title: SH-SY5Y TDP-43 knockdown with cycloheximide treatment - TDP-43 control no cycloheximide - 1

description:
Library name: SHSY5Y_CTRL_CTRL_1
SH-SY5Y cells were transduced with a SmartVector lentivirus (V3IHSHEG_6494503) containing a doxycycline-inducible shRNA cassette for TDP-43. Transduced cells were selected with puromycin (1 μg/ml) for one week, before being plated as single cells and expanded to obtain a clonal population. Cells were grown in DMEM/F12 + Glutamax (Thermo) supplemented with 10% FBS (Thermo) and 1% PenStrep (Thermo). For induction of shRNA against TDP-43, cells were treated with 25 ng/ml doxycyline hyclate (Sigma). After 10 days, cells were treated  either with 100 µM cycloheximide (CHX) or DMSO (Pereverzev et al., 2015) for 6 h before isolating the RNA. Overall, 4 conditions were generated: Control for TDP-43 knockdown and DMSO (control/control), Control for TDP-43 knockdown and CHX-treated (control/CHX), Positive for TDP-43 knockdown and DMSO (TDP43/control) and Positive for TDP-43 knockdown and CHX-treated (TDP43/CHX). RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s protocol, including the on-column DNA digestion step.  RNA concentrations were measured by Nanodrop and 1,000 ng of RNA was used for reverse transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x100bp, > 40 million reads per sample).


title: TBI054_1VST_CD4

status: Public on May 30 2025

type: SRA

city: Chapel Hill

first: Kristina

last: De Paris

state: North Carolina

address: Mary Ellen Jones Building , 116 manning Drive

department: Department of Microbiology and Immunology

country: USA

institute: University of North Carolina at Chapel Hill

description:
Library name: TBI054_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0

library_source: transcriptomic

data_processing:
Raw FASTAQ files after RNA sequencing were processed using the CLC Genomics Workbench 22.0 (CLC Bio, QIAGEN, Aarhus, Denmark).
The raw reads were  trimmed for universal and small RNA adaptors.
Subsequently mapped and aligned to the hg38 reference genome using built-in RNA-seq.
Differential gene expression analysis workflow in CLC genomics with default settings
Assembly: The hg38 reference genome was used to align
Supplementary files format and content: CSV files including  log2 fold change, p value and FDR value.

molecule: total RNA

organism: Homo sapiens

extract_protocol:
PBMC were separated by Ficoll-Hypaque gradient centrifugation and stored as viable PBMC in liquid nitrogen.Later PBMC were thawed, stained with anti-CD3, CD4, CD14, CD16, CD20, and CD56 antibodies and cells were sorted as CD4 and monocytes.otal RNA was extracted from sorted CD4 T cells and monocytes using the Quick RNA Microprep kit (Zymo research CA, USA) according to the manufacturer’s instructions. RNA concentration and quality were assessed using a Qubit fluorometer (Life Technologies, USA) and TapeStation 2200 system (Agilent Technologies, USA).
Libraries were prepared using the Ovation Solo RNA sequence system (NuGen Technology, CA USA) following manufactures protocol and RNA sequencing was performed using the Illumina MiSeq 2x150 bp paired-end sequencing platform.

treatment_protocol: Infants peripheral blood samples was collected at the time of BCG vaccination and 2 to 13 (average: 7.8 weeks±2.9 weeks) post-BCG vaccination when the infants returned for routine pediatric visits.

tag: cell type

tag: treatment

value: Pre BCG vaccination


title: PN0456_0005

city: Milan

first: Edoardo

last: Pedrini

state: Lombardy

address: Via Olgettina 60

department: Division of Neuroscience

country: Italy

institute: Hospital San Raffaele

description: Library name: PN0456_0005_S32

data_processing:
Reads have been trimmed with Cutadapt for adapter sequences and quality and Aligned to the reference genome with STAR
Bam files were counted against the Genecode V31 human GTF annoation of genes (exon and gene_name) using featureCount
Assembly: hg38
Supplementary files format and content: raw count matrices from featureCount with multimapping fractional counts

source_name: umbilical cord blood

growth_protocol: Endothelial colony forming cells (ECFCs) were isolated from umbilical cord blood of full-term pregnancies. Cord-blood mononuclear cells were extracted by density gradient centrifugation and plated on collagen-coated plates (Corning) in EGM-2 medium (Lonza) supplemented with 10% fetal bovine serum (FBS). Plates were cultured in 37oC incubator with 5% CO2. ECFC colonies appeared after 2-3 weeks in culture with cobblestone morphology and immunophenotype confirmed by flow cytometry.

extract_protocol:
Total RNA was isolated using the Maxwell RSC simplyRNA Cells Kit (Promega) and a Maxwell RSC instrument (Promega) following the manufacturer’s instructions. RNA quantity and purity were assessed using the Nanodrop (Thermo Fisher Scientific)and the quality was assessed using the Fragment Analyzer (Agilent Technologies).
Libraries were prepared using KAPA RNA HyperPrep Kit with Poly A enrichment (Roche). Samples were sequenced on a NextSeq 2000 instrument (Illumina) to yield at least 40 million reads per sample, read length 2x50bp PE.

treatment_protocol: ECFCs were transfected with 10nM Silencer select PROCR SiRNA or Scrambled SiRNA control (Thermo Fisher Scientific) at 70% confluency using Lipofectamine RNAiMAX in EGM-2 without antibiotics, for 16 hours. RNA was extracted 48 hrs after transfection.

tag: tissue

value: umbilical cord blood

tag: replicate

tag: clone

value: Scrambled SiRNA control


title: SH-SY5Y TDP-43 knockdown - doxycycline 0 ng/ml - 2

city: Leuven

first: Sam

last: Bryce-Smith

address: Herestraat 49

department: VIB-KU Leuven Center for Brain and Disease Research

country: Belgium

institute: KU Leuven

description:
Library name: SHSY5Y_DOX_0_2
SH-SY5Y cells were transduced with a SmartVector lentivirus (V3IHSHEG_6494503) containing a doxycycline-inducible shRNA cassette for TDP-43. Transduced cells were selected with puromycin (1 μg/ml) for one week, before being plated as single cells and expanded to obtain a clonal population. Cells were grown in DMEM/F12 + Glutamax (Thermo) supplemented with 10% FBS (Thermo) and 1% PenStrep (Thermo). For induction of shRNA against TDP-43, cells were treated with the following amounts of doxycycline hyclate (Sigma) and collected after 10 days: 12.5 ng/ml, 18.75 ng/ml, 21 ng/ml, 25 ng/ml, and 75 ng/ml. RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s protocol, including the on-column DNA digestion step.  RNA concentrations were measured by Nanodrop and 1,000 ng of RNA was used for reverse transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x100bp, > 40 million reads per sample).

data_processing:
Raw reads in FASTQ format were quality trimmed for a minimum Phred score of 10 and otherwise default parameters using fastp (v0.20.1). Quality trimmed reads were aligned to the GRCh38 genome build using gene models from GENCODE v40 with STAR (v2.7.8a).  The  alignment  pipeline is implemented in Snakemake and is available at  https://github.com/frattalab/rna_seq_snakemake  .
Gencode v40 transcripts were quantified by Salmon using a decoy-aware transcriptome index. Salmon v1.8.0 was used with the --posBias, --gcBias and --seqBias flags enabled (otherwise default parameters). Tximport (default parameters) was used to summarise estimated transcript counts to the gene-level and produce raw count matrices.
Assembly: hg38
Supplementary files format and content: comma-delimited text file containing raw gene counts for each sample

molecule: polyA RNA

extract_protocol:
RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription.
Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (SH-SY5Y = 2x100bp, > 40 million reads per sample).

tag: cell line

value: neuroblast


title: TBI053_1VST_CD4

description:
Library name: TBI053_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: SH-SY5Y TDP-43 knockdown - doxycycline 0 ng/ml - 1

description:
Library name: SHSY5Y_DOX_0_1
SH-SY5Y cells were transduced with a SmartVector lentivirus (V3IHSHEG_6494503) containing a doxycycline-inducible shRNA cassette for TDP-43. Transduced cells were selected with puromycin (1 μg/ml) for one week, before being plated as single cells and expanded to obtain a clonal population. Cells were grown in DMEM/F12 + Glutamax (Thermo) supplemented with 10% FBS (Thermo) and 1% PenStrep (Thermo). For induction of shRNA against TDP-43, cells were treated with the following amounts of doxycycline hyclate (Sigma) and collected after 10 days: 12.5 ng/ml, 18.75 ng/ml, 21 ng/ml, 25 ng/ml, and 75 ng/ml. RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s protocol, including the on-column DNA digestion step.  RNA concentrations were measured by Nanodrop and 1,000 ng of RNA was used for reverse transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x100bp, > 40 million reads per sample).


