATAC_Seq. Bigwig files of normalized  ATAC_Seq.

extract_protocol:
Flash-frozen tissue was thawed by addition of 700ul of lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40), homogenized by trituration 10x with a p1000 pipet set to 500ul, dounced in an ice cold RNase-free 2mL glass dounce 10x with a loose pestle and 10x with tight pestle, and transferred back to original 1.5mL LoBind tube (Eppendorf). Dounce was washed with an additional 700ul ice cold lysis buffer and buffer was transferred to tube with sample for a final volume of 1.4mL. Sample was then centrifuged in a microcentrifuge at 4C for 10min at 500 rcf. The pellet was re-eluted in 1.4mL ice cold lysis buffer, transferred to a pre-chilled dounce, homogenized again 12x with tight pestle, and transferred back to 1.5mL LoBind tube. Nuclei were counted on a hemocytometer and the volume of lysis buffer/nuclei suspension needed for 20,000 nuclei was aliquoted into a separate 1.5mL LoBind tube. Samples were centrifuged along with tube containing remaining nuclei (for nuclear localized RNA) at 4C for 5min at 500g (RCF). The supernatant was removed from samples and remaining nuclei. Subsequent ATAC libraries were generated with 20,000 nuclei per sample using the Nextera DNA library prep kit (FC-121-1030; Illumina, San Diego, CA USA).
ATAC-Seq libraries were prepared as described above and sequenced on the Illumina NextSeq 500 platform with 75 bp single-end reads.

tag: developmental stage

value: Adult

tag: genotype/variation

value: Gabra3 knockout

value: neural retinal


title: Gabra3_3WT_Retina_ATAC


title: H3K4me3 ChIP-seq Lin- WT Rep2

source_name: H3K4me3 ChIP-seq Lin- WT

tag: cell type

value: Bone marrow Lin- cells

value: Female


title: Monte022AA

tag: age


title: Monte024

tag: phase


title: ATAC-seq LSK+ WT Rep2

source_name: ATAC-seq LSK+ WT

value: Bone marrow LSK+ cells

value: 129Sv4/Jae x B6


title: ATAC-seq LSK+ XistΔ/Δ Rep2

source_name: ATAC-seq LSK+ XistΔ/Δ

value: Xist knockout


title: H3K4me3 ChIP-seq Lin- WT Rep1

value: Wild type


title: Monte023

source_name: Whole Blood

value: whole blood

tag: gender

value: F


title: H3K27me3 ChIP-seq Lin- XistΔ/Δ Rep2

source_name: H3K27me3 ChIP-seq Lin- XistΔ/Δ

value: anti-H3K27me3 (Abcam, 6002)


title: ESCs_si_neg_1

data_processing:
fastq files were obtained, as provided by sequencing facility (functional genomics center zurich)
Adapters were initially trimmed using TrimGalore with arguments “--stringency 3 –illumina”
Next, trimmed reads were polyA-tail-trimmed using TrimGalore with argument “--polyA”
Finally, processed files were mapped and analyzed using snakePipes' RNA-seq pipeline (v2.3.1) with arguments “--libraryType 1 –featureCountOptions ‘--primary’ –mode alignment,alignment-free,deepTolls_qc –sampleSheet mir290_vs_wt.conf”
Genome_build: ENSEMBL GRCm38 v98

value: 129/Ola background

value: Blastocyst

value: MiR-290-295 knockout cells

value: Embryonic stem cells


title: Monte-025-RM-Clinic

value: M


title: Monte010AA


title: BD Rhapsody WTA [Bdofficial]

city: Chiba

first: Shigeyuki

last: Shichino

address: 2641, Yamasaki, Noda-shi

department: Department of Molecular regulation of Inflammatory and Immune Diseases

country: Japan

institute: Tokyo University of Science

data_processing:
Adapter trimming of sequencing data was performed by using cutadapt 2.10. Filtered reads were chunked into 16 parts for parallel processing by using Seqkit 0.9.0. Filtered cell barcode reads were annotated by Python script provided by BD Biosciences with minor modification for compatibility to Python 3.7. Reference RNA sequences were built by concatenating cDNA and ncRNA fasta files of Ensembl database (build GRCm38 release-101 for mouse data and GRCh38 release-101 for human data). Associated cDNA reads were mapped to reference RNA by using bowtie2-2.4.2 by the following parameters: -p 2 –very-sensitive-local -N 1 -norc -seed 656565 -reorder. Then, cell barcode information of each read was added to the bowtie2-mapped BAM files by the python script and pysam 0.15.4, and read counts of each gene in each cell barcode were counted by using mawk.
Resulted count data was converted to a single-cell gene-expression matrix file and the inflection point of the knee-plot (total read count versus the rank of the read count) was detected by using DropletUtils package in R 3.6.3 (https://cran.r-project.org/). Cells of which total read count was over inflection point were considered as valid cells.
Genome_build: GRCm38
Supplementary_files_format_and_content: counts