title: SK-N-BE(2) TDP-43 knockdown - doxycycline 1000 ng/ml - 3

description:
Library name: SKNBE2_DOX_1_3
SK-N-BE(2) cells were transduced with a SmartVector lentivirus (V3IHSHEG_6494503) containing a doxycycline-inducible shRNA cassette for TDP-43. Transduced cells were selected with puromycin (1 μg/ml) for one week, before being plated as single cells and expanded to obtain a clonal population. Cells were grown in DMEM/F12 + Glutamax (Thermo) supplemented with 10% FBS (Thermo) and 1% PenStrep (Thermo). For induction of shRNA against TDP-43, cells were treated with the following amounts of doxycycline hyclate (Sigma) and collected after 10 days: 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 75 ng/ml, 100 ng/ml, and 1000 ng/ml. RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s protocol, including the on-column DNA digestion step.  RNA concentrations were measured by Nanodrop and 1,000 ng of RNA was used for reverse transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp, > 40 million reads per sample).

data_processing:
Raw reads in FASTQ format were quality trimmed for a minimum Phred score of 10 and otherwise default parameters using fastp (v0.20.1). Quality trimmed reads were aligned to the GRCh38 genome build using gene models from GENCODE v40 with STAR (v2.7.8a).  The  alignment  pipeline is implemented in Snakemake and is available at  https://github.com/frattalab/rna_seq_snakemake  .
Gencode v40 transcripts were quantified by Salmon using a decoy-aware transcriptome index. Salmon v1.4.0 was used with the --gcBias and --seqBias flags enabled (otherwise default parameters). Tximport (default parameters) was used to summarise estimated transcript counts to the gene-level and produce raw count matrices.
Assembly: hg38
Supplementary files format and content: comma-delimited text file containing raw gene counts for each sample

extract_protocol:
RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription.
Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (SK-N-BE(2) = 2x150bp, > 40 million reads per sample).


title: TBI049_2VST_CD4

description:
Library name: TBI049_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0

value: Post BCG vaccination


title: TBI049_1VST_CD4

description:
Library name: TBI049_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: PN0456_0003

description: Library name: PN0456_0003_S30

value: Endothelial colony forming cells (ECFCs)


title: SK-N-BE(2) TDP-43 knockdown - doxycycline 1000 ng/ml - 2

description:
Library name: SKNBE2_DOX_1_2
SK-N-BE(2) cells were transduced with a SmartVector lentivirus (V3IHSHEG_6494503) containing a doxycycline-inducible shRNA cassette for TDP-43. Transduced cells were selected with puromycin (1 μg/ml) for one week, before being plated as single cells and expanded to obtain a clonal population. Cells were grown in DMEM/F12 + Glutamax (Thermo) supplemented with 10% FBS (Thermo) and 1% PenStrep (Thermo). For induction of shRNA against TDP-43, cells were treated with the following amounts of doxycycline hyclate (Sigma) and collected after 10 days: 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 75 ng/ml, 100 ng/ml, and 1000 ng/ml. RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s protocol, including the on-column DNA digestion step.  RNA concentrations were measured by Nanodrop and 1,000 ng of RNA was used for reverse transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp, > 40 million reads per sample).


title: TBI043_1VST_CD4

description:
Library name: TBI043_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI043_2VST_CD4

description:
Library name: TBI043_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: PN0456_0001

description: Library name: PN0456_0001_S28


title: SK-N-BE(2) TDP-43 knockdown - doxycycline 1000 ng/ml - 1

description:
Library name: SKNBE2_DOX_1_1
SK-N-BE(2) cells were transduced with a SmartVector lentivirus (V3IHSHEG_6494503) containing a doxycycline-inducible shRNA cassette for TDP-43. Transduced cells were selected with puromycin (1 μg/ml) for one week, before being plated as single cells and expanded to obtain a clonal population. Cells were grown in DMEM/F12 + Glutamax (Thermo) supplemented with 10% FBS (Thermo) and 1% PenStrep (Thermo). For induction of shRNA against TDP-43, cells were treated with the following amounts of doxycycline hyclate (Sigma) and collected after 10 days: 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 75 ng/ml, 100 ng/ml, and 1000 ng/ml. RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s protocol, including the on-column DNA digestion step.  RNA concentrations were measured by Nanodrop and 1,000 ng of RNA was used for reverse transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp, > 40 million reads per sample).


title: TBI047_2VST_CD4

description:
Library name: TBI047_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI048_2VST_CD4

description:
Library name: TBI048_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: PN0456_0002

description: Library name: PN0456_0002_S29

value: PROCR SiRNA


title: SK-N-BE(2) TDP-43 knockdown - doxycycline 0 ng/ml - 3

description:
Library name: SKNBE2_DOX_0_3
SK-N-BE(2) cells were transduced with a SmartVector lentivirus (V3IHSHEG_6494503) containing a doxycycline-inducible shRNA cassette for TDP-43. Transduced cells were selected with puromycin (1 μg/ml) for one week, before being plated as single cells and expanded to obtain a clonal population. Cells were grown in DMEM/F12 + Glutamax (Thermo) supplemented with 10% FBS (Thermo) and 1% PenStrep (Thermo). For induction of shRNA against TDP-43, cells were treated with the following amounts of doxycycline hyclate (Sigma) and collected after 10 days: 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 75 ng/ml, 100 ng/ml, and 1000 ng/ml. RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s protocol, including the on-column DNA digestion step.  RNA concentrations were measured by Nanodrop and 1,000 ng of RNA was used for reverse transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp, > 40 million reads per sample).


title: TBI038_2VST_CD4

description:
Library name: TBI038_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI042_1VST_CD4

description:
Library name: TBI042_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI042_2VST_CD4

description:
Library name: TBI042_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI038_1VST_CD4

description:
Library name: TBI038_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: SK-N-BE(2) TDP-43 knockdown - doxycycline 0 ng/ml - 2

description:
Library name: SKNBE2_DOX_0_2
SK-N-BE(2) cells were transduced with a SmartVector lentivirus (V3IHSHEG_6494503) containing a doxycycline-inducible shRNA cassette for TDP-43. Transduced cells were selected with puromycin (1 μg/ml) for one week, before being plated as single cells and expanded to obtain a clonal population. Cells were grown in DMEM/F12 + Glutamax (Thermo) supplemented with 10% FBS (Thermo) and 1% PenStrep (Thermo). For induction of shRNA against TDP-43, cells were treated with the following amounts of doxycycline hyclate (Sigma) and collected after 10 days: 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 75 ng/ml, 100 ng/ml, and 1000 ng/ml. RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s protocol, including the on-column DNA digestion step.  RNA concentrations were measured by Nanodrop and 1,000 ng of RNA was used for reverse transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp, > 40 million reads per sample).


title: TBI053_2VST_CD4

description:
Library name: TBI053_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI036_2VST_CD4

description:
Library name: TBI036_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: SK-N-BE(2) TDP-43 knockdown - doxycycline 0 ng/ml - 1

description:
Library name: SKNBE2_DOX_0_1
SK-N-BE(2) cells were transduced with a SmartVector lentivirus (V3IHSHEG_6494503) containing a doxycycline-inducible shRNA cassette for TDP-43. Transduced cells were selected with puromycin (1 μg/ml) for one week, before being plated as single cells and expanded to obtain a clonal population. Cells were grown in DMEM/F12 + Glutamax (Thermo) supplemented with 10% FBS (Thermo) and 1% PenStrep (Thermo). For induction of shRNA against TDP-43, cells were treated with the following amounts of doxycycline hyclate (Sigma) and collected after 10 days: 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 75 ng/ml, 100 ng/ml, and 1000 ng/ml. RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s protocol, including the on-column DNA digestion step.  RNA concentrations were measured by Nanodrop and 1,000 ng of RNA was used for reverse transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp, > 40 million reads per sample).


title: PN0456_0004

description: Library name: PN0456_0004_S31


title: TBI036_1VST_CD4

description:
Library name: TBI036_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: i3Neuron TDP-43/UPF1 knockdown - TDP-43 knockdown UPF1 control - rep4

description:
Library name: i3Neuron_TDP43_CTRL_4
Human iPS cells used in this study were engineered (Fernandopulle et al., 2018; Wang et al., 2017) to express mouse or human neurogenin-2 under a doxycycline-inducible promoter, as well as an enzymatically dead Cas9 (+/− CAG-dCas9-BFP-KRAB) (Tian et al., 2019). To achieve knockdown, sgRNAs targeting either a non-targeting control guide (GTCCACCCTTATCTAGGCTA), UPF1 (GGCCAGACGCAGACGCCCCC), or TARDBP (GGGAAGTCAGCCGTGAGACC) were delivered to iPS cells by lentiviral transduction (Lois et al., 2002), for a total of 4 groups: Control for TDP-43 and UPF1 knockdown (control/control), Control for TDP-43 knockdown and positive for UPF1 knockdown (control/UPF1KD), Positive for TDP-43 knockdown and control for UPF1 knockdown (TDP43KD/control), Positive for TDP-43 and UPF1 knockdown (TDP43KD/UPF1KD). After lentiviral transduction, cells were plated in tissue culture dishes coated with human embryonic stem cell-qualified Matrigel (Corning). The following morning, cells were washed with PBS and the media was changed to E8 alone or (depending on cell number) E8 supplemented with 10 µM ROCK inhibitor in a 37°C, 5% CO2 incubator. Cells were passaged with accutase (Life Technologies) for 5–10 min at 37 °C and then washed with PBS before re-plating. Two days after lentiviral delivery, cells were selected overnight with either puromycin (10 µg/ml) or blasticidin (50–100 µg/ml). iPS cells were then expanded for 2 days before initiating neuronal differentiation. To initiate neuronal differentiation, 20–25 million iPS cells per 15 cm plate were individualised using accutase on day 0 and re-plated onto Matrigel-coated tissue culture dishes in N2 differentiation media containing: knockout DMEM/F12 media (Life Technologies Corporation) with N2 supplement (Life Technologies Corporation), 1× GlutaMAX (Thermofisher Scientific), 1× MEM nonessential amino acids (Thermofisher Scientific), 10 µM ROCK inhibitor (Selleckchem) and 2 µg/ml doxycycline (Clontech). Media was changed daily during this stage. On day 3 pre-neuron cells were replated onto dishes coated with freshly made poly-L-ornithine (0.1 mg/ml; Sigma), 6-well dishes (2 million per well), in i3Neuron Culture Media: Brain-Phys media (Stemcell Technologies) supplemented with 1× B27 Plus Supplement (ThermoFisher Scientific), 10 ng/ml BDNF (PeproTech), 10 ng/ml NT-3 (PeproTech), 1 µg/ml mouse laminin (Sigma), and 2 µg/ml doxycycline (Clontech). i3Neurons were then fed three times a week by half media changes. i3Neurons were then collected on day 7 after the addition of doxycycline. RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp,  > 40 million reads per sample).

extract_protocol:
RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription.
Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (iPSC-derived cortical neurons = 2x150bp, > 40 million reads per sample)

value: iPSC-derived cortical neuron


title: TBI035_1VST_CD4

description:
Library name: TBI035_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI035_2VST_CD4

description:
Library name: TBI035_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI032_1VST_CD4

description:
Library name: TBI032_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: i3Neuron TDP-43/UPF1 knockdown - TDP-43 knockdown UPF1 control - rep3

description:
Library name: i3Neuron_TDP43_CTRL_3
Human iPS cells used in this study were engineered (Fernandopulle et al., 2018; Wang et al., 2017) to express mouse or human neurogenin-2 under a doxycycline-inducible promoter, as well as an enzymatically dead Cas9 (+/− CAG-dCas9-BFP-KRAB) (Tian et al., 2019). To achieve knockdown, sgRNAs targeting either a non-targeting control guide (GTCCACCCTTATCTAGGCTA), UPF1 (GGCCAGACGCAGACGCCCCC), or TARDBP (GGGAAGTCAGCCGTGAGACC) were delivered to iPS cells by lentiviral transduction (Lois et al., 2002), for a total of 4 groups: Control for TDP-43 and UPF1 knockdown (control/control), Control for TDP-43 knockdown and positive for UPF1 knockdown (control/UPF1KD), Positive for TDP-43 knockdown and control for UPF1 knockdown (TDP43KD/control), Positive for TDP-43 and UPF1 knockdown (TDP43KD/UPF1KD). After lentiviral transduction, cells were plated in tissue culture dishes coated with human embryonic stem cell-qualified Matrigel (Corning). The following morning, cells were washed with PBS and the media was changed to E8 alone or (depending on cell number) E8 supplemented with 10 µM ROCK inhibitor in a 37°C, 5% CO2 incubator. Cells were passaged with accutase (Life Technologies) for 5–10 min at 37 °C and then washed with PBS before re-plating. Two days after lentiviral delivery, cells were selected overnight with either puromycin (10 µg/ml) or blasticidin (50–100 µg/ml). iPS cells were then expanded for 2 days before initiating neuronal differentiation. To initiate neuronal differentiation, 20–25 million iPS cells per 15 cm plate were individualised using accutase on day 0 and re-plated onto Matrigel-coated tissue culture dishes in N2 differentiation media containing: knockout DMEM/F12 media (Life Technologies Corporation) with N2 supplement (Life Technologies Corporation), 1× GlutaMAX (Thermofisher Scientific), 1× MEM nonessential amino acids (Thermofisher Scientific), 10 µM ROCK inhibitor (Selleckchem) and 2 µg/ml doxycycline (Clontech). Media was changed daily during this stage. On day 3 pre-neuron cells were replated onto dishes coated with freshly made poly-L-ornithine (0.1 mg/ml; Sigma), 6-well dishes (2 million per well), in i3Neuron Culture Media: Brain-Phys media (Stemcell Technologies) supplemented with 1× B27 Plus Supplement (ThermoFisher Scientific), 10 ng/ml BDNF (PeproTech), 10 ng/ml NT-3 (PeproTech), 1 µg/ml mouse laminin (Sigma), and 2 µg/ml doxycycline (Clontech). i3Neurons were then fed three times a week by half media changes. i3Neurons were then collected on day 7 after the addition of doxycycline. RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp,  > 40 million reads per sample).


title: TBI026_2VST_CD4

description:
Library name: TBI026_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI025_2VST_CD4

description:
Library name: TBI025_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: i3Neuron TDP-43/UPF1 knockdown - TDP-43 knockdown UPF1 control - rep2

description:
Library name: i3Neuron_TDP43_CTRL_2
Human iPS cells used in this study were engineered (Fernandopulle et al., 2018; Wang et al., 2017) to express mouse or human neurogenin-2 under a doxycycline-inducible promoter, as well as an enzymatically dead Cas9 (+/− CAG-dCas9-BFP-KRAB) (Tian et al., 2019). To achieve knockdown, sgRNAs targeting either a non-targeting control guide (GTCCACCCTTATCTAGGCTA), UPF1 (GGCCAGACGCAGACGCCCCC), or TARDBP (GGGAAGTCAGCCGTGAGACC) were delivered to iPS cells by lentiviral transduction (Lois et al., 2002), for a total of 4 groups: Control for TDP-43 and UPF1 knockdown (control/control), Control for TDP-43 knockdown and positive for UPF1 knockdown (control/UPF1KD), Positive for TDP-43 knockdown and control for UPF1 knockdown (TDP43KD/control), Positive for TDP-43 and UPF1 knockdown (TDP43KD/UPF1KD). After lentiviral transduction, cells were plated in tissue culture dishes coated with human embryonic stem cell-qualified Matrigel (Corning). The following morning, cells were washed with PBS and the media was changed to E8 alone or (depending on cell number) E8 supplemented with 10 µM ROCK inhibitor in a 37°C, 5% CO2 incubator. Cells were passaged with accutase (Life Technologies) for 5–10 min at 37 °C and then washed with PBS before re-plating. Two days after lentiviral delivery, cells were selected overnight with either puromycin (10 µg/ml) or blasticidin (50–100 µg/ml). iPS cells were then expanded for 2 days before initiating neuronal differentiation. To initiate neuronal differentiation, 20–25 million iPS cells per 15 cm plate were individualised using accutase on day 0 and re-plated onto Matrigel-coated tissue culture dishes in N2 differentiation media containing: knockout DMEM/F12 media (Life Technologies Corporation) with N2 supplement (Life Technologies Corporation), 1× GlutaMAX (Thermofisher Scientific), 1× MEM nonessential amino acids (Thermofisher Scientific), 10 µM ROCK inhibitor (Selleckchem) and 2 µg/ml doxycycline (Clontech). Media was changed daily during this stage. On day 3 pre-neuron cells were replated onto dishes coated with freshly made poly-L-ornithine (0.1 mg/ml; Sigma), 6-well dishes (2 million per well), in i3Neuron Culture Media: Brain-Phys media (Stemcell Technologies) supplemented with 1× B27 Plus Supplement (ThermoFisher Scientific), 10 ng/ml BDNF (PeproTech), 10 ng/ml NT-3 (PeproTech), 1 µg/ml mouse laminin (Sigma), and 2 µg/ml doxycycline (Clontech). i3Neurons were then fed three times a week by half media changes. i3Neurons were then collected on day 7 after the addition of doxycycline. RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp,  > 40 million reads per sample).