source_name: spleen

extract_protocol:
The spleen was mushed on cell strainer with 5 ml of preparation medium and resulted cells from three spleen were harvested and pooled. Then, spleen cells were suspended with ACK lysis buffer [155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3] and incubated for 10 min at room temperature. The cell suspension was washed two times with PBS, filtered with 40 μm strainer, suspended with CellBanker 1, and gradually freezed at -80 ºC by using CoolCell (Thermo Fisher Scientific) for long term storage. Stored spleen cells were thawed and washed just before use for processing by BD Rhapsody.
2000 mouse spleen cells were loaded onto BD Rhapsody cartrige. Cell load, cell lysis, mRNA trap, cDNA synthesis, and primer digestion were performed according to the manufacture's instruction.
Half of the resulted beads were subjected to cDNA amplification by TAS-Seq, and the remained half of the beads were subjected to library generation by BD official WTA workflow. Sequencing libraries from amplified cDNAs were produced by using NEB UltraII FS Kit (New England Biolabs) with some modifications. Sequencing was performed by using a Novaseq SP and a Novaseq 6000 sequencer (Illumina) according to the manufacture's instructions. 13% PhiX spike-in was used for sequencing.

value: spleen single cell suspension

value: female

tag: model

value: lewis lung carcinoma, anti-CD4 treated

tag: organ

value: spleen


title: H3K27me3 ChIP-seq Lin- XistΔ/Δ Rep1

value: Bone marrow Lin- cells

value: Female


title: ESCs_si_zic2_2


title: H3K27me3 ChIP-seq Lin- WT Rep2

source_name: H3K27me3 ChIP-seq Lin- WT

value: 129Sv4/Jae x B6


title: ESCs_si_zic2_1

source_name: Embryonic stem cells


title: ESCs_si_neg_2


title: ESCs_si_wiz_2


title: H3K27me3 ChIP-seq Lin- WT Rep1


title: Single cell ATAC-seq - embryonic tdTomato MPs, Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato Embryonic Day 14.5

data_processing:
Samples were demultiplexed using cellranger-atac 1.1.0 mkfastq with bcl2fastq.
Samples were aligned and output matrices generated using cellranger-atac 1.1.0 count for alignment and peak calling to cell clustering.
cellranger-atac aggregate was used to aggregate multiple samples for combined analysis
Genome_build: mm10
Supplementary_files_format_and_content: peak matrices, cell barcode and peak list are included as .mtx, .tsv and .tsv files, respectively

extract_protocol:
FACS purified cells were counted and inspected by hemocytometer before input to the 10X chromium nuclei preperation pipeline.
nuclei were lysed for five minutes according to 10x standard protocol and subjected to the transposition reaction followed by library preparation pipeline and subsequent NGS.
Chromium Single Cell ATAC Reagent Kits User Guide (10x Genomics; CG000168 Rev B)

treatment_protocol: To label mesenchymal progenitors of the murine forelimb, adult dams >7 weeks of age were paired in timed matings and subsequently gavaged with 7.5mg tamoxifen in 300uL sunflower oil at embryonic day ten before forelimbs were dissociated and reporter positive cells isolated for interrogation.

value: Embryonic Day 14.5

value: Embryonic forelimb


title: BD Rhapsody WTA [UTlung]

data_processing:
Adapter trimming of sequencing data was performed by using cutadapt 2.10. Filtered reads were chunked into 16 parts for parallel processing by using Seqkit 0.9.0. Filtered cell barcode reads were annotated by Python script provided by BD Biosciences with minor modification for compatibility to Python 3.7. Reference RNA sequences were built by concatenating cDNA and ncRNA fasta files of Ensembl database (build GRCm38 release-101 for mouse data and GRCh38 release-101 for human data). Associated cDNA reads were mapped to reference RNA by using bowtie2-2.4.2 by the following parameters: -p 2 –very-sensitive-local -N 1 -norc -seed 656565 -reorder. Then, cell barcode information of each read was added to the bowtie2-mapped BAM files by the python script and pysam 0.15.4, and read counts of each gene in each cell barcode were counted by using mawk.
Resulted count data was converted to a single-cell gene-expression matrix file and the inflection point of the knee-plot (total read count versus the rank of the read count) was detected by using DropletUtils package in R 3.6.3 (https://cran.r-project.org/). Cells of which total read count was over inflection point were considered as valid cells.
Genome_build: GRCm38 or GRCh38
Supplementary_files_format_and_content: txt