title: TBI026_1VST_CD4

description:
Library name: TBI026_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI025_1VST_CD4

description:
Library name: TBI025_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: EV, PDAC 3

type: RNA

city: Asahikawa

first: Kenji

last: Takahashi

state: Hokkaido

address: 2-1-1-1, Midorigaokahigashi

department: Department of Medicine

country: Japan

institute: Asahikawa Medical University

description: EV RNA was isolated from serum EVs

hyb_protocol: Affymetrix GeneChip WT PLUS Reagent Kit User Manuall（Affymetrix 703174 Rev.2）

scan_protocol: GeneChip were scanned by GeneChip Scaner 3000(Affymetrix 690036) using GeneChip Command Console Software ver3.2(Affymetrix 702569). The procedure of scan is followed by  Affymetrix GeneChip WT PLUS Reagent Kit User Manual（Affymetrix 703174 Rev.2）

data_processing: Data is processed by RMA-Skech algorythm using Expression ConsoleTM Softwere ver.1.3（Affymetrix 702387）

label: biotin

source_name: serum

label_protocol: The procedure of labeling is followed by Affymetrix GeneChip WT PLUS Reagent Kit User Manual（Affymetrix 703174 Rev.2）. 100ng Total RNA was used for this assay.

extract_protocol: serum was isolated from blood samples

tag: individual

value: PDAC patient

tag: sample type

value: serum EVs

tag: fraction

value: EV RNA


title: EV, PDAC 1

description: EV RNA was isolated from serum EVs


title: i3Neuron TDP-43/UPF1 knockdown - TDP-43 control UPF1 control - rep4

description:
Library name: i3Neuron_CTRL_CTRL_4
Human iPS cells used in this study were engineered (Fernandopulle et al., 2018; Wang et al., 2017) to express mouse or human neurogenin-2 under a doxycycline-inducible promoter, as well as an enzymatically dead Cas9 (+/− CAG-dCas9-BFP-KRAB) (Tian et al., 2019). To achieve knockdown, sgRNAs targeting either a non-targeting control guide (GTCCACCCTTATCTAGGCTA), UPF1 (GGCCAGACGCAGACGCCCCC), or TARDBP (GGGAAGTCAGCCGTGAGACC) were delivered to iPS cells by lentiviral transduction (Lois et al., 2002), for a total of 4 groups: Control for TDP-43 and UPF1 knockdown (control/control), Control for TDP-43 knockdown and positive for UPF1 knockdown (control/UPF1KD), Positive for TDP-43 knockdown and control for UPF1 knockdown (TDP43KD/control), Positive for TDP-43 and UPF1 knockdown (TDP43KD/UPF1KD). After lentiviral transduction, cells were plated in tissue culture dishes coated with human embryonic stem cell-qualified Matrigel (Corning). The following morning, cells were washed with PBS and the media was changed to E8 alone or (depending on cell number) E8 supplemented with 10 µM ROCK inhibitor in a 37°C, 5% CO2 incubator. Cells were passaged with accutase (Life Technologies) for 5–10 min at 37 °C and then washed with PBS before re-plating. Two days after lentiviral delivery, cells were selected overnight with either puromycin (10 µg/ml) or blasticidin (50–100 µg/ml). iPS cells were then expanded for 2 days before initiating neuronal differentiation. To initiate neuronal differentiation, 20–25 million iPS cells per 15 cm plate were individualised using accutase on day 0 and re-plated onto Matrigel-coated tissue culture dishes in N2 differentiation media containing: knockout DMEM/F12 media (Life Technologies Corporation) with N2 supplement (Life Technologies Corporation), 1× GlutaMAX (Thermofisher Scientific), 1× MEM nonessential amino acids (Thermofisher Scientific), 10 µM ROCK inhibitor (Selleckchem) and 2 µg/ml doxycycline (Clontech). Media was changed daily during this stage. On day 3 pre-neuron cells were replated onto dishes coated with freshly made poly-L-ornithine (0.1 mg/ml; Sigma), 6-well dishes (2 million per well), in i3Neuron Culture Media: Brain-Phys media (Stemcell Technologies) supplemented with 1× B27 Plus Supplement (ThermoFisher Scientific), 10 ng/ml BDNF (PeproTech), 10 ng/ml NT-3 (PeproTech), 1 µg/ml mouse laminin (Sigma), and 2 µg/ml doxycycline (Clontech). i3Neurons were then fed three times a week by half media changes. i3Neurons were then collected on day 7 after the addition of doxycycline. RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp,  > 40 million reads per sample).


title: i3Neuron TDP-43/UPF1 knockdown - TDP-43 knockdown UPF1 control - rep1

description:
Library name: i3Neuron_TDP43_CTRL_1
Human iPS cells used in this study were engineered (Fernandopulle et al., 2018; Wang et al., 2017) to express mouse or human neurogenin-2 under a doxycycline-inducible promoter, as well as an enzymatically dead Cas9 (+/− CAG-dCas9-BFP-KRAB) (Tian et al., 2019). To achieve knockdown, sgRNAs targeting either a non-targeting control guide (GTCCACCCTTATCTAGGCTA), UPF1 (GGCCAGACGCAGACGCCCCC), or TARDBP (GGGAAGTCAGCCGTGAGACC) were delivered to iPS cells by lentiviral transduction (Lois et al., 2002), for a total of 4 groups: Control for TDP-43 and UPF1 knockdown (control/control), Control for TDP-43 knockdown and positive for UPF1 knockdown (control/UPF1KD), Positive for TDP-43 knockdown and control for UPF1 knockdown (TDP43KD/control), Positive for TDP-43 and UPF1 knockdown (TDP43KD/UPF1KD). After lentiviral transduction, cells were plated in tissue culture dishes coated with human embryonic stem cell-qualified Matrigel (Corning). The following morning, cells were washed with PBS and the media was changed to E8 alone or (depending on cell number) E8 supplemented with 10 µM ROCK inhibitor in a 37°C, 5% CO2 incubator. Cells were passaged with accutase (Life Technologies) for 5–10 min at 37 °C and then washed with PBS before re-plating. Two days after lentiviral delivery, cells were selected overnight with either puromycin (10 µg/ml) or blasticidin (50–100 µg/ml). iPS cells were then expanded for 2 days before initiating neuronal differentiation. To initiate neuronal differentiation, 20–25 million iPS cells per 15 cm plate were individualised using accutase on day 0 and re-plated onto Matrigel-coated tissue culture dishes in N2 differentiation media containing: knockout DMEM/F12 media (Life Technologies Corporation) with N2 supplement (Life Technologies Corporation), 1× GlutaMAX (Thermofisher Scientific), 1× MEM nonessential amino acids (Thermofisher Scientific), 10 µM ROCK inhibitor (Selleckchem) and 2 µg/ml doxycycline (Clontech). Media was changed daily during this stage. On day 3 pre-neuron cells were replated onto dishes coated with freshly made poly-L-ornithine (0.1 mg/ml; Sigma), 6-well dishes (2 million per well), in i3Neuron Culture Media: Brain-Phys media (Stemcell Technologies) supplemented with 1× B27 Plus Supplement (ThermoFisher Scientific), 10 ng/ml BDNF (PeproTech), 10 ng/ml NT-3 (PeproTech), 1 µg/ml mouse laminin (Sigma), and 2 µg/ml doxycycline (Clontech). i3Neurons were then fed three times a week by half media changes. i3Neurons were then collected on day 7 after the addition of doxycycline. RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp,  > 40 million reads per sample).


title: TBI024_2VST_CD4

description:
Library name: TBI024_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI022_1VST_CD4

description:
Library name: TBI022_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI019_1VST_CD4

description:
Library name: TBI019_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: EV, PDAC 2

description: EV RNA was isolated from serum EVs


title: EV, control 3

description: EV RNA was isolated from serum EVs

value: control


title: TBI019_2VST_CD4

description:
Library name: TBI019_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI017_2VST_CD4

description:
Library name: TBI017_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI017_1VST_CD4

description:
Library name: TBI017_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: i3Neuron TDP-43/UPF1 knockdown - TDP-43 control UPF1 control - rep3

description:
Library name: i3Neuron_CTRL_CTRL_3
Human iPS cells used in this study were engineered (Fernandopulle et al., 2018; Wang et al., 2017) to express mouse or human neurogenin-2 under a doxycycline-inducible promoter, as well as an enzymatically dead Cas9 (+/− CAG-dCas9-BFP-KRAB) (Tian et al., 2019). To achieve knockdown, sgRNAs targeting either a non-targeting control guide (GTCCACCCTTATCTAGGCTA), UPF1 (GGCCAGACGCAGACGCCCCC), or TARDBP (GGGAAGTCAGCCGTGAGACC) were delivered to iPS cells by lentiviral transduction (Lois et al., 2002), for a total of 4 groups: Control for TDP-43 and UPF1 knockdown (control/control), Control for TDP-43 knockdown and positive for UPF1 knockdown (control/UPF1KD), Positive for TDP-43 knockdown and control for UPF1 knockdown (TDP43KD/control), Positive for TDP-43 and UPF1 knockdown (TDP43KD/UPF1KD). After lentiviral transduction, cells were plated in tissue culture dishes coated with human embryonic stem cell-qualified Matrigel (Corning). The following morning, cells were washed with PBS and the media was changed to E8 alone or (depending on cell number) E8 supplemented with 10 µM ROCK inhibitor in a 37°C, 5% CO2 incubator. Cells were passaged with accutase (Life Technologies) for 5–10 min at 37 °C and then washed with PBS before re-plating. Two days after lentiviral delivery, cells were selected overnight with either puromycin (10 µg/ml) or blasticidin (50–100 µg/ml). iPS cells were then expanded for 2 days before initiating neuronal differentiation. To initiate neuronal differentiation, 20–25 million iPS cells per 15 cm plate were individualised using accutase on day 0 and re-plated onto Matrigel-coated tissue culture dishes in N2 differentiation media containing: knockout DMEM/F12 media (Life Technologies Corporation) with N2 supplement (Life Technologies Corporation), 1× GlutaMAX (Thermofisher Scientific), 1× MEM nonessential amino acids (Thermofisher Scientific), 10 µM ROCK inhibitor (Selleckchem) and 2 µg/ml doxycycline (Clontech). Media was changed daily during this stage. On day 3 pre-neuron cells were replated onto dishes coated with freshly made poly-L-ornithine (0.1 mg/ml; Sigma), 6-well dishes (2 million per well), in i3Neuron Culture Media: Brain-Phys media (Stemcell Technologies) supplemented with 1× B27 Plus Supplement (ThermoFisher Scientific), 10 ng/ml BDNF (PeproTech), 10 ng/ml NT-3 (PeproTech), 1 µg/ml mouse laminin (Sigma), and 2 µg/ml doxycycline (Clontech). i3Neurons were then fed three times a week by half media changes. i3Neurons were then collected on day 7 after the addition of doxycycline. RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp,  > 40 million reads per sample).


title: i3Neuron TDP-43/UPF1 knockdown - TDP-43 control UPF1 control - rep2

description:
Library name: i3Neuron_CTRL_CTRL_2
Human iPS cells used in this study were engineered (Fernandopulle et al., 2018; Wang et al., 2017) to express mouse or human neurogenin-2 under a doxycycline-inducible promoter, as well as an enzymatically dead Cas9 (+/− CAG-dCas9-BFP-KRAB) (Tian et al., 2019). To achieve knockdown, sgRNAs targeting either a non-targeting control guide (GTCCACCCTTATCTAGGCTA), UPF1 (GGCCAGACGCAGACGCCCCC), or TARDBP (GGGAAGTCAGCCGTGAGACC) were delivered to iPS cells by lentiviral transduction (Lois et al., 2002), for a total of 4 groups: Control for TDP-43 and UPF1 knockdown (control/control), Control for TDP-43 knockdown and positive for UPF1 knockdown (control/UPF1KD), Positive for TDP-43 knockdown and control for UPF1 knockdown (TDP43KD/control), Positive for TDP-43 and UPF1 knockdown (TDP43KD/UPF1KD). After lentiviral transduction, cells were plated in tissue culture dishes coated with human embryonic stem cell-qualified Matrigel (Corning). The following morning, cells were washed with PBS and the media was changed to E8 alone or (depending on cell number) E8 supplemented with 10 µM ROCK inhibitor in a 37°C, 5% CO2 incubator. Cells were passaged with accutase (Life Technologies) for 5–10 min at 37 °C and then washed with PBS before re-plating. Two days after lentiviral delivery, cells were selected overnight with either puromycin (10 µg/ml) or blasticidin (50–100 µg/ml). iPS cells were then expanded for 2 days before initiating neuronal differentiation. To initiate neuronal differentiation, 20–25 million iPS cells per 15 cm plate were individualised using accutase on day 0 and re-plated onto Matrigel-coated tissue culture dishes in N2 differentiation media containing: knockout DMEM/F12 media (Life Technologies Corporation) with N2 supplement (Life Technologies Corporation), 1× GlutaMAX (Thermofisher Scientific), 1× MEM nonessential amino acids (Thermofisher Scientific), 10 µM ROCK inhibitor (Selleckchem) and 2 µg/ml doxycycline (Clontech). Media was changed daily during this stage. On day 3 pre-neuron cells were replated onto dishes coated with freshly made poly-L-ornithine (0.1 mg/ml; Sigma), 6-well dishes (2 million per well), in i3Neuron Culture Media: Brain-Phys media (Stemcell Technologies) supplemented with 1× B27 Plus Supplement (ThermoFisher Scientific), 10 ng/ml BDNF (PeproTech), 10 ng/ml NT-3 (PeproTech), 1 µg/ml mouse laminin (Sigma), and 2 µg/ml doxycycline (Clontech). i3Neurons were then fed three times a week by half media changes. i3Neurons were then collected on day 7 after the addition of doxycycline. RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp,  > 40 million reads per sample).