source_name: lung

extract_protocol:
Murine and human Lungs were cut into small pieces with a razor blade and digested enzymatically using 0.25 mg/ml Liberase TM (Roche) and 20 kU/mL DNase I (Sigma-Aldrich, St Louis, MO) for 60 min at 37°C. Erythrocytes and dead cells were removed by 25%/65% Percoll PLUS (Cytiva) density separation. Resulting cells were stained with Calcein AM for 5 min at 37°C, immediately chilled on ice, and filtered through 40 μm cell strainer (BD).
10000 mouse cells and 6000 human cells were loaded onto BD Rhapsody cartrige. Cell load, cell lysis, mRNA trap, cDNA synthesis, and primer digestion were performed according to the manufacture's instruction.
Half of the resulted beads were subjected to cDNA amplification by TAS-Seq. Sequencing libraries from amplified cDNAs were produced by using NEB UltraII FS Kit (New England Biolabs) with some modifications. Sequencing was performed by using a Novaseq S2 and a Novaseq 6000 sequencer (Illumina) according to the manufacture's instructions. 20% PhiX spike-in was used for sequencing.

value: lung single cell suspension

value: bleomycin-induced lung fibrosis

value: lung


title: BD Rhapsody WTA [TASSeq]


title: HeLa SMN CRISPR mutant clone 2  (5A)

city: Rome

first: Grazia Daniela

last: Raffa

address: P.le Aldo Moro 5

country: Italy

institute: Sapienza Università di Roma

data_processing:
Reads were aligned against the reference genome (GRCh38) using STAR (version 2.7.0f).
Transcript were assembled from raw reads using the Scallop software (version 0.10.3). These results were merged with the reference Gencode annotation (release 34) using the gtfmerge program (part of the rnaseqtools suite, version 1.0.2; https://github.com/Kingsford-Group/rnaseqtools).
Genome_build: GRCh38
Supplementary_files_format_and_content: Scallop_Transcriptome.gtf lists the transcripts assembled from Illumina reads (GTF format).

source_name: HeLa

growth_protocol: HeLa S3 cells were cultured in DMEM supplemted with 10% Fetal Bovine Serum and Penicillin-Streptomycin at 37°C in 5% CO2

extract_protocol:
Total RNA was extracted with Trizol reagent (Ambion), treated with Ambion™ DNase I (RNase-free) extracted with phenol/chloroform. RNA samples were quantified and quality tested by Agilent 2100 Bioanalyzer RNA assay (Agilent technologies, Santa Clara, CA)
TruSeq Stranded mRNA kit (Illumina, San Diego, CA) has been used for library preparation following the manufacturer’s instructions (library type: fr-firstrand). Final libraries were checked with both Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and Agilent Bioanalyzer DNA assay.

value: Cervical adenocarcinoma

value: SMN -/-


title: RNA-seq LSK+ XistΔ/Δ Rep2

source_name: RNA-seq LSK+ XistΔ/Δ

value: Bone marrow LSK+ cells

value: Xist knockout


title: ESCs_si_wiz_1

value: 129/Ola background

value: Blastocyst

value: MiR-290-295 knockout cells

value: Embryonic stem cells


title: ESCs_si_tfap4_2


title: ChIP-seq on AP2-EXP in the AP2-EXP::HA NF54 background at 30 hpi replicate 3 immunoprecipitate

city: STATE COLLEGE

middle: J

last: Russell

state: PA

address: W122 Millennium Science Complex

institute: Pennsylvania State University

data_processing:
Reads in each DNA library were trimmed to remove low quality base calls and Illumina adaptor sequences using Trimmomatic 0.32.3.
Reads were mapped to the P. falciparum 3D7 genome (Pf 3D7 v36) using BWA-MEM 0.4.1.
Duplicate reads were removed using samtools 1.1.2.
MACS2 (version 2.1.1.20160309) was used to call peaks with a q value cutoff of 0.01.
Deeptools BamCoverage 3.5.1.0.0 was used to generate bigwig files from mapped .bam files.
Assembly: PlasmoDB-36_Pfalciparum3D7_Genome.fasta
Supplementary files format and content: bigwig for all libraries, narrowpeak calls for immunoprecipitate over input