title: EV, control 2

description: EV RNA was isolated from serum EVs


title: i3Neuron TDP-43/UPF1 knockdown - TDP-43 control UPF1 control - rep1

description:
Library name: i3Neuron_CTRL_CTRL_1
Human iPS cells used in this study were engineered (Fernandopulle et al., 2018; Wang et al., 2017) to express mouse or human neurogenin-2 under a doxycycline-inducible promoter, as well as an enzymatically dead Cas9 (+/− CAG-dCas9-BFP-KRAB) (Tian et al., 2019). To achieve knockdown, sgRNAs targeting either a non-targeting control guide (GTCCACCCTTATCTAGGCTA), UPF1 (GGCCAGACGCAGACGCCCCC), or TARDBP (GGGAAGTCAGCCGTGAGACC) were delivered to iPS cells by lentiviral transduction (Lois et al., 2002), for a total of 4 groups: Control for TDP-43 and UPF1 knockdown (control/control), Control for TDP-43 knockdown and positive for UPF1 knockdown (control/UPF1KD), Positive for TDP-43 knockdown and control for UPF1 knockdown (TDP43KD/control), Positive for TDP-43 and UPF1 knockdown (TDP43KD/UPF1KD). After lentiviral transduction, cells were plated in tissue culture dishes coated with human embryonic stem cell-qualified Matrigel (Corning). The following morning, cells were washed with PBS and the media was changed to E8 alone or (depending on cell number) E8 supplemented with 10 µM ROCK inhibitor in a 37°C, 5% CO2 incubator. Cells were passaged with accutase (Life Technologies) for 5–10 min at 37 °C and then washed with PBS before re-plating. Two days after lentiviral delivery, cells were selected overnight with either puromycin (10 µg/ml) or blasticidin (50–100 µg/ml). iPS cells were then expanded for 2 days before initiating neuronal differentiation. To initiate neuronal differentiation, 20–25 million iPS cells per 15 cm plate were individualised using accutase on day 0 and re-plated onto Matrigel-coated tissue culture dishes in N2 differentiation media containing: knockout DMEM/F12 media (Life Technologies Corporation) with N2 supplement (Life Technologies Corporation), 1× GlutaMAX (Thermofisher Scientific), 1× MEM nonessential amino acids (Thermofisher Scientific), 10 µM ROCK inhibitor (Selleckchem) and 2 µg/ml doxycycline (Clontech). Media was changed daily during this stage. On day 3 pre-neuron cells were replated onto dishes coated with freshly made poly-L-ornithine (0.1 mg/ml; Sigma), 6-well dishes (2 million per well), in i3Neuron Culture Media: Brain-Phys media (Stemcell Technologies) supplemented with 1× B27 Plus Supplement (ThermoFisher Scientific), 10 ng/ml BDNF (PeproTech), 10 ng/ml NT-3 (PeproTech), 1 µg/ml mouse laminin (Sigma), and 2 µg/ml doxycycline (Clontech). i3Neurons were then fed three times a week by half media changes. i3Neurons were then collected on day 7 after the addition of doxycycline. RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturer’s  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp,  > 40 million reads per sample).


title: TBI016_1VST_CD4

description:
Library name: TBI016_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI016_2VST_CD4

description:
Library name: TBI016_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: EV, control 1

description: EV RNA was isolated from serum EVs


title: TBI015_2VST_CD4

description:
Library name: TBI015_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI013_2VST_CD4

description:
Library name: TBI013_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI015_1VST_CD4

description:
Library name: TBI015_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: Subj.PNMB233_Day7

city: Palo Alto

first: Dmitri

middle: A

last: Kazmin

state: CA

address: 240 Pasteur Dr

department: Institute for Immunity, Transplantation and Infection

institute: Stanford University

hyb_protocol: Samples were hybridized on  HG-133-plus-PM plates

scan_protocol: Plates were scanned on GeneTitan multichannel instrument (Affymetrix)

data_processing: Probe level processing was done by RMA with log2 transform and probe summary by median polish

label: Biotin

source_name: PBMC

label_protocol: Fragemented cRNA was lebeled with biotin

extract_protocol: RNA was isolated from whole blood Paxgene tubes using RNAeasy kit (Quagen)

tag: subject

tag: time point


title: Subj.PNMB233_Day0


title: Subj.PNMB232_Day0


title: Subj.PNMB232_Day7


title: Jurkat cells, BMP4+BMP7, PHA-activated, rep1

city: Wuhan

first: Zilun

last: RUAN

state: Hubei

address: Donghu Road, No. 115, Wuchang District

department: Department of Infectious Diseases, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University

country: China

institute: Wuhan University

description: Library name: E7

data_processing:
FASTQ files were first trimmed using Trim_galore (v0.6.9) to remove sequencing adapters and low-quality reads (Q<15). Trimmed sequencing reads were aligned to the human Hg38 reference genome (GENCODE, GRCh37.p13) using STAR (v2.7.10a). SAM files were subsequently converted to BAM files, sorted, and indexed using samtools (v1.15). BAM files were used to generate bigwig files using bamCoverage (part of the Deeptools package; v3.5.1). Read counting across genomic features was performed using featureCounts (part of the subread package; v2.0.1).
Assembly: hg38
Supplementary files format and content: csv file includes raw counts for each sample

source_name: Blood

extract_protocol:
Approximately 1×107 cells were used for RNA extraction, and DNA digestion was carried out by DNaseI after RNA extraction. The quality of RNA was determined by examining A260/A280 and 1.5% agarose gel electrophoresis.
Qualified RNAs were finally quantified by Qubit3.0 with QubitTM RNA Broad Range Assay kit (Life Technologies). Total RNAs (2 mg) were used for stranded RNA sequencing library preparation, PCR products corresponding to 200-500 bps were enriched, quantified and finally sequenced on DNBSEQ-T7 sequencer (MGI Tech Co., Ltd. China) with PE150 model.

value: Blood

value: Jurkat

value: Human T lymphocyte cells

tag: genotype

value: Wild type


title: TBI008_1VST_CD4

description:
Library name: TBI008_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: Jurkat cells, untreated, PHA-activated, rep2

description: Library name: E6


title: TBI004_2VST_CD4

description:
Library name: TBI004_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: TBI000_2VST_CD4

description:
Library name: TBI000_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: Subj.PNMC330_Day0


title: TBI000_1VST_CD4

description:
Library name: TBI000_1VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0


title: Primary Human Hepatocytes, Replicate1

city: Sao Paulo

first: Kayque

last: Alves Telles Silva

address: 106 Matao St.

country: Brazil

institute: University of Sao Paulo

description: Library name: Sample 7

data_processing:
Raw data (raw reads) of fastq format were processed through in-house NovoGene's perl scripts.
Assembly: hg38
Supplementary files format and content: Tab-delimited text files include raw counts for each sample.

source_name: Human Plateable Hepatocytes, 5-Donor

extract_protocol:
RNA was harvested using the PureLink RNA Mini Kit (Thermo Fisher Scientific). 1 ug of RNA was used for the construction of the sequencing libraries.
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification.

value: Human Plateable Hepatocytes, 5-Donor


title: Hepatocyte-like cells, Etoposide, Day5, Replicate2

description: Library name: Sample 5

value: Hepatocyte-like cells


title: Primary Human Hepatocytes, Replicate3

description: Library name: Sample 9


title: Jurkat cells, gALK3, PHA, rep2

description: Library name: BA2

value: ALK3 knockout


title: Jurkat cells, untreated, PHA-activated, rep1

description: Library name: E5


title: Primary Human Hepatocytes, Replicate2

description: Library name: Sample 8


title: Hepatocyte-like cells, Etoposide, Day5, Replicate3

description: Library name: Sample 6

source_name: WTCII

value: WTCII


title: Hepatocyte-like cells, Etoposide, Day5, Replicate1

description: Library name: Sample 4


title: Jurkat cells, gALK3, PHA, rep1

description: Library name: BA1


title: Jurkat cells, BMP4+BMP7, PHA-activated, rep2

description: Library name: E8


title: Subj.PNMC328_Day7


title: Jurkat cells, gALK2, PHA, rep2

description: Library name: AA2

value: ALK2 knockout


title: TBI008_2VST_CD4

description:
Library name: TBI008_2VST_CD4
Post BCG vaccinated sequencing files vs Pre BCG vaccinated files  were processed  using the CLC genomics workbench 22.0

value: Post BCG vaccination


title: Hepatocyte-like cells, Control, Day5, Replicate3

description: Library name: Sample 3


title: Subj.PNMC323_Day7


title: Subj.PNMC326_Day0


title: Subj.PNMC326_Day7


title: Hepatocyte-like cells, Control, Day5, Replicate1

description: Library name: Sample 1


title: Subj.PNMC330_Day7


title: Subj.PNMC328_Day0


title: Jurkat cells, gALK2, PHA, rep1

description: Library name: AA1


title: Hepatocyte-like cells, Control, Day5, Replicate2

description: Library name: Sample 2


title: Jurkat cells, gNc, PHA, rep2

description: Library name: NA2

value: NC


title: KO_5

city: Lanzhou

first: Xiaoqiang

last: Liu

address: No. 82, Cuiyingmen, Linxia Road, Chengguan District

institute: The Second Clinical Medical College of Lanzhou University

description:
Library name: sample 11
SK-MEL-2, COMMD4-knockout, KO group, replicate biosample 2

data_processing:
CLC Genomics Workbench v 11.0.1
Sequence reads were trimmed for adaptor sequence/low-quality sequence using CLC genomic benchwork (parameter- Quality limit: 0.05)
Trimmed sequence reads were mapped to GrCM38 using CLC genomic benchwork (parameters- mismath cost: 2 insertion cost: 3 deletion cost: 3 length fraction: 0.8 similarity fraction: 0.8)
Read count extraction and normalization were performed using CLC genomic benchwork
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes gene counts for each sample
Supplementary files format and content: tab-delimited text file includes fpkm value for each sample

extract_protocol:
Niche cells were enriched using a Lineage Cell Depletion Kit and isolated by flow cytometry after labeling with antibodies for the detection of CD45-PE-Cy7, lineage mix (CD3e, CD4, CD8, B220, Mac-1, Gr-1, Ter-119) and CD71. Calcein AM and DAPI were used to identify and eliminate dead cells. Samples with a cell viability of greater than 80%, a cell concentration ranging from 700 cells/μL to 1,200 cells/μL, and cell diameters ranging from 5 μm to 40 μm were collected and loaded for sequencing.
The cell suspension and gel beads contained barcode-sequencing-generated single-cell gel bead-in emulsions for reverse transcription, and library preparation was performed using a Chromium Single Cell 30, version 2, Reagent Kit (10X Genomics). Sequencing was performed on an Illumina HiSeq PE150 with 50,000–100,000 reads per cell and a 70%–80% saturation level, as recommended by 10X Genomics.


title: KO_6

description:
Library name: sample 12
SK-MEL-2, COMMD4-knockout, KO group, replicate biosample 3


title: Jurkat cells, gNc, PHA, rep1

description: Library name: NA1


title: Jurkat cells, gNc, unstimulated, rep1

description: Library name: NM1


title: control_5

description:
Library name: sample 5
SK-MEL-2, control group, replicate biosample 2


title: KO_4

description:
Library name: sample 10
SK-MEL-2, COMMD4-knockout, KO group, replicate biosample 1


title: Subj.PNMC321_Day7


title: control_6

description:
Library name: sample 6
SK-MEL-2, control group, replicate biosample 3


title: KO_2

description:
Library name: sample 8
A375, COMMD4-knockout, KO group, replicate biosample 2


title: KO_1

description:
Library name: sample 7
A375, COMMD4-knockout, KO group, replicate biosample 1


title: Subj.PNMC323_Day0


title: control_4

description:
Library name: sample 4
SK-MEL-2, control group, replicate biosample 1


title: KO_3

description:
Library name: sample 9
A375, COMMD4-knockout, KO group, replicate biosample 3


title: Jurkat cells, gNc, unstimulated, rep2

description: Library name: NM2


title: control_3

description:
Library name: sample 3
A375, control group, replicate biosample 3


title: Subj.PNMC318_Day7


title: Subj.PNMC318_Day0


title: Subj.PNMC321_Day0


title: Subj.PNMC317_Day7


title: control_1

description:
Library name: sample 1
A375, control group, replicate biosample 1


title: 250nM estradiol, rep3

city: Beijing

first: Li

last: Jin

state: -- None selected --

address: Department of Obstetrics and Gynecology

institute: peking union medical college hospital

data_processing:
The quality of the samples, experiment, and the analysis data were completed by the Genesky Biotechnologies Inc.
The known mature miRNA expression profile was generated by using the quantifier module of the miRDeep2 package, which gives read counts for the known miRNAs.
The raw reads’ expression profiles generated for all the replicates of the samples were subjected to trimmed mean of M-values’ (TMM) normalization using the Bioconductor package edgeR.
Assembly: oryCun2
Supplementary files format and content: tab-delimited text files include raw counts for each Sample

source_name: endometrium

growth_protocol: ECC cells were cultured in MEM containing NEAA ( 11140050, Gibco), and 10% FBS (10099141, Gibco), at 37℃ in a 5% CO2 incubator. Cells were seeded at a density of 2 × 105 viable cells /mL, and passaged every 3–4 days.

extract_protocol:
For RNA isolation, total RNA was isolated using TRIzol reagent.
A total of 2 μg of RNA/sample was used as input material for the miRNA library. The miRNA sequencing libraries were generated using a TruSeq Small RNA Sample Preparation Kit, and index codes were added to attribute sequences to each sample.

treatment_protocol: β-estradiol power (E2257,Sigma) was dissolved in DMSO with 3.7% filtered sodium hydrogen carbonate. After incubation of the final concentration of β-estradiol with 250nmol/L, cells were incubated at 37°C and 5% CO2 for 48 h before harvested.

value: endometrium

value: endometrioid endometrial carcinoma

value: estradiol


title: 250nM estradiol, rep2


title: control_2

description:
Library name: sample 2
A375, control group, replicate biosample 2


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 24hr 4sU - 2

description:
Library name: TDPKD_2_24h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP

data_processing:
Using the ‘rna_seq_snakemake’ alignment pipeline ( https://github.com/frattalab/rna_seq_snakemake ), raw FASTQ files were quality trimmed using fastp with the parameter “qualified_quality_phred: 10”, and aligned without soft-clipping to the GRCh38 genome build using STAR (v2.7.0f) with gene models from GENCODE v34. GRAND-SLAM (v2.0.7b) was run on the aligned data using gene models from GENCODE v34 using the “-trim5p 10 -trim3p 10” parameter to ignore mismatches at the ends of reads.
Assembly: hg38
Supplementary files format and content: Tab-delimited file containing GRAND-SLAM 2.0 outputs for per-gene estimated new RNA fractions and number of reads with T-C conversions. For per-sample values, column names are space-separated, with the first field corresponding to the sample name. Each sample has the following columns: 'Readcount' (The total number of reads mapped to gene, non-normalised, multimapping reads counted fractionally), '0.05 quantile' (lower bound/5% quantile of the posterior distribution of the new-to-total RNA ratio for the gene), 'Mean' (mean of posterior distribution of new-to-total RNA ratio for the gene), 'MAP' (mode of posterior distribution of new-to-total RNA ratio for the gene), '0.95 quantile' (upper bound/95 % quantile of posterior distribution of new-to-total RNA ratio for the gene), 'alpha' (alpha parameter of the beta approximation of posterior distribution of new-to-total RNA ratio for the gene), 'beta' (beta parameter of the beta approximation of posterior distribution of new-to-total RNA ratio for the gene), 'Conversions' (total number of T-C conversions in this gene), 'Coverage' (total number of uridines covered by any reads), 'Double-Hits' (total number of conversions for gene, but only counting in positions where sequenced by both mates of paired-end reads ), 'Double-Hit Coverage' (total number of  uridines covered by any reads, counting in positions where sequenced by both mates of paired-end reads) and 'min2' (number of reads with at least 2 T-C mismatches). The table has 3 'standard' columns: 'Gene' (Ensembl gene ID), 'Symbol' (the gene symbol/name) and 'Length' (length of longest transcript of gene in nt).
Library strategy: SLAM-seq

extract_protocol:
RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher).
Iodoacetamide (IAA) treatment was applied to each sample, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2 x  250 bp) on an Illumina NovaSeq SP.

value: iPSC-derived cortical neuron


title: 250nM estradiol, rep1


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 12hr 4sU - 2

description:
Library name: TDPKD_2_12h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 8hr 4sU - 2

description:
Library name: TDPKD_2_8h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 4hr 4sU - 2

description:
Library name: TDPKD_2_4h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: Control, rep3


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 1hr 4sU - 2

description:
Library name: TDPKD_2_1h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 0hr 4sU - 2

description:
Library name: TDPKD_2_0h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: Control, rep1


title: Control, rep2


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 8hr 4sU - 2

description:
Library name: Ctrl_2_8h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: Subj.PNMC317_Day0


title: Subj.PNMC315_Day7


title: Subj.PNMC315_Day0


title: Subj.PNMC314_Day7


title: TSB503_BJ_Young_shJMJD6-53

city: La Jolla

first: Tom

last: Suter

address: 9500 Gilman Dr

institute: UCSD

data_processing:
Data was aligned to hg18 using STAR-aligner, with parameters adjusted to account for spliced alignment.
Aligned reads were processed for downstream analysis using the HOMER (Heinz et al., 2010) “makeTagDirectory” program, filtering for tags that were uniquely mapped and only selecting at most one sequencing read per position per length of read.
bigWig files were generated using the makeBigWig.pl function in HOMER, using the -rnaseq style parameter.
Genome_build: hg18
Supplementary_files_format_and_content: .bigWig: display of ChIP-seq data on genome browser as a graph

source_name: Foreskin dermal fibroblast

growth_protocol: BJ fibroblasts (ATCC® CRL-2522) were cultured at 37 °C in 20% O2 and 5% CO2 in 4.5 g/L DMEM (Gibco 10566-016) in 10% FBS.  Cell lines were initially plated at PD26, grown to confluence, and then split 1:8.  Population doubling was recorded and incremented by 3 upon each 1:4 8 split.  BJ fibroblasts were considered to be “young” when collected below population doubling (PD) 35, intermediate fibroblasts were harvested between PD 35-50, and fibroblasts were considered to be senescent when cells failed to divide after at least one week of passaging, which typically occurred after at least PD64 and was initially confirmed using beta galactosidase staining.  To mitigate the effects of cell cycle upon our results, non-senescent, still dividing fibroblasts were grown to confluence prior to harvesting for any experiments, in order to ensure contact inhibited quiescence and similar growth arrest among all populations of cells.    Long term shRNA experiments were done by transducing either pLKO.1 or GIPZ lentivirus into young (<PD30) BJ fibroblasts.  For both pLKO.1 and GIPZ constructs in young fibroblasts, cells were maintained under selection by 0.6ug/ml of puromyin and expanded for 2 weeks until they were frozen down.  Frozen stable lines were then thawed and grown continuously until reaching senescence, and the timing of their population doublings were recorded over time.