organism: Plasmodium falciparum

growth_protocol: Parasites were grown at 37°C, 5% O2, 7% CO2 using RPMI1640 media supplemented with hypoxanthine and .5% Albumax II (Thermo). AP2-EXP::GFP and AP2-EXP::HA were maintained in media supplemented with 2.5nM WR99210

extract_protocol:
ChIP was performed as described in PMID: 32198457. Briefly, nuclear material was extracted following cross-linking in 1% formaldehyde. Chromatin was sonicated using an M220 ultrasonicator (Covaris) at 5% duty cycle, 75 W peak incident power, 200 cycles per burst, treatment time 300 seconds. Input chromatin was set aside to use as a control, and the remaining material was immunoprecipitated overnight using anti-HA or anti-GFP antibody. RNA and protein were removed by enzymatic digestion and DNA was isolated using the Qiagen MinElute kit.
ChIP-seq DNA libraries were prepared as described in PMID: 32198457 using the NEBNext II DNA library kit (New England Biolabs) according to the manufacturer’s instructions. Quality was assessed using an Agilent 2100 Bioanalyzer or TapeStation. AMPure XP beads (Beckmann Coulter) were used to size select and purify DNA between NEBNext II library preparation steps.

tag: Stage

value: trophozoite

tag: cell line

value: Sigma 3F10 anti-HA


title: HeLa SMN CRISPR mutant clone 1  (4A)

tag: strain


title: RNA-seq LSK+ WT Rep2

source_name: RNA-seq LSK+ WT

tag: genotype

value: Wild type

value: Female


title: RNA-seq LSK+ XistΔ/Δ Rep1

value: 129Sv4/Jae x B6


title: HeLa TGS1 CRISPR mutant clone 1 (2A)


title: HeLa parental cell line rep 2              (1B)

value: wild type


title: ChIP-seq on AP2-EXP in the AP2-EXP::HA NF54 background at 30 hpi replicate 3 input


title: ESCs_si_tfap4_1


title: HeLa parental cell line rep 1              (1A)


title: RNA-seq LSK+ WT Rep1


title: WT_2_mESC

value: WT mESCs


title: BD Rhapsody WTA [RAILD1-F]

value: lung single cell suspension

tag: disease

value: lung cancer with RA-ILD

tag: region

value: fibrotic area


title: HeLa TGS1 CRISPR mutant clone 2 (3A)

value: Cervical adenocarcinoma


title: WT_1_mESC


title: AML12, Chrna4-2

city: Nanjing

first: Jun

last: Liu

address: Longmiandadao

institute: CPU

description: Transfected with PAV-TBG-P2A-GFP-Chrna4 plasmid

data_processing:
Basecalling based on Illumina Casava1.7 software
After RNA sequencing reads quality checking, the reads were  mapped to the mouse genome (mm10) using tophat2 by requiring 3 edit distance (mismatch+indel) at maximum
The gene expression abundance was estimated by counting number of reads mapped to the cognate gene exons using HTSeq
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: File includes gene ID and expression level in reads count for each sample

source_name: Hepatocytes

growth_protocol: Mouse hepatocyte cell line AML12 cells (ATCC, CRL-2254) were cultured in DMEM/F12 medium (Gibco, 11330-032, USA) with 5 mg.ml-1 insulin, 5 mg.ml-1 transferrin, 5 ng.ml-1 selenium, 40 ng.ml-1 dexamethasone, 10% FBS (Hyclone, SH30396.03, USA) and 1% anti-anti (Gibco, 15240-062, USA)

extract_protocol:
Total RNA was extracted with RNAiso Plus (TaKaRa) from cultured cells.
A cDNA library was generated from polyA+ RNA according to the Illumina RNAseq protocol. The cDNA library was sequenced on an Illumina Hiseq4000 by a 2X150 bp run.

treatment_protocol: AML12 cells were classified into two groups, one was transfected with pCMV6-AC-GFP plasmid, the other was transfected with PAV-TBG-P2A-GFP-Chrna4 plasmid. We aimed to identify key factors promoting this transition.

value: Hepatocytes from a mouse (CD1 strain, line MT42)

tag: passages

value: Chrna4 over-expression


title: BD Rhapsody WTA [RAILD1-NF]

value: non-fibrotic area

value: lung


title: AML12, Chrna4-1


title: MiR-290-295_KO2_mESC

value: 129/Ola background

value: Blastocyst

value: MiR-290-295 knockout cells


title: MiR-290-295_KO1_mESC

value: Embryonic stem cells


title: AML12, Ctrl-3

description: Transfected with pCMV6-AC-GFP plasmid

value: control


title: NuMA (TDIF) ChIP-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:
IG = Immunoglobulin G, MH = NuMA (TDIF), IN = Input sample, IP = ChIP sample
TDIF_ChIP-seq_TSS_TTS_Metagene_Matrix.txt
TDIF_ChIP-seq_Upstream_Exons_Downstream_Metagene_Matrix.txt
TDIF_ChIP-seq_Promoter_Counts_Matrix.txt

data_processing:
Data was mapped using BWA 0.7.17-r1188 with parameters -mem -M -k 25
Metagene matrices indicating coverage over the TSS/TTS and gene upstream, exonic, and downstream regions were generated using bam2geneprofile from the CGAT suite of tools with the parameters --reporter = gene, -m = tssprofile/geneprofile --merge-pairs
Counts of reads overlapping promoter regions were determined by creating a window 1000 base pairs upstream and downstream of the TSSs of protein coding genes using bedtools slop on a TSS bed, converting the resulting bed file to a GTF file and running featureCounts (parameters: -t exons -g gene) on the bam files over the promoter gtf
The mapped bam files were converted into bigwig files showing fold change relative to input by first performing narrow peak calling using MACS2 - parameters -g hs -B SPMR with the inputs as the control files. The sample and control lambda (derived from inputs) BEDgraph signal tracks were then compared using MACS2 bdgcomp with the option -m FE in order to generate sample BEDgraph files showing fold change relative to input, which were then converted to bigwigs.
Genome_build: hg38
Supplementary_files_format_and_content: bigwig file for visualising fold change in coverage relative to input, TSS/TTS and gene upstream, exonic, and gene downstream region metagene matrices assessing read density over various genomic features and a matrix containing read counts over promoter regions.
Supplementary_files_format_and_content: Read counts over promoter regions matrix
Supplementary_files_format_and_content: Metagene profile matrix for TSS/TTS regions
Supplementary_files_format_and_content: Metagene profile matrix for gene upstream/exonic/gene downstream regions

source_name: Lung fibroblast cell line

extract_protocol:
DNA was extracted from cells through phenol chloroform extraction followed by ethanol precipitation
ChIP libraries were prepared for sequencing using standard Illumina protocols

value: Lung


title: AII_Que_5

city: Fuzhou

last: Peng

state: Fujian

address: 1 Qiuyang Road, Minhou Shangjie

department: Academy of Integrative Medicine

institute: Fujian University of Traditional Chinese Medicine

description: WT mice with 28-day Angiotensin-II (500ng/kg/min) and Que (5mg/kg/d) treatment

data_processing:
Data were filtered using the following specifications fastq software，The filtering standards from raw data to clean reads are:：  Check the sample for contamination and, if there is pollution, remove it  If the reads contains a joint sequence, the joint sequence is cut off first  a base having an average base mass of less than 15 on both ends of the readth window is cut with four base lengths as a sliding window;  Remove the number of threads with a high N content. If the N content in a read is more than 5%, the whole read is removed, and the read pair with high low-quality base content is removed. If the low-quality base in one read (the mass value is not higher than the base of 20) exceeds 30%, the whole pair of the reads is removed;  if the truncated read length is less than 50 bp, the entire read is removed  Remove unmatched reads  Q30，Q20 explanation：  Q30 indicates that the sequencing error rate of the base is 0.1%.  Q20 indicates that the sequencing error rate of the base is 1%.
Using String Tie for readcount and FPKM calculation.
Differential expression analyses were performed using the limma package.
Assembly: GCRmm38/mm10(UCSC)
Supplementary files format and content: tab_delimited text files include FPKM values for each Sample

source_name: renal

extract_protocol:
The renal were dissected and  stored in liquid nitrogen and stored in low temperature.Total RNA was extracted with TRIzol® Reagent (Invitrogen,Carlsbad, CA, USA). RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA concentration was measured by KAPA Library Quantification Kit (KAPA Biosystem, Wilmington, MA, USA).
A total amount of 1 μg RNA per sample was used. In brief, the NEB Next rRNA Depletion Kit was used to remove rRNA from the total RNA. The NEB Next Ultra RNA Library Prep Kit  (NEB,Ipswich, MA, USA)for Illumina was used to construct the libraries for sequencing following the manufacturer’s instructions.

value: renal

value: WT

tag: treatment 1

value: Angiotensin-II

tag: treatment 2

value: Quercetin

tag: group

value: AngII+Que


title: Single cell RNA-seq - embryonic  MPs, both Hic1;tdTomato+ and Hic1;tdTomato- Day 11.5 replicate 2

source_name: Mesenchymal Progenitors

treatment_protocol: To label mesenchymal progenitors of the murine forelimb, adult dams >7 weeks of age were paired in timed matings and subsequently gavaged with 7.5mg tamoxifen in 300uL sunflower oil at embryonic day ten before forelimbs were dissociated and reporter positive and negative cells isolated for interrogation.

tag: tissue

value: Embryonic forelimb

tag: developmental stage

value: Embryonic Day 11.5


title: Single cell RNA-seq - embryonic  MPs, both Hic1;tdTomato+ and Hic1;tdTomato- Day 11.5 replicate 1


title: HK2 24h Urea

type: RNA

city: Bonn

first: Sibylle

last: von Vietinghoff

address: Venusbergcampus 1

country: Germany

institute: University Clinic Bonn

hyb_protocol: cRNA fragmentation, hybridisation and washing steps were carried-out as recommended in the ‘One-Color Microarray-Based Gene Expression Analysis Protocol V5.7’, except that 500ng of each fluorescently labelled cRNA population were used for hybridization.

scan_protocol: Slides were scanned on the Agilent Micro Array Scanner G2565CA (pixel resolution 3 µm, bit depth 20).

data_processing: Data extraction was performed with the ‘Feature Extraction Software V10.7.3.1’ by use of the extraction protocol file ‘GE1_107_Sep09.xml’.  Measurements of on-chip replicates were averaged using the geometric mean of processed intensity values of the green channel, ‘gProcessedSignal’ (gPS) to retrieve one resulting value per unique non-control probe. Single Features were excluded from averaging, if they i) were manually flagged, ii) were identified as Outliers by the Feature Extraction Software, iii) lay outside the interval of ‘1.42 x interquartile range‘ regarding the normalized gPS distribution of the respective on-chip replicate population, or, iv) showed a coefficient of variation of pixel intensities per Feature that exceeded 0.5.   Averaged gPS values were normalized by global linear scaling: All gPS values of one sample were multiplied by an array-specific scaling factor. This factor was calculated by dividing a ‘reference 75th Percentile value’ (set as 1500 for the whole series) by the 75th Percentile value of the particular Microarray to be normalized (‘Array I’ in the formula shown below). Accordingly, normalized gPS values for all samples (microarray data sets) were calculated by the following formula:   normalized gPSArray i = gPSArray i x (1500 / 75th PercentileArray i)    Finally, a lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed at 5 normalized gPS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 5.

label: Alexa Fluor 555

label_protocol: 200ng of total RNA were used to prepare aminoallyl-UTP-modified (aaUTP) cRNA (Amino Allyl MessageAmp™ II Kit; #AM1753; Thermo Fisher Scientific) applying one-round of amplification as directed by the company, except for a twofold downscaling of all reaction volumes. Just one half of the generated cDNA was used for aaUTP-cRNA synthesis. The labeling of aaUTP-cRNA was performed by use of Alexa Fluor 555 Reactive Dye (#A32756; Thermo Fisher Scientific) as recommended in the manual of the Amino Allyl MessageAmp™ II Kit (twofold downscaled reaction volumes).

growth_protocol: HK2 human tubular cells (ATCC, Manassas, VA) were cultured in full Dulbecco's Modified Eagle Medium: F12 (DMEM, 328mOsm/L, Lonza, Walkersville, MD) supplemented with 10% FCS.

extract_protocol: RNA was isolated using NucleoSpin RNA Plus Kit (Macherey-Nagel, Duren, Germany), yield and purity were determined in a Colibri Microvolume Spectrometer (Titertek-Berthold, Pforzheim, Germany).

treatment_protocol: NaCl, urea, and mannitol (Sigma-Aldrich, Saint Louis, MO) were added in the indicated concentrations.

tag: cell type

value: Renal tubular epithelium

tag: treatment

value: 24h Urea


title: IgG ChIP-seq rep1

value: wild type


title: NuMA (TDIF) ChIP-seq rep1


title: Single cell RNA-seq - embryonic tdTomato MPs, Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato Embryonic Day 16.5

value: Embryonic forelimb segment of the distal stylopod and proximal zeugopod and their articulation (elbow joint).

value: Embryonic Day 16.5


title: AII_Que_3


title: AII_Que_2


title: AII_Que_6


title: HK2 24h NaCl

source_name: HK2 human tubular cells (ATCC, Manassas, VA)

value: 24h NaCl


title: HK2 24h Mannitol

value: 24h Mannitol


title: NuMA (TDIF) Input rep1


title: hiPS brain, several timepoints of neural differentiation

city: Mannheim

first: Philipp

last: Koch

state: Deutschland

institute: Hector Institute for Translational Brain Research

description: boost

library_source: transcriptomic single cell

data_processing:
demultiplexing and feature counting with cellranger count 3.0.1
Assembly: refdata-cellranger-GRCh38-3.0.0
Supplementary files format and content: barcodes.tsv: Barcode sequences correspond to column indices
Supplementary files format and content: features.tsv.gz: Features correspond to row indices. For each feature, its feature ID and name are stored in the first and second column of the (unzipped) features.tsv.gz file, respectively. The third column identifies the type of feature;
Supplementary files format and content: matrix.mtx.gz: Each element of the feature-barcode matrix is the number of UMIs associated with a feature (row) and a barcode (column)

source_name: hiPSC brain

growth_protocol: IPSCs and NPCs were cultured on dishes coated with Geltrex™ (Life Technologies). IPSCs were maintained in DMEM/F12 with glutamine and HEPES (Life Technologies) supplemented with 1% Pen/Strep, 14 ng/ml sodium selenite (Sigma-Aldrich, S5261-10G), 64 µg/ml LAAP (Sigma-Aldrich, A8960-5G), 20 µg/ml insulin (Sigma-Aldrich, 91077C-1G), 2 ng/ml TGF-β1 (Cell Guidance Systems, GFH109), 200 ng/ml FGF-2 (Cell Guidance Systems, GFH146-1000) and 11 µg/ml transferrin (Sigma-Aldrich, T3705-5G). Base medium for NPCs and neurons was DMEM/F12 with glutamine (Life Technologies) containing N2 and B27 supplement (Life Technologies), 1% Pen/Strep, 1x GlutaMAX, 1x NEAA, 800 µg/ml D-glucose. For neural induction, monolayers of iPSCs were cultivated for 8-11 days in NPC base medium supplemented with 500 nM A83-01 (Biomol GmbH, Cay9001799-25), 200 nM LDN193189 (StemCell Technologies, 72148), 2 µM XAV939 (Cell Guidance Systems, SM38-50) and 1 mM cyclopamine (Biomol GmbH, Cay11321-10). NPCs were maintained in neural induction medium without cyclopamine. Neuronal differentiation of NPCs was initiated by switching to neuronal base medium supplemented with 1.8 mM CaCl2, 200 µM ascorbic acid, 1 µM LM22A (Sigma-Aldrich, SML0848-25MG), 1 µM LM22B (Tocris Bioscience, 6037), 2 µM PD-0332991 (Selleck Chemicals, 1116) and 5 µM DAPT (Cell Guidance Systems, SM15-50). The day of this medium change marked day 0 of differentiation. On day 3, cultures were dissociated using TrypLE™ Express (Life Technologies) and re-plated on dishes coated with 0.1 mg/ml poly-L-lysine and 3.75 µg/ml laminin (Sigma-Aldrich, L2020-1MG). Also on day 3, the previous neuronal medium was further supplemented with 3 µM CHIR99021 (Cell Guidance Systems, SM13-50), 10 µM forskolin (Cell Guidance Systems, SM18-100), 300 µM GABA (Sigma-Aldrich, A5835-10G). At day 11, DAPT and GABA and at day 18 CHIR99021 was removed. Subsequently, neurons matured in neuronal base medium containing 1.8 mM CaCl2, 200 µM ascorbic acid, 2 µM PD-0332991, 1 µM LM22A and 1 µM LM22B. All experiments in this study were performed after cultivation of the cells in neuronal base media (without additional additives) for 48 hours.

extract_protocol:
Single cell suspensions were generated by expure to Tryple Express for 10 min.
following instructions from 10X libary preparation protocol for single cell seqencing

value: hiPSC brain

value: pooled


title: hiPSC, d28, CIMHi068, replicate1

data_processing:
Sequencing data was run through an RNAseq processing workflow by the Omics IT and Data Management Core Facility, German Cancer Research Center (DKFZ), as detailed in the 'data_processing_readme.txt' Series supplementary file.
Assembly: 1KGRef_PhiX
Supplementary files format and content: featureCounts.tsv: a matrix containing genes (features) as rows, and count information in columns

extract_protocol: RNA was isolated using TriFast™ (VWR, 30-2010) phenol:chloroform extraction.

tag: time point


title: AML12, Ctrl-1

description: Transfected with pCMV6-AC-GFP plasmid

value: Hepatocytes from a mouse (CD1 strain, line MT42)

value: control


title: hiPSC, d28, CIMHi069, replicate2


title: Single cell RNA-seq - embryonic tdTomato MPs, Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato Embryonic Day 14.5

value: Embryonic Day 14.5


title: hiPSC, d28, CIMHi069, replicate1


title: NuMA (TDIF)-KD Input rep2

description:
ND = No Doxycyclin (WT), PD = Post-doxycyclin (NuMA (TDIF)-KD), In = Input
AP-seq_TSS_TTS_Metagene_Profiles_Matrix.txt
AP-seq_Upstream_Exons_Downstream_Metagene_Profiles_Matrix.txt
AP-seq_Promoter_Counts_Matrix.txt

data_processing:
Data was mapped using BWA 0.7.17-r1188 with parameters -mem -M -k 25
Metagene matrices indicating coverage over the TSS/TTS and gene upstream, exonic, and downstream regions were generated using bam2geneprofile from the CGAT suite of tools with the parameters --reporter = gene, -m = tssprofile/geneprofile --merge-pairs
Counts of reads overlapping promoter regions were determined by creating a window 1000 base pairs upstream and downstream of the TSSs of protein coding genes using bedtools slop on a TSS bed, converting the resulting bed file to a GTF file and running featureCounts (parameters: -t exons -g gene) on the bam files over the promoter gtf
The mapped bam files were converted into bigwig files showing fold change relative to input by first performing narrow peak calling using MACS2 - parameters -g hs -B SPMR with the inputs as the control files. The sample and control lambda (derived from inputs) BEDgraph signal tracks were then compared using MACS2 bdgcomp with the option -m FE in order to generate sample BEDgraph files showing fold change relative to input, which were then converted to bigwigs.
Genome_build: hg38
Supplementary_files_format_and_content: bigwig file for visualising fold change in coverage relative to input, TSS/TTS and gene upstream, exonic, and gene downstream region metagene matrices assessing read density over various genomic features and a matrix containing read counts over promoter regions.
Supplementary_files_format_and_content: Read counts over promoter regions matrix
Supplementary_files_format_and_content: Metagene profile matrix for TSS/TTS regions

source_name: Retinal epithelium cell line

extract_protocol:
DNA was extracted from cells using the Qiagen Blood and Tissue Kit
ChIP libraries were prepared for sequencing using standard Illumina protocols

value: Retina

value: NuMA (TDIF)-KD


title: AII_6

description: WT mice with 28-day Angiotensin-II (500ng/kg/min) treatment

value: renal

value: WT

value: Angiotensin-II

value: Saline

value: AngII


title: IgG ChIP-seq rep2

value: Lung

value: wild type


title: AII_Que_1

value: Quercetin

value: AngII+Que


title: AII_Que_4


title: hiPSC, d28, CIMHi001, replicate2


title: Single cell RNA-seq - embryonic tdTomato MPs, Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato Embryonic Day 13.5

value: Embryonic forelimb

value: Embryonic Day 13.5


title: HK2 24h Ctrl

value: Renal tubular epithelium

value: 24h Ctrl


title: Single cell RNA-seq - embryonic tdTomato MPs, Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato Embryonic Day 12.5

value: Embryonic Day 12.5


title: NuMA (TDIF)-KD Input rep1


title: AII_5


title: Single cell RNA-seq - embryonic tdTomato MPs, Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato Embryonic Day 11.5

value: Embryonic Day 11.5


title: hiPSC, d28, CIMHi001, replicate1

value: hiPSC brain


title: IgG Input rep1


title: AII_4


title: NuMA (TDIF)-KD OGAP-seq rep1

value: Retina

value: NuMA (TDIF)-KD


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


title: hiPSC, d45, replicate2


title: Single cell ATAC-seq - embryonic tdTomato MPs, Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato Embryonic Day 16.5

value: Embryonic Day 16.5

value: Embryonic forelimb segment of the distal stylopod and proximal zeugopod and their articulation (elbow joint).


title: WT Input rep1