extract_protocol:
Total cellular RNA was harvested from cells using the RNeasy Mini Kit (QIAGEN-74104), making use of the optional DNase treatment step.
Samples were then prepared for sequencing with the Illumina HiSeq 2500 system using the TruSeq Stranded Total RNA LT Sample Prep Kit (RS-122-2201/RS-122-2202) for ribosomal RNA depleted libraries.  Samples were run on the Illumina HiSeq 2500 as single-read reactions of 50 cycles.

treatment_protocol: Lentivirus was produced through transfection of packaging plasmids into 293T (ATCC® CRL-3216) cells grown to ~80% confluency.  All transfections were done using Lipofectamine 2000 (Thermo Fisher 12566014), following the manufacturer’s protocol, with the ratios of plasmids and Lipofectamine 2000 reagent listed below.  GIPZ stable knockdown lines were generated using a 667ul : 1333ul ratio of GIPZ viral supernatant to fresh culture media.  pLKO.1 stable knockdown lines were generated using a 48 ul : 1952 ul ratio of pLKO.1 viral supernatant with fresh culture media.  All transduction mixtures were given to cells and allowed to incubate overnight with polybrene added at a final concentration of 8ug/ml.  Knockdown cells were then maintained in 0.6ug/ml puromycin starting 4 days after transduction, until they were harvested at the senescent stage.

value: BJ Fibroblast

tag: Stage

value: Young

tag: shRNA

value: shRNA - JMJD6-construct 53


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 0hr 4sU - 2

description:
Library name: Ctrl_2_0h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: TSB501_BJ_Young_shNS

value: shRNA - Non-silencing - Control


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 24hr 4sU - 1

description:
Library name: TDPKD_1_24h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: TSB429_BJ_Senescent_anti_H3K79me3

description: Replicate 2

library_source: genomic

data_processing:
Data was aligned to hg18 using STAR-aligner with parameters adjusted avoid spliced alignments.
Aligned reads were processed for downstream analysis using the HOMER (Heinz et al., 2010) “makeTagDirectory” program, filtering for tags that were uniquely mapped and only selecting at most one sequencing read per position per length of read.
bigWig files were generated using the makeBigWig.pl function in HOMER, using the -chipseq style parameter
Genome_build: hg18
Supplementary_files_format_and_content: .bigWig: display of ChIP-seq data on genome browser as a graph

molecule: genomic DNA

growth_protocol: BJ fibroblasts (ATCC® CRL-2522) were cultured at 37 °C in 20% O2 and 5% CO2 in 4.5 g/L DMEM (Gibco 10566-016) in 10% FBS.  Cell lines were initially plated at PD26, grown to confluence, and then split 1:8.  Population doubling was recorded and incremented by 3 upon each 1:8 split.  BJ fibroblasts were considered to be “young” when collected below population doubling (PD) 35, intermediate fibroblasts were harvested between PD 35-50, and fibroblasts were considered to be senescent when cells failed to divide after at least one week of passaging, which typically occurred after at least PD64 and was initially confirmed using beta galactosidase staining.  To mitigate the effects of cell cycle upon our results, non-senescent, still dividing fibroblasts were grown to confluence prior to harvesting for any experiments, in order to ensure contact inhibited quiescence and similar growth arrest among all populations of cells.

extract_protocol:
Fixed chromatin immunoprecipitation assays were performed as previously described (Li et al., 2013), with the following modifications: Samples prepared for ChIP of histone modifications were fixed in 1% formaldehyde for 10 minutes at room temperature, while samples prepared for ChIP of transcription factors were double cross-linked in 2mM DSG alone for 45 minutes at room temperature followed by 10 minute fixation with 1% formaldehyde.  After neutralization with 0.125M glycine, samples underwent nuclear extraction, and were afterwards resuspended in nuclear lysis buffer (50mM Tris/HCl pH7.4, 1% SDS, 10mM EDTA, 1x dissolved cOmplete Protease Inhibitor Cocktail (Sigma 11836145001)), and samples were sonicated using the Bioruptor (Diagenode).  The concentration of chromatin in each sample was detected using the Qubit Fluorometer (Thermo Fisher), and samples were then diluted 1:10 in dilution buffer (20mM Tris/HCl pH7.4, 100mM NaCl, 0.5% Triton-X-100, 2mM EDTA, 1x dissolved cOmplete Protease Inhibitor Cocktail).  Between 20-50 ug of chromatin from each sample was then used for each histone or transcription factor ChIP assay, and the volume of each sample was raised to 0.5 mL using 1:10 sonication:dilution buffer mixture.  Antibodies were then incubated with each sample overnight.    Antibody:chromatin complexes were then immunoprecipitated as described using 20 uL Protein G Dynabeads (Thermo Fisher), which were then washed using the following washes:  ChIPs of histone modifications were washed with the following buffers in order: 2xWB1 (20mM Tris/HCl pH7.4, 150mM NaCl, 1% Triton-X-100, 0.1% SDS, 2mM EDTA), 1xWB2 (20mM Tris/HCl pH7.4, 500mM NaCl, 1% Triton-X-100, 2mM EDTA), 1xWB3 (10mM Tris/HCl pH7.4, 250mM LiCl, 1% NP-40, 1% Deoxycholate, 1mM EDTA), 2xTE (10mM Tris/HCl pH 8.0, 1mM EDTA).  ChIPs of transcription factors were washed with the following buffers in order: 2xWB1 (20mM Tris/HCl pH7.4, 150mM NaCl, 1% Triton-X-100, 2mM EDTA), 1xWB2 (20mM Tris/HCl pH7.4, 300mM NaCl, 1% Triton-X-100, 2mM EDTA), 1xWB3 (20mM Tris/HCl pH7.4, 400mM NaCl, 1% Triton-X-100, 2mM EDTA), 2xTE (10mM Tris/HCl pH 8.0, 1mM EDTA).  After washing, samples were eluted overnight at 65 °C as described, purified using the QIAquick PCR Purification Kit (QIAGEN), and were eluted in EB (1% SDS, 0.1M NaHCO3).  Samples were then further sonicated for 10 minutes to ensure the majority of purified chromatin fell between 100-300bp.
Purified chromatin was then prepared for deep sequencing with the Illumina HiSeq 2500 system using either the KAPA LTP Library Preparation Kit (KK8232) or KAPA Hyper Prep Kit (KK8500) for histone or transcription factor ChIPs, respectively, following manufacturer’s instructions.  Samples were run on the Illumina HiSeq 2500 as single-read reactions of 50 cycles.

value: Senescent

tag: antibody

value: H3K79me3:abcam ab2621


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 12hr 4sU - 1

description:
Library name: TDPKD_1_12h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 8hr 4sU - 1

description:
Library name: TDPKD_1_8h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: Subj.PNMC311_Day7


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 4hr 4sU - 1

description:
Library name: TDPKD_1_4h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 12hr 4sU - 2

description:
Library name: Ctrl_2_12h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 1hr 4sU - 1

description:
Library name: TDPKD_1_1h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: TSB421_BJ_Young_anti_H3K79me3

description: Replicate 2

value: Young - Quiescent


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 0hr 4sU - 1

description:
Library name: TDPKD_1_0h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP