title: Scramble (control) biological replicate 3

city: Milan

first: Nico

last: Mitro

state: MI

address: Via Balzaretti 9

department: Pharmacological and biomolecular sciences

country: Italy

institute: Università degli studi di Milano

hyb_protocol: An aliquot (100 ng) of RNA was used for the preparation of targets for Affymetrix® MoGene 2.0 ST arrays, according to the Ambion WT Expression kit manual. Affymetrix® MoGene 2.0 ST arrays (which contain 41073 genes), were purchased from Affymetrix (Affymetrix, USA). The staining, washing and scanning of the arrays were conducted using the Fluidics 450 station, Command Console Software and GeneChip® Scanner 3000 7G, generating .CEL files for each array (Affymetrix, USA).

scan_protocol: The images were scanned by Affymetrix GeneChip Command Console (AGCC) and analyzed with the Affymetrix GeneChip Expression Console.

data_processing: The quality control of the scanned data was first estimated by confirming the order of the signal intensities of the Poly-A and Hybridization controls using Expression Console Software (Affymetrix, USA). Raw expression values were imported as Affymetrix.CEL files into Partek Genomics Suite 6.6 (Partek Inc., St. Louis, MO, USA). Raw expression values from the Affymetrix MoGene 2.0 ST arrays were analyzed and normalized using Partek Genomics Suite 6.6, which includes the Preprocessing, Differentially Expressed Genes (DEGs) Finding and Clustering modules. A total of 10 .CEL files were uploaded and normalized in PM (perfect match)-only conditions as a PM intensity adjustment.

label: biotin

source_name: Scramble shRNA

label_protocol: Affymetrix

growth_protocol:
Cells were maintained in growth medium (high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) (v/v) (Gibco), 2% glutamine (v/v) (Thermo Scientific), 2% pen/strep (v/v) (Thermo Scientific) and in 37°C, 5% CO2 and 90% humidified atmosphere.
Zc3h10 targeted silencing used a validated adenoviral construct contained specific short hairpin RNA sequence under a U6 promoter (shADV-276549), while control vector contained a scrambled sequence (Vector Biolabs, 1122). Both constructs were transduced using a MOI of 50 in C3H10T1/2 cells carried out by 72h exposure.

extract_protocol:
Cells were rinsed in 1 ml of ice-cold PBS. Total RNA was isolated using silica column approach (NucleoSpin® RNA extraction kit, Macherey-Nagel, 740955.250). Briefly, cells were washed in ice-cold PBS and lysed in 350 of lysis buffer supplemented with 1% β-mercaptoethanol. After adding 100µl of chloroform, mouse samples were centrifuged at 10,000g at 4°C for 15 min. Upper polar phases and cell samples were then transferred to silica columns and spun 11,000g for 30 seconds. Total RNA was then isolated by following manufacturer’s instructions and eluted from columns with 50µl of RNase free water.
3µg of RNA were used for the analyses. Total RNA concentration and purity was assessed by UV spectrophotometry (NanoDrop 1000 Spectrophotometer, Thermo Scientific). Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated before differential gene expression evaluation.

tag: age

value: 9 days of differentiation

value: C3H10T1/2 adipocyte

value: Scramble short hairpin RNA


title: Zc3h10 shRNA biological replicate 3

status: Public on Feb 14 2023

type: RNA

molecule: total RNA

organism: Mus musculus

source_name: Zc3h10 shRNA

tag: tissue

tag: genotype

value: Zc3h10 specific short hairpin RNA


title: PA21A092, Time 32h, Rep 2

city: Frederick

first: Shivendra

middle: G

last: Tewari

state: MD

address: 2405 Whittier Drive, Suite 200

country: USA

institute: Biotechnology HPC Software Applications Institute

description: Gene expression data

hyb_protocol: The eight time course samples were hybridized (along with the manufacturer’s RNA spike-in controls) according to standard protocol to the 8 sectors of an Agilent 8x15K platform microarray (AMADID 037237) with separate microarray slides used for each biological replicate.

scan_protocol: Arrays were scanned with the Agilent G2600D SureScan microarray scanner using scan protocol AgilentHD_GX_1color. Agilent’s Feature Extraction Software was used to assign grids, provide raw image files per array, and generate QC metric reports from the microarray scan data.

data_processing: Txt files from Agilent’s Feature Extraction Software were transferred to Partek Genomics Suite software (v7.0). Within Partek, the gProcessedSignal was imported and the intensity values were normalized using Quantile Normalization and Log Tranformation Base 2.0 after import.

organism: Plasmodium falciparum

source_name: PA92-treated Dd2-A211V, Time 32, Rep 2

label_protocol: From 100 ng of total RNA, Cyanine-3 labeled cRNA was prepared using the Low Input Quick Amp Labeling Kit One-Color (Agilent).

extract_protocol: For each data point, RNA was extracted from 50 µL of packed RBC culture with Trizol Reagent and the PureLink Mini Kit (Invitrogen/Thermo Fisher Scientific). 1ml Trizol was added to each sample, followed by high-speed disruption in tubes containing Lysing Matrix D (MP Biomedical) in a FastPrep 120 Instrument at speed 6 for 20 seconds. Homogenates were subsequently processed according to the PureLink Mini Kit manufacturer’s (Invitrogen/Thermo Fisher Scientific) protocol, including on-column DNase treatment.

treatment_protocol: Sublethal doses of KAE609 (30 pM) or PA21A092 (400 nM) was added to control RPMI medium at 0 h of the experiment, which was determined using blood smears of the parasite cultures (described in the paper).

value: Blood-stage Dd2-A211V P. falciparum parasites cultured in human erythrocytes at 2% hematocrit and 400 nM PA21A092

tag: time

tag: replicate

value: Rep 2


title: Zc3h10 shRNA biological replicate 2


title: Scramble (control) biological replicate 2


title: Scramble (control) biological replicate 1


title: rip_ago2_shints11_24h_r2

type: SRA

city: Miami

first: Felipe

last: Beckedorff

state: FL

address: 1501 NW 10th Avenue room 731.G , Miami, FL

institute: University of Miami

description:
shINTS11
s4u_rip_shints11_mirna_quantification.tsv

library_source: transcriptomic

data_processing:
Adapter trimming:   Small_RNA:  FASTQ data were processed with Cutadapt v1.18, adapter trimmed (AAAAAAAAAA) and reads smaller 17 bp were discarded.  Total RNA-seq: FASTQ data were processed with Trimmomatic v0.32 to remove adapters and low-quality reads.   eCLIP: Double adapter trimming was performed using Cutadapt v1.14.
Small_RNA: Reads were aligned against human elements in RepBase (v23.08) with STAR37 (v2.5.3a) and the unmapped output then mapped against the human genome (hg19), allowing three mismatches and keeping all uniquely aligned reads.   RNA-seq: reads were aligned to the hg19 human genome using STAR aligner v2.5.3a with default parameters and RSEM v1.2.31 to obtain expected gene counts against the human Ensembl reference (release 87).   eCLIP: Data was processed according to Van Nostrand, E., Pratt, G., Shishkin, A. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508–514 (2016). https://doi.org/10.1038/nmeth.3810 - https://github.com/YeoLab/eclip
s4U small RNA-seq and RIP small rna-seq were treated as described in Reichholf B, Herzog VA, Fasching N, Manzenreither RA, Sowemimo I, Ameres SL. Time-Resolved Small RNA Sequencing Unravels the Molecular Principles of MicroRNA Homeostasis. Mol Cell. 2019 Aug 22;75(4):756-768.e7. doi: 10.1016/j.molcel.2019.06.018. Epub 2019 Jul 23. PMID: 31350118; PMCID: PMC6713562. )https://github.com/breichholf/smRNAseq)
Genome_build: hg19
Supplementary_files_format_and_content: Small_RNA and RNA-seq: the processed data files are in bigWig format normalized by Count Per Million (CPM) produced from DeepTools2.
Supplementary_files_format_and_content: eCLIP: strand specific bigwig files;  clipper v1.1 output (bed files, col1: chrom, col2: chromStart, col3: chromEnd, col4: -log10 pvalue, col5: log2 fold enrichment above input, col6: strand)) contains clusters of Ints11 binding

organism: Homo sapiens

source_name: HeLa

growth_protocol: HeLa cell lines containing stable shRNA were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. DMEM medium was supplemented with 10% FBS and puromycin (2 μg/ml). Knockdowns were induced by adding of 1 μg/ml doxycycline (Dox) into the culture medium daily for 2 days. In addition, 4-Thiouridine (Cayman chemical, 16373) was added for the indicated timepoints to 200 µM.

extract_protocol:
RIP was performed in the dark from s4U treated cells at the indicated timepoints. Before RNA extraction, 2 pmol synthetic ath-miR-159a was spiked-in and RNA was extracted using Trizol reagent (Thermo Fisher Scientific, #15596026) according to the manufacturer’s instructions. All samples were treated with Iodoacetamide (+IAA) for thiol modification. Genomic DNA was removed by Turbo DNAse treatment (Invitrogen, #AM1907).
Small RNA libraries were prepared using the SMARTer smRNA-seq kit (Takara, #635030) with RNA resulting from RIP (typically 200-400 ng)

tag: cell type

value: cervical adenocarcinoma

tag: cell source

tag: modification

value: Tet-pLKO_shINTS11 stable cells

tag: ip antibody

value: Ago2 (abcam, ab57113)

tag: nucleotide alteration

value: s4U, IAA


title: 𝚫ppdA/B  24-h mycelium 1

city: Jena

first: Matthew

middle: George

last: Blango

address: Adolf-Reichwein-Str. 23

department: Junior Resaerch Group RNA Biology of Fungal Infections

country: Germany

institute: Leibniz Institute for Natural Product Research and Infection Biology

description:
Biological Replicate 1
B7

data_processing:
Preprocessing of raw reads including quality control and gene abundance estimation was done with the GEO2RNaseq pipeline (v0.9.12; (https://doi.org/10.1101/771063)) in R (version 3.5.1). Quality analysis was done with FastQC (v0.11.8) before and after trimming. Read-quality trimming was done with Trimmomatic (v0.36).
Reads were rRNA-filtered using SortMeRNA (v2.1) with a single rRNA database combining all rRNA databases shipped with SortMeRNA.
Reference annotation was created by extracting and combining exon features from corresponding annotation files. Reads were mapped against the reference genome of A. fumigatus (Af293, ASM265v1) using HiSat2 (v2.1.0, paired-end mode). Gene abundance estimation was done with featureCounts (v1.28.0) in paired-end mode with default parameters. MultiQC version 1.7 was finally used to summarize and assess the quality of the output of FastQC, Trimmomatic, HiSat, featureCounts and SAMtools. The count matrix with gene abundance data without and with median-of-ratios normalization (MRN; (https://doi.org/10.1186/gb-2010-11-10-r106)) were extracted.
Assembly: A. fumigatus (Af293, ASM265v1)
Supplementary files format and content: The processed data file is an excel .xlsx file containing four tabs, including read counts for each gene, MRN-normalized counts, RPKM-normalized counts, and TPM-normalized counts.

molecule: polyA RNA

organism: Aspergillus fumigatus

source_name: Mycelium

growth_protocol: A. fumigatus strains were grown on Aspergillus minimal medium (AMM) agar plates (70 mM NaNO3, 11.2 mM KH2PO4, 7 mM KCl, 2 mM MgSO4, 1% (w/v) glucose and 1 μl/ml trace element solution (pH 6.5)). The trace element solution was composed of 1 g FeSO4 • 7 H2O, 8.8 g ZnSO4 • 7 H2O, 0.4 g CuSO4 • 5 H2O, 0.15 g MnSO4 • H2O, 0.1 g NaB4O7 • 10 H2O, 0.05 g (NH4)6Mo7O24 • 4 H2O, and ultra-filtrated water to 1000 ml. Spores were harvested after 5 days with 10 ml of sterile distilled water using a T-shaped inoculation spreader and spore suspensions were filtered through a 30-µm cell strainer (MACS, Miltenyi Biotec GmbH, Germany) to exclude mycelium.

extract_protocol:
Total RNA was isolated from three biological replicates of CEA17ΔakuBKU80, ΔppdA/B, ΔdclA/B, and ΔrrpA/B knockout strains from conidia, 24-h-, and 48-h-old mycelium. For A. fumigatus conidia, the spores were first homogenized in a bead beater (0.5 mm beads; FastPrep-24, MP Biomedicals) in Trizol. Fungal mycelium was collected from liquid culture using Miracloth (Millipore) and disrupted in liquid nitrogen using a precooled mortar and pestle. Roughly 0.5 g of homogenized mycelium was transferred into a 2-ml Eppendorf tube. 800 µl TRIzol was added to the ground mycelia and vortexed vigorously. Tubes were frozen briefly for 5 sec in liquid nitrogen and allowed to thaw on ice. To all samples, chloroform was added to the fungal samples in TRIzol, vortexed, and centrifugated for 5 min at 4°C at full speed. The aqueous upper phase was transferred to a fresh 2-ml tube without disturbing the interphase. RNA extraction from aqueous phase was done with 1 volume of phenol/chloroform/isoamyl alcohol (25:24:1, v/v). Brief vortexing preceded centrifugation for 5 min at 4°C. RNA was precipitated using 400 µl isopropanol for 20 min, followed by pelleting by centrifugation for 20 min at 4°C. The pellet was washed with 700 µl 70% ethanol and air dried at 37°C for 5 min prior to resuspension in RNase free water. The RNA isolation was followed by a DNase treatment using 2 units of TURBO DNase (Thermo Fisher) per 10 µg RNA for 30 min at 37°C in 100 µl total volume. Total RNA was then collected using the RNA Clean and Concentrator-25 kit (Zymo Research) according to the manufacturer’s instructions.
Poly-(A) selection and directional sequencing of mRNA was performed by Novogene using paired-end 150-bp read sequencing on a NovaSeq 6000.

value: Mycelium

tag: cell line

value: CEA17 delta_akuB::KU80

tag: growth condition

value: Aspergillus minimal media


title: 𝚫dclA/B  24-h mycelium 3

description:
Biological Replicate 3
B6


title: Zc3h10 shRNA biological replicate 1


title: 𝚫dclA/B  24-h mycelium 2

description:
Biological Replicate 2
B5


title: rip_ago2_shints11_0h_r2


title: PA21A092, Time 24h, Rep 2

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 24, Rep 2


title: rip_ago2_shints11_0h_r1


title: Wild-type 24-h mycelium 3

description:
Biological Replicate 3
B3

value: Wild type


title: PA21A092, Time 16h, Rep 2

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 16, Rep 2


title: PA21A092, Time 24h, Rep 1

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 24, Rep 1

value: Rep 1


title: Wild-type 24-h mycelium 2

description:
Biological Replicate 2
B2


title: PA21A092, Time 8h, Rep 2

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 8, Rep 2


title: PA21A092, Time 32h, Rep 1

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 32, Rep 1


title: PA21A092, Time 48h, Rep 1

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 48, Rep 1


title: rip_ago2_shints11_24h_r1


title: Wild-type 24-h mycelium 1

description:
Biological Replicate 1
B1


title: rip_ago2_shints6_24h_r2

description:
shINTS6
s4u_rip_shints6_mirna_quantification.tsv

value: Tet-pLKO_shINTS6 stable cells


title: rip_ago2_shints6_24h_noS4U_r1

description:
shINTS6
control_nos4u_rip_shints6_mirna_quantification.tsv

value: IAA


title: rip_ago2_shints6_24h_r1


title: 𝚫dclA/B  24-h mycelium 1

description:
Biological Replicate 1
B4


title: PA21A092, Time 8h, Rep 1

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 8, Rep 1


title: PA21A092, Time 40h, Rep 1

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 40, Rep 1


title: PA21A092, Time 0h, Rep 1

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 0, Rep 1


title: PA21A092, Time 0h, Rep 2

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 0, Rep 2


title: KAE609, Time 24h, Rep 4

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 24, Rep 4

value: Blood-stage Dd2-A211V P. falciparum parasites cultured in human erythrocytes at 2% hematocrit and 30 pM KAE609

value: Rep 4


title: KAE609, Time 32h, Rep 4

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 32, Rep 4


title: KAE609, Time 48h, Rep 4

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 48, Rep 4


title: PA21A092, Time 16h, Rep 1

description: Gene expression data

source_name: PA92-treated Dd2-A211V, Time 16, Rep 1


title: rip_ago2_shints6_24h_noS4U_r2


title: rip_ago2_shints6_0h_r2


title: KAE609, Time 40h, Rep 4

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 40, Rep 4


title: 𝚫rrpA/B conidia 2

description:
Biological Replicate 2
A11

source_name: Conidia

value: Conidia

value: 0 h


title: 𝚫rrpA/B conidia 1

description:
Biological Replicate 1
A10


title: 𝚫ppdA/B conidia 3

description:
Biological Replicate 3
A9


title: KAE609, Time 16h, Rep 4

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 16, Rep 4


title: 𝚫rrpA/B conidia 3

description:
Biological Replicate 3
A12


title: KAE609, Time 8h, Rep 4

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 8, Rep 4


title: KAE609, Time 48h, Rep 3

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 48, Rep 3

value: Rep 3


title: rip_ago2_shcontrol_24h_noS4U_r2

description:
shGFP
control_nos4u_rip_shcontrol_mirna_quantification.tsv

source_name: HeLa

value: Tet-pLKO_shGFP stable cells


title: 𝚫ppdA/B conidia 2

description:
Biological Replicate 2
A8


title: 𝚫ppdA/B conidia 1

description:
Biological Replicate 1
A7


title: rip_ago2_shcontrol_24h_noS4U_r1

value: cervical adenocarcinoma


title: KAE609, Time 32h, Rep 3

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 32, Rep 3


title: KAE609, Time 24h, Rep 3

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 24, Rep 3


title: rip_ago2_shcontrol_24h_r2

description:
shGFP
s4u_rip_shcontrol_mirna_quantification.tsv


title: 𝚫dclA/B conidia 2

description:
Biological Replicate 2
A5


title: rip_ago2_shcontrol_0h_r2


title: KAE609, Time 8h, Rep 3

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 8, Rep 3


title: 𝚫dclA/B conidia 3

description:
Biological Replicate 3
A6


title: rip_ago2_shcontrol_24h_r1


title: KAE609, Time 16h, Rep 3

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 16, Rep 3


title: KAE609, Time 0h, Rep 4

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 0, Rep 4


title: 𝚫dclA/B conidia 1

description:
Biological Replicate 1
A4


title: KAE609, Time 0h, Rep 3

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 0, Rep 3


title: rip_ago2_shints6_0h_r1


title: KAE609, Time 48h, Rep 2

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 48, Rep 2

value: Rep 2


title: KAE609, Time 16h, Rep 2

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 16, Rep 2


title: KAE609, Time 40h, Rep 3

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 40, Rep 3


title: KAE609, Time 24h, Rep 2

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 24, Rep 2


title: Wild-type conidia 3

description:
Biological Replicate 3
A3


title: rip_ago2_shcontrol_0h_r1

value: Ago2 (abcam, ab57113)


title: KAE609, Time 0h, Rep 2

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 0, Rep 2


title: KAE609, Time 40h, Rep 2

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 40, Rep 2


title: shints11_24h_noIAA_r2

description:
shINTS11
timecourse_mergedReplicates_shINTS11set_shINTS11_miRNA_quantification.tsv

extract_protocol:
Protected from light. Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, #15596026) according to the manufacturer’s instructions. Indicated samples were treated with Iodoacetamide (+IAA) for thiol modification. Genomic DNA was removed by Turbo DNAse treatment (Invitrogen, #AM1907).
Small RNA libraries were prepared using the SMARTer smRNA-seq kit (Takara, #635030) with 800 ng of total RNA; 5% (40 ng) of Drosophila RNA were added as spike-in

value: Tet-pLKO_shINTS11 stable cells


title: shints11_24h_noIAA_r1


title: Wild-type conidia 2

description:
Biological Replicate 2
A2

source_name: Conidia


title: Wild-type conidia 1

description:
Biological Replicate 1
A1


title: Microglia IP, male, stroke, 3 days (IP_98)

city: Stanford

first: Marion

last: Buckwalter

state: California

address: 1201 Welch Rd

department: Neurology & Neurological Sciences

institute: Stanford University

data_processing:
Reads were trimmed of adapter sequences, low quality bases, and very short reads using trimGalore! (v0.4.5), a wrapper script which utilizes cutadapt and FastQC (v0.11.7).
Remaining reads were aligned to the mouse mm10 genome (GRCm38.p6) using the STAR aligner (v2.7.1).
Transcript abundances were then annotated and quantified using the RSEM software package (v1.3.1)
Assembly: mm10
Supplementary files format and content: text files include length, effective length, expected count, TPM and FPKM values for each sample

source_name: Brain

extract_protocol:
RNA was extracted according to manufacturer’s protocols using an RNeasy Micro Kit (Qiagen) for IP and RNeasy Mini Kit (Qiagen) for input samples
Microglia sample RNA libraries for RNA-seq were prepared using the Smart-Seq2 method, and then libraries were modified for RNA-sequencing using the Nextera XT DNA Sample Preparation Kit (Illumina). Astrocyte sample RNA libraries were sent to Azenta (formerly GeneWiz) for library preparation, using the NEBNext Ultra RNA Library Preparation Kit with polyA selection.

treatment_protocol: Mice underwent a distal middle artery occlusion (stroke) or sham control surgery prior to sample extraction.

value: Brain

tag: Sex

value: Male

value: Microglia

value: Cx3cr1-creERT2 hemi; Ribotag homo

tag: treatment

value: Stroke

value: 3 days

tag: fraction

value: IP


title: KAE609, Time 24h, Rep 1

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 24, Rep 1

value: Blood-stage Dd2-A211V P. falciparum parasites cultured in human erythrocytes at 2% hematocrit and 30 pM KAE609

value: Rep 1


title: shints11_24h_noS4U_r2

value: IAA


title: KAE609, Time 32h, Rep 2

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 32, Rep 2


title: KAE609, Time 16h, Rep 1

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 16, Rep 1


title: shints11_24h_noS4U_r1

source_name: HeLa


title: Microglia IP, male, sham, 3 days (IP_97)

value: Sham


title: KAE609, Time 40h, Rep 1

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 40, Rep 1


title: KAE609, Time 32h, Rep 1

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 32, Rep 1


title: sal_K1_dsRed

city: Richmond

first: Hengbin

last: Wang

state: VA

address: 401 College Street

department: Massey Cancer Institute

institute: Virginia Commonwealth University

library_source: genomic

data_processing:
library strategy: CUT&RUN
Illumina Casava1.7 software used for basecalling.
Sequencing reads were deduplicated based on barcode. Deduplicated reads were next aligned to the yeast genome (R64-1-1) using bowtie2. Remaining sequencing reads were aligned with the STAR aligner to versions of the Flybase R6.18 modified to contain transgene sequences as necessary.
Aligned reads were separated into nucleosome (>150bp) and subnucleosome (<120bp) resolution fragments.  Spike-in normalized bigwigs were prepared.
Genome_build: dm6
Supplementary_files_format_and_content: bigwig files

molecule: genomic DNA

organism: Drosophila melanogaster

source_name: salivary glands

growth_protocol: Drosophila larvae were grown at room temperature on standard media

extract_protocol:
CUT&RUN was performed using a modified version of a Drosophila specific CUT&RUN protocol (Ahmed, 2019). Approximately 100 pairs of dissected L3 larvae salivary glands were utilized for Cut&Run per genotype.
CUT&RUN sequencing libraries were prepared using the NEXTFLEX RAPID DNA-seq Kit 2.0 (Bioo NOVA 5188-12) following the manufacturer’s protocol with modification. Briefly, the thermal cycler program for End-Repair and Adenylation was modified (30min at 20C, 75 min at 58C), adapter ligation proceeded at 8C overnight, and post-ligation NEXTFLEX bead clean-up was not size selective to retain small fragments.

value: L3 larvae

value: salivary glands


title: Microglia IP, female, stroke, 3 days (IP_93)

value: Female


title: sal_mCh-K1_dsRed


title: Microglia IP, male, sham, 3 days (IP_91)


title: KAE609, Time 8h, Rep 1

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 8, Rep 1


title: KAE609, Time 48h, Rep 1

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 48, Rep 1


title: KAE609, Time 0h, Rep 1

description: Gene expression data

source_name: KA09-treated Dd2-A211V, Time 0, Rep 1


title: shints11_24h_r2

value: cervical adenocarcinoma

value: s4U, IAA


title: 48_h_3

description: small RNA

data_processing:
Manatee Small RNA Analysis
Assembly: Aspergillus fumigatus A1163
Supplementary files format and content: sRNA_list.xlsx, an excel file containing two sheets. First, a list of unique sRNAs identified using the Manatee software package and secondly, a list of all loci identified by Manatee (https://doi.org/10.1038/s41598-020-57495-9), which includes duplicate regions found in multiple samples.

source_name: mycelium

growth_protocol: Total RNA was isolated from three separate biological experiments using A. fumigatus CEA10 conidia (5 days on AMM agar plates at 37°C) and mycelium after 24 and 48 h of growth in AMM liquid culture at 37°C.

extract_protocol:
Total RNA was isolated from three separate biological experiments using A. fumigatus conidia and mycelium after 24 and 48 hours of growth using the Universal RNA Kit (Roboklon) with slight modifications. Namely, conidia and mycelia at different time points were harvested and resuspended in RL buffer followed by bead beating 3x for 30 s each using 0.5 mm glass beads (Carl Roth). The total RNA was then isolated according to the yeast protocol provided by the manufacturer (Roboklon) using an on-column DNase treatment (Lucigen Baseline-ZERO DNase). The RNA isolation was followed by an additional DNase treatment using 4 units of TURBO DNase (Thermo Fisher) for 30 min at 37°C in 100 µl total volume. The small RNAs were then collected using the RNA Clean and Concentrator-25 kit (Zymo Research) according to the manufacturer’s instructions for small RNA enrichment.
Since the 5’ end of A. fumigatus small RNAs are not well-defined, the isolated RNA was treated with 1 µl (5 units) RppH enzyme (New England Biolabs) in NEB Buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM DTT at pH 7.9) for 1 h at 37°C to facilitate removal of a pyrophosphate from the 5’ end of triphosphorylated RNA. The reaction was ended with EDTA for 5 min at 65°C followed by cleanup using the RNA Clean and Concentrator-25 kit (Zymo Research) as described above. RNA was quantified using the Qubit 4 Fluorometer (Thermo Fisher). Small RNA libraries were then prepared from 10 ng of small RNA using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs #E7300S) according to the manufacturer’s recommendation using NEBNext Multiplex Oligos for Illumina (Index Primer Set 1, #E7335S; Table S5). Size selection was performed according to the manufacturer using AMPure XP beads (Beckman Coulter). Libraries were validated for the correct size by polyacrylamide gel electrophoresis. The barcoded libraries were pooled without further adjustment, followed by 50-cycle-single-end sequencing using the Illumina HiSeq 4000 platform (Eurofins Genomics).

value: mycelium

value: fungus

value: wild type


title: Microglia IP, female, sham, 3 days (IP_88)

value: Brain

value: Microglia


title: 48_h_2

description: small RNA


title: shints11_24h_r1

value: Tet-pLKO_shINTS11 stable cells


title: 48_h_1

description: small RNA


title: shints11_12h_r2


title: sal_STI-K1_dsRed


title: Microglia IP, female, sham, 4 hours (IP_257)

value: Cx3cr1-creERT2 hemi; Ribotag homo

value: 4 hours


title: sal_STI-mCh-K1_dsRed


title: 24_h_3

description: small RNA


title: Microglia IP, male, sham, 3 days (IP_253)

value: Male

value: 3 days

value: IP


title: sal_STI150_K1_K9m3


title: 24_h_2

description: small RNA


title: sal_STI150-mCh-K1_K9m3


title: 24_h_1

description: small RNA


title: Conidia_3

description: small RNA

source_name: conidia

value: conidia


title: Microglia IP, male, stroke, 4 hours (IP_251)

value: Stroke


title: shints11_6h_r2


title: Microglia IP, male, stroke, 3 days (IP_252)


title: sal_STIdel-mCh-K1_dsRed

value: L3 larvae

value: salivary glands


title: sal_STI150-mCh-K1_H3K4m3


title: sal_STI150_K1_H3K4m3


title: Microglia IP, female, sham, 3 days (IP_243)

value: Female

value: Sham


title: shints11_6h_r1

value: cervical adenocarcinoma

value: s4U, IAA


title: shints11_3h_r2


title: Microglia IP, male, sham, 4 hours (IP_248)


title: Conidia_1

description: small RNA

value: fungus

value: wild type


title: Conidia_2

description: small RNA


title: Liver CD45+ CD11b+ cells - RES

city: Shinjuku-ku

first: Yohei

last: Mikami

state: Tokyo

address: Shinanomachi 35

country: Japan

institute: Keio University School of Medicine

description: gene expression

data_processing:
BCL conversion and demultiplexing: Bcl2fastq and CellRanger
Mapping: CellRanger
Genome_build: mm10
Supplementary_files_format_and_content: CellRanger output files (barcodes.tsv, features.tsv, matrix.mtx)

source_name: Liver

growth_protocol: Liver mononuclear cells were separated by Histopaque solution (Sigma-Aldrich) from liver tissue suspension,  then samples from five mice were pooled. Liver CD45+CD11b+ non-parenchymal cells were separated by FACS sorting using FACS (BD Aria III).

extract_protocol:
Target cells (CD45+CD11b+) were isolated from liver mononuclear cells via FACS.
scRNA-seq: Freshly sorted liver CD45+CD11b+ cells were encapsulated into droplets, and libraries were prepared using Chromium Single Cell 3’ Reagent Kits v2 according to manufacturer’s protocol (10X Genomics). Indexed libraries were multiplexed and sequenced on Illumina HiSeq X instruments according to the manufacturer's instructions.

tag: strain

value: Liver

value: CD45+ CD11b+ cells

value: Liver fibrosis resolved


title: shints11_3h_r1

value: Tet-pLKO_shINTS11 stable cells


title: Liver CD45+ CD11b+ cells - Ctrl

description: gene expression

value: Uninjured control


title: shints11_1h_r1


title: Microglia IP, female, stroke, 3 days (IP_241)

value: Brain

value: Microglia

value: Cx3cr1-creERT2 hemi; Ribotag homo

value: 3 days


title: Microglia IP, female, sham, 4 hours (IP_240)

value: 4 hours

value: IP


title: Ect1 cells, IFN epsilon, R4

city: Melbourne

first: Jamie

last: Gearing

address: 27-31 Wright St

country: Australia

institute: Hudson Institute of Medical Research

data_processing:
Base calling was perfromed using bcl2fastq (v2.20.0.422).
RNA-seq analysis was performed in R (v3.5.1 and v3.6.1). The scPipe package (v1.2.1) was employed to process and de-multiplex the data. A combined multiplexed FASTQ file was created from the R1 and R2 FASTQ files, by trimming the sample index and UMI sequences and storing them in the read header, using the sc_trim_barcode function (with bs2 = 0, bl2 = 8, us = 8, ul = 10).
Additionally, the original multiplexed R1 and R2 FASTQ files were de-multiplexed using cutadapt v3.0 (with error rate 0 and action none).
Read alignment was performed on the combined multiplexed FASTQ file using the RSubread package (v1.30.9). An index was built using the Ensembl Homo sapiens GRCh38 primary assembly genome file and alignment was performed with default settings. Aligned reads were mapped to exons using the sc_exon_mapping function with the Ensembl Homo sapiens GRCh38 v98 GFF3 genome annotation file.
The resulting BAM file was de-multiplexed and reads mapping to exons were associated with each individual sample using the sc_demultiplex function, taking the UMI into account, and an overall count for each gene for each was sample was generated using the sc_gene_counting function.
UMI gene counts were processed using the edgeR package (v3.28.0). Lowly expressed genes were removed using the filterByExpr function and normalisation factors were calculated using the TMM method.
Differential gene expression analyses were performed using the limma package (v3.42.0), with the voom, lmFit, contrasts.fit and treat functions.
Assembly: hg38
Supplementary files format and content: Matrix table with UMI gene counts for every gene and every sample

source_name: Ect1 ectocervical cell line

growth_protocol: Ect1 and VK2 cells were cultured at 37°C with 5% CO2 in Keratinocyte Serum Free Media supplemented with 0.1 ng/mL human recombinant EGF, 0.05 mg/mL bovine pituitary extract, additional calcium chloride 44.1 mg/mL and 1% Penicillin/Streptomycin.

extract_protocol:
Cells were harevested in RLT buffer (Qiagen) with added β-mercaptoethanol. Lysates were homogenized by passing through a 20-gauge needle attached to a sterile plastic syringe. Total RNA was extracted from lysates using the Qiagen RNeasy kit, including removal of DNA contamination using the Qiagen RNase-free DNase set as per the manufacturer’s instructions.
Libraries were generated using an in-house multiplex RNA-seq method (MHTP, Medical Genomics Facility) and were prepared using 20 ng of total RNA input. An 8 bp sample index and a 10 bp unique molecular identifier (UMI) were added during initial poly(A) priming and pooled samples were amplified using a template switching oligonucleotide. The Illumina P5 and P7 sequences were added by PCR and Nextera transposase, respectively. The library was designed so that the forward read (R1) utilised a custom primer (5' GCC TGT CCG CGG AAG CAG TGG TAT CAA CGC AGA GTA C 3') to sequence directly into the index and then the 10 bp UMI. The reverse read (R2) used the standard R2 primer to sequence the cDNA in the sense direction for transcript identification. Paired-end sequencing (R1 19 bp; R2 72 bp) was performed on the NextSeq 550 (Illumina), using the v2.5 High Output Kit.

treatment_protocol: Cells were seeded to 90% confluency in 6-well plates and treated with hIFNε, hIFNα-2a or hIFNλ-3 (100 ng/mL) diluted in fresh culture media or left untreated (n = 4) for 6 h.

value: Epsilon


title: Ect1 cells, IFN epsilon, R3


title: shints11_30min_r2


title: shints11_12h_r1


title: Microglia IP, female, sham, 4 hours (IP_239)

value: Female

value: Sham


title: P5_scRNAseq

city: Leipzig

first: Bilal

last: Sheikh

state: Sachsen

address: Philipp-Rosenthal-Str. 27

department: HI-MAG

institute: Helmholtz Zentrum München

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
De-multiplexing and mapping to the mouse genome (build mm10) was done with CellRanger v5.0 (10x Genomics).
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol:
The brains of 6 female WT mice (6 to 8 weeks old) were dissected and thoroughly minced with a scalpel. Tissue was incubated with 3 ml of enzymatic solution (30 U/mL papain and 40 ug/mL DNase I in Earl’s balanced salt solution) for 70 min at 37 °C while being mixed every 10 min with tube inversions. The digested brain was then passed 10 times through an 18 gauge needle and subsequently 10 times through a 21 gauge needle. Then, the brain was transferred to a 15 mL Falcon tube and layered on top of 5 ml of 22% BSA. In order to separate the myelin, the solution was centrifuged at 1000 g for 10 min and the top lipid layer was carefully removed. The cell pellet was resuspended in 5 mL of endothelial cell growth media (10% FCS, 25 ug/mL heparin, 1ug/mL Ascorbic acid, 2mM GlutaMax, 1x Pen/Strep and 30 ug/mL ECGS in Hams F12) and centrifuged for 5 min at 300g.
Single cell RNA-seq was performed using 10x Next GEM Single Cell 3’ GEM kit v3.1 (10x Genomics; Pleasanton, CA, USA according to the manufacturer’s protocol. Briefly, passage 5 and passage 20 PCs were spun down (4°C, 300 g, for 5 min), resuspended at ~1000 cells/µl, and immediately loaded into the 10x Chromium controller.

value: Pericytes

value: 6-8 weeks old


title: P20_scRNAseq

description: 10x Genomics


title: Ect1 cells, IFN epsilon, R2


title: Microglia IP, male, sham, 3 days (IP_223)

value: Male


title: Ect1 cells, IFN epsilon, R1


title: shints11_15min_r2

value: cervical adenocarcinoma

value: s4U, IAA


title: Ect1 cells, IFN alpha, R3

value: Alpha


title: Gold2

city: Seoul

first: Chaemin

last: Lim

address: 221 HeukSuk-Dong DongJak-Gu, 102 dong, 315 ho (Chung-Ang university, R&D center)

department: College of Pharmacy

country: South Korea

institute: Chung-Ang University

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

source_name: GNC treatment with NIR irradiation

extract_protocol:
RNA was harvested using Trizol reagent (Invitrogen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using the NEBNext Ultra II Directional RNA-Seq Kit standard protocols.

value: tumor

value: murine fibrosarcoma

value: WT

value: GNC treatment at day 1 and 7


title: shints11_30min_r1

value: Tet-pLKO_shINTS11 stable cells


title: Microglia IP, female, sham, 3 days (IP_219)

value: Brain

value: Microglia

value: Cx3cr1-creERT2 hemi; Ribotag homo

value: 3 days


title: shints11_0min_r2


title: Control3

source_name: Ctrl

value: No treatment


title: Gold3


title: shints11_15min_r1


title: Microglia IP, male, stroke, 3 days (IP_221)

source_name: Brain

value: Stroke

value: IP


title: Ect1 cells, IFN alpha, R2


title: Microglia IP, female, stroke, 4 hours (IP_217)

value: Female

value: 4 hours


title: shints11_0min_r1

value: cervical adenocarcinoma


title: shints11_1h_r2

value: s4U, IAA


title: Ect1 cells, IFN alpha, R1

value: Alpha


title: Microglia IP, female, sham, 3 days (IP_218)

value: Sham


title: Ect1 cells, IFN alpha, R4


title: Control2

value: tumor

value: murine fibrosarcoma


title: shcontrol_forints11_24h_noIAA_r2

description:
shGFP
timecourse_mergedReplicates_shINTS11set_shControl_miRNA_quantification.tsv

value: Tet-pLKO_shGFP stable cells


title: Ect1 cells, IFN lambda, R4

value: Lambda


title: shcontrol_forints11_24h_noIAA_r1


title: Control1

value: WT


title: Ect1 cells, IFN lambda, R3


title: Gold1

value: GNC treatment at day 1 and 7


title: shcontrol_forints11_24h_noS4U_r2

value: IAA


title: shcontrol_forints11_24h_noS4U_r1


title: Ect1 cells, IFN lambda, R2


title: shcontrol_forints11_24h_r2


title: Microglia IP, male, sham, 4 hours (IP_200)

value: Brain

value: Male

value: Microglia

value: Cx3cr1-creERT2 hemi; Ribotag homo


title: Microglia IP, female, stroke, 4 hours (IP_192)

value: Female

value: Stroke

value: IP


title: Microglia IP, female, sham, 4 hours (IP_216)

value: 4 hours


title: Ect1 cells, IFN lambda, R1


title: Microglia IP, male, stroke, 3 days (IP_196)

value: 3 days


title: Microglia IP, male, stroke, 4 hours (IP_188)


title: Ect1 cells, no IFN, R4

value: Untreated


title: shcontrol_forints11_24h_r1

value: cervical adenocarcinoma

value: s4U, IAA


title: KPP-EGP Pre-Implantation - GEX [SAM24405162]

city: South San Francisco

first: Joaquin

last: Pechuan jorge

state: CA

address: 1 dna way

department: Cancer Immunology

institute: Genentech

data_processing:
Single-cell RNA-seq fastq files were processed with cellranger count (CellRanger 4.0.2 from 10x Genomics)using a custom reference based on the mouse genome GRCm38 with GENCODE35 gene  models  together  with  the  sequences  for  the  transgenes  employed  in  our  experiments  (tdTomatoand GFP). To demonstrate the lack of deterministic correlation between the transcriptional heterogeneity of the KPP-EGFP cell line in vitro and the immune phenotypes in vivo,  paired samples of the cell line before and after implantation were single cell sequenced. Tumors were cell-hashed with antibodies and then were pooled prior to sequencing. The samples were demultiplexed after centered log ratio transformation normalization on the hashing antibody counts using Seurat::HTODemux() and doublets and negative cells were removed.
Assembly: GRCm38
Supplementary files format and content: PreImplantation_Seurat.Rdata: Seurat object wit the KPP-EGFP cell pre-implantations
Supplementary files format and content: PostImplantation_Seurat.Rdata: Seurat Object with the Demultiplexed sorted tumor cells after clustering

source_name: Skin

extract_protocol:
FOXN1-deficient nude mice with adoptively transferred tdTomato+ CD3+ T cells bearing KPP-EGFP STAMP microtumors were biopsied 8 days post tumor implantation using a 1 mm Miltex sterile disposable biopsy punch with plunger (Integra Biosciences, NH, USA). 3-6 pooled tissue biopsies were moved into a precooled 1.5 mL tube (Eppendorf, Germany) containing 300µL digestion cocktail consisting of Gibco RPMI 1640 (Thermo Fisher Scientific, MA, USA), 0.1% Gibco fetal calf serum (Thermo Fisher Scientific, MA, USA), 0.1mg/ml Liberase TM (Roche, Switzerland), 0.1mg/ml DNAse I (Roche, Switzerland), 32µM Gibco Actinomycin D (Thermo Fisher Scientific, MA, USA). Tissues were incubated for 30 min at 37°C and 950x RPM on a Thermoblock (Eppendorf, Germany) and mechanically dissociated every 10 min with a pipette. To quench the digestion, the cell suspension was filtered through a 40 µm mesh into pre-cooled FACS filter tube containing quenching buffer of Gibco fetal calf serum (Thermo Fisher Scientific, MA, USA) with 32 µM Gibco Actinomycin D (Thermo Fisher Scientific, MA, USA). The cell suspension was centrifuged at 350xg for 8 min and resuspended in 400 µL Gibco RPMI 1640 (Thermo Fisher Scientific, MA, USA) with 5 µM CalceinBlue (Invitrogen, CA, USA) and a 1:200 dilution of Molecular Probes Fixable Live/Dead Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific, MA, USA). FACS was performed to isolate Calcein Blue-positive and L/D NearIR-negative cells into a precooled 1.5ml Eppendorf tube containing 750µL collection buffer consisting of Gibco RPMI 1640 (Thermo Fisher Scientific, MA, USA), 10% Gibco fetal calf serum (Thermo Fisher Scientific, MA, USA), 32 µM Gibco Actinomycin D (Thermo Fisher Scientific, MA, USA). The cell number and viability were determined on a Vi-Cell XR cell viability analyzer (Beckman Coulter, CA, USA) and scRNAseq library preparation was performed using dual single cell mouse kit 5’/TCR according to the manufacturer’s instructions (10x Genomics, CA, USA).
scRNA Seq HTO multiplex for SAM24405163  with Antibody counts in LIB5446558_SAM24405163 (B0301 for Desert, B0303 for Inflamed, B0302 for Excluded) and Gene expression in LIB5446560_SAM24405163; SAM24405162 Contains gene expression data only

value: Tumor

value: KPP-EGFP

value: Skin

value: Cultured Cells


title: Ect1 cells, no IFN, R1


title: Microglia IP, male, stroke, 4 hours (IP_185)


title: shcontrol_forints11_6h_r2

value: Tet-pLKO_shGFP stable cells


title: KPP-EGP Post-Implantation - HTO/Ab [LIB5446558_SAM24405163]

description: LIB5446558_SAM24405163 (B0301 for Desert, B0303 for Inflamed, B0302 for Excluded)

value: Excluded, Inflamed and Desert tumor cells


title: Ect1 cells, no IFN, R2


title: Microglia IP, female, stroke, 4 hours (IP_215)

value: Brain


title: shcontrol_forints11_12h_r1


title: VK2 cells, IFN epsilon, R4

source_name: VK2 vaginal keratinocyte cell line

value: Epsilon


title: shcontrol_forints11_6h_r1


title: shcontrol_forints11_12h_r2


title: R9A10_H9

city: Guangzhou

first: Jinyong

state: GuangDong

address: 190 Kai Yuan Avenue, Science Park, Guangzhou, China

country: China

institute: Guangzhou Institutes of Biomedicine and Health (GIBH)

data_processing:
Bcl2fastq (verison 2.16.0.10) convert .bcl files into fastq files
Fastq files were aligned to mm10 genome.*.ht2 files to generate *.sam file by HISAT2 (version 2.1.0). And the used samtools (version 0.1.19) to convert and sort the sam file to bam file. StringTie (version 1.3.4) were used to calculated genes expression level in TPM.
Gene expression levels in TPM of different samples were normalized by Stringtie.
Assembly: mm10
Supplementary files format and content: Matrix table with TPM value for every gene and every cell

source_name: iHPCs

growth_protocol: We identified that synergistic and inducible expression of Runx1, Hoxa9, and Hoxa10 in pluripotent stem cells gave rise to engraftable iHPC capable of developing into mature blood cells in C57BL/6 recipients.

extract_protocol:
The procedure of RNA extraction and reverse transcription into double-stranded DNA were prepared using Discover-sc WTA Kit V2 (Vazyme, N711)
Libraries were prepared according to instruction of TruePrepTM  DNA Library Prep Kit V2 (Vazyme,TD503).

treatment_protocol: The single iHPCs were sorted for RNAseq analysis.

value: iHPCs


title: Microglia IP, female, stroke, 3 days (IP_182)

value: Female

value: Microglia

value: Cx3cr1-creERT2 hemi; Ribotag homo


title: Microglia IP, female, stroke, 3 days (IP_210)

value: Stroke

value: IP


title: Microglia IP, female, stroke, 4 hours (IP_181)

value: 4 hours


title: shcontrol_forints11_3h_r2

source_name: HeLa

value: cervical adenocarcinoma


title: KPP-EGP Post-Implantation - GEX [LIB5446560_SAM24405163]

description: LIB5446560_SAM24405163 (B0301 for Desert, B0303 for Inflamed, B0302 for Excluded)


title: R9A10_H8


title: Ect1 cells, no IFN, R3

value: Untreated


title: R9A10_H7


title: VK2 cells, IFN epsilon, R2


title: shcontrol_forints11_3h_r1

value: s4U, IAA


title: Microglia IP, male, sham, 4 hours (IP_153)

value: Male

value: Sham


title: R9A10_H6


title: VK2 cells, IFN epsilon, R1


title: VK2 cells, IFN alpha, R4

value: Alpha


title: R9A10_H5


title: Microglia IP, female, stroke, 3 days (IP_151)

value: 3 days


title: VK2 cells, IFN alpha, R3


title: shcontrol_forints11_1h_r1

value: Tet-pLKO_shGFP stable cells


title: Microglia IP, female, stroke, 4 hours (IP_143)

value: Brain


title: Microglia IP, male, stroke, 3 days (IP_149)


title: VK2 cells, IFN alpha, R2


title: R9A10_H3


title: shcontrol_forints11_30min_r2


title: shcontrol_forints11_30min_r1


title: shcontrol_forints11_15min_r2


title: Microglia IP, female, stroke, 3 days (IP_110)


title: VK2 cells, IFN epsilon, R3

value: Epsilon


title: VK2 cells, IFN alpha, R1


title: R9A10_H2

value: iHPCs


title: VK2 cells, IFN lambda, R4

value: Lambda


title: Microglia IP, male, stroke, 4 hours (IP_166)

value: Microglia


title: shcontrol_forints11_15min_r1


title: Microglia IP, male, sham, 4 hours (IP_108)

value: Cx3cr1-creERT2 hemi; Ribotag homo


title: Microglia IP, male, sham, 4 hours (IP_109)


title: R9A10_H10


title: shcontrol_forints11_1h_r2


title: R9A10_H4


title: Microglia IP, male, stroke, 3 days (IP_107)

value: Stroke


title: shcontrol_forints11_0min_r2


title: Microglia IP, male, stroke, 4 hours (IP_105)

value: IP


title: shints6_24h_noIAA_r2

description:
shINTS6
timecourse_mergedReplicates_shINTS6set_shINTS6_miRNA_quantification.tsv

value: Tet-pLKO_shINTS6 stable cells


title: R9A10_G7


title: R9A10_G9


title: VK2 cells, IFN lambda, R2


title: R9A10_G8


title: Microglia IP, male, sham, 3 days (IP_104)


title: VK2 cells, IFN lambda, R1


title: VK2 cells, no IFN, R4


title: Microglia IP, female, stroke, 4 hours (IP_101)

value: Female

value: 4 hours


title: VK2 cells, no IFN, R3

value: Untreated


title: R9A10_H11


title: Microglia IP, female, sham, 3 days (IP_100)


title: R9A10_G6


title: shints6_24h_noIAA_r1

value: cervical adenocarcinoma


title: shints6_24h_noS4U_r2

value: IAA


title: R9A10_G5


title: VK2 cells, no IFN, R2


title: Microglia whole brain input, male, stroke, 3 days (input_98)

value: Male

value: Input


title: shints6_24h_noS4U_r1


title: R9A10_G4


title: VK2 cells, IFN lambda, R3


title: VK2 cells, no IFN, R1


title: Microglia whole brain input, male, sham, 3 days (input_97)

value: Sham

value: 3 days


title: shints6_24h_r2

value: s4U, IAA


title: R9A10_G3

value: iHPCs


title: shints6_24h_r1


title: shcontrol_forints11_0min_r1

value: Tet-pLKO_shGFP stable cells


title: Microglia whole brain input, female, stroke, 3 days (input_93)

value: Brain

value: Microglia


title: R9A10_G2


title: R9A10_F9


title: shints6_12h_r2


title: shints6_12h_r1


title: shints6_6h_r2


title: R9A10_G10


title: Microglia whole brain input, male, sham, 3 days (input_91)

value: Cx3cr1-creERT2 hemi; Ribotag homo


title: Microglia whole brain input, female, sham, 3 days (input_92)


title: shints6_6h_r1


title: shints6_3h_r1


title: Microglia whole brain input, female, sham, 3 days (input_88)


title: Microglia whole brain input, female, sham, 4 hours (input_239)


title: shints6_3h_r2


title: shints6_1h_r2


title: R9A10_F6


title: R9A10_F8


title: Microglia whole brain input, female, stroke, 4 hours (input_217)

value: Stroke


title: R9A10_F7


title: Microglia whole brain input, male, stroke, 4 hours (input_185)


title: R9A10_F4


title: R9A10_F3


title: shints6_1h_r1

value: Tet-pLKO_shINTS6 stable cells


title: Microglia whole brain input, male, stroke, 4 hours (input_166)


title: R9A10_F2


title: Microglia whole brain input, male, stroke, 3 days (input_89)


title: mESC cells, T7, STM2457, rep4

city: Würzburg

first: Julian

last: Konig

address: Am Hubland

institute: Würzburg University

description: male

data_processing:
Quality control: FastQC (v0.11.8)
Sequencing data was processed using SLAM-DUNK (v0.4.3) (Neumann et. Al., 2019) with the follwing parameeters:
Mapping: Mapping was performed allowing multiple mapping to up to 100 genomic positions for a given read (-n 100).
Filter: Reads were filtered using SLAM-DUNK -filter with default parameters.
SNP annotation/filter: For annotation of single nucleotide polymorphisms (SNPs), all unlabelled samples were merged and SNPs were called using SLAM-DUNK snp with default parameters and -f 0.2.
Count T-to-C transitions: Transition rates were calculated using SLAM-DUNK count with default parameters, providing the SNP annotation of unlabelled samples (-v).
Collapse: If more than one 3’ UTR per gene remained, they were collapsed using SLAM-DUNK collapse
Assembly: GRCm38.p6 (Gencode release M23)

source_name: mESC

growth_protocol: Male mESC were taken from a previous publication (PMID: 25569111).  Cells were cultured in 2i/LIF medium. Briefly, 500 ml medium was generated by adding 235 ml of each DMEM/F12 and neurobasal (Gibco; 21331020, 21103049), 7.5 ml BSA solution (7.5%, ThermoFisher; 11500496), 5 ml penicillin-streptomycin (Gibco; 15140-122), 2 mM L-Glutamin (ThermoSci; 25030024), 100 µM β-mercaptoethanol (Gibco; 21985023), 5 ml mM nonessential amino acids (Gibco; 11140050), 2.5 ml N2 supplement (Gibco; 17502048), 5 ml B27 supplement (Gibco; 17504044), 3 µM CHIR99021 (Sigma; SML1046), 1 µM PD 0325901 (Biomol; 13034-1), 10 ng/ml LIF (IMB Protein Production core facility). Cells were cultured at 37°C  with 5% CO2. Cell culture dishes were coated using gelatin (Sigma; ES-006-B). The medium was exchanged every day and cells were passaged every second day.  For human primary fibroblasts: xxx#

extract_protocol:
After the metabolic labelling, cells were washed twice with 1x PBS and the fresh medium was supplemented with a 100x excess of Uridine. At timepoints increasing in a 1.5x rate, medium was removed and cells were directly lysed in TRIzol reagent in reducing conditions following Lexogens recommended protocol. Total RNA was resuspended in an elution buffer. The iodoacetamide treatment was performed using 5 µg of RNA. The library preparation for sequencing was performed using the QuantSeq 3‘ mRNA-Seq Library Prep Kit for Illumina (FWD)) from Lexogen following the recommended protocol.
NGS library prep was performed with Lexogen`s QuantSeq 3´mRNA-Seq Library Prep Kit FWD following Lexogen`s standard protocol (015UG009V0260). Libraries were prepared with a starting amount of 147ng and amplified in 15 PCR cycles.  Libraries were profiled in a High Sensitivity DNA on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies).   All 71 Libraries  were pooled together in equimolar ratio and sequenced on 3 NextSeq 500 Highoutput Flowcells, SR for 1x84 cycles plus 7 cycles for the index read.

treatment_protocol: mES cells were seeded one day prior to the experiment in a 24-well plate to reach full confluency, according to the doubling time, at the time of sample collection. The metabolic labelling was performed by addition of 100 µM s4U to the mESC medium for 24 h. The medium was exchanged every 3 h. The uridine chase was started after 24 h of labelling. STM2457 or DMSO treatment was performed 6 h prior the uridine chase and continued within the uridine chase.

value: mESC

value: mouse embryonic stem cells (mESC)


title: R9A10_F11


title: shints6_30min_r1


title: mESC cells, T7, STM2457, rep3

description: male


title: Microglia whole brain input, female, stroke, 4 hours (input_143)

value: Female

value: 4 hours


title: Microglia whole brain input, female, sham, 4 hours (input_240)


title: shints6_15min_r2

value: cervical adenocarcinoma


title: mESC cells, T7, STM2457, rep2

description: male


title: R9A10_F10


title: R9A10_F5


title: shints6_15min_r1


title: shints6_0min_r2


title: Astrocyte IP, male, sham, 4 hours (542_IP)

value: Male

value: Astrocyte

value: Aldh1l1-creERT2 hemi; Ribotag homo

value: IP


title: Microglia whole brain input, male, sham, 4 hours (input_109)

value: Sham

value: Input


title: Astrocyte whole brain input, male, sham, 4 hours (542_Input)


title: mESC cells, T7, STM2457, rep1

description: male


title: R9A10_E9

value: iHPCs


title: shints6_30min_r2

value: s4U, IAA


title: shcontrol_forints6_24h_noIAA_r2

description:
shGFP
timecourse_mergedReplicates_shINTS6set_shControl_miRNA_quantification.tsv

value: Tet-pLKO_shGFP stable cells


title: mESC cells, T7, DMSO, rep4

description: male

value: DMSO


title: shints6_0min_r1


title: R9A10_E8


title: Astrocyte IP, female, stroke, 4 hours (541_IP)

value: Brain

value: Stroke


title: Astrocyte IP, male, stroke, 4 hours (536_IP)


title: mESC cells, T7, DMSO, rep3

description: male

value: mESC


title: R9A10_E6


title: shcontrol_forints6_24h_noIAA_r1


title: mESC cells, T7, DMSO, rep2

description: male

value: mouse embryonic stem cells (mESC)


title: R9A10_E5


title: Astrocyte whole brain input, male, stroke, 4 hours (536_Input)


title: shcontrol_forints6_24h_noS4U_r2

value: IAA


title: R9A10_E7


title: mESC cells, T7, DMSO, rep1

description: male


title: Astrocyte IP, male, stroke, 3 days (535_IP)

value: 3 days


title: mESC cells, T6, STM2457, rep4

description: male


title: Astrocyte IP, male, sham, 3 days (534_IP)


title: R9A10_E4


title: R9A10_E3


title: shcontrol_forints6_24h_noS4U_r1

value: cervical adenocarcinoma


title: shcontrol_forints6_24h_r2


title: R9A10_E2


title: mESC cells, T6, STM2457, rep3

description: male


title: shcontrol_forints6_24h_r1


title: mESC cells, T6, STM2457, rep1

description: male


title: Astrocyte whole brain input, male, sham, 3 days (531_Input)

value: Male


title: Astrocyte IP, male, sham, 3 days (531_IP)

value: Astrocyte


title: R9A10_E11


title: shcontrol_forints6_12h_r1


title: mESC cells, T6, STM2457, rep2

description: male


title: R9A10_E10


title: Astrocyte IP, female, sham, 3 days (530_IP)

value: Female

value: Aldh1l1-creERT2 hemi; Ribotag homo


title: Astrocyte IP, female, stroke, 3 days (529_IP)

value: IP


title: mESC cells, T6, DMSO, rep4

description: male


title: R9A10_D9


title: shcontrol_forints6_12h_r2


title: shcontrol_forints6_6h_r2


title: R9A10_D8


title: mESC cells, T6, DMSO, rep3

description: male


title: Astrocyte IP, male, stroke, 3 days (527_IP)


title: shcontrol_forints6_6h_r1


title: shcontrol_forints6_3h_r2


title: mESC cells, T6, DMSO, rep2

description: male


title: Astrocyte whole brain input, male, stroke, 3 days (527_Input)

value: Input


title: mESC cells, T6, DMSO, rep1

description: male


title: Astrocyte IP, female, sham, 3 days (523_IP)

value: Sham


title: shcontrol_forints6_1h_r2

value: s4U, IAA


title: mESC cells, T5, STM2457, rep3

description: male


title: shcontrol_forints6_1h_r1

value: Tet-pLKO_shGFP stable cells


title: R9A10_D5

value: iHPCs


title: Astrocyte IP, male, sham, 4 hours (506_IP)

value: Brain

value: 4 hours


title: R9A10_D4


title: R9A10_D3


title: Astrocyte IP, female, sham, 4 hours (499_IP)


title: shcontrol_forints6_30min_r2


title: Astrocyte IP, female, stroke, 4 hours (502_IP)

value: Stroke


title: mESC cells, T5, STM2457, rep1

description: male

value: mESC

value: mouse embryonic stem cells (mESC)


title: R9A10_D2


title: shcontrol_forints6_30min_r1


title: mESC cells, T5, DMSO, rep4

description: male

value: DMSO


title: R9A10_D7


title: Astrocyte IP, female, stroke, 3 days (526_IP)

value: 3 days


title: Astrocyte IP, male, sham, 4 hours (497_IP)

value: Male


title: R9A10_D11


title: shcontrol_forints6_15min_r2

value: cervical adenocarcinoma


title: mESC cells, T5, DMSO, rep3

description: male


title: mESC cells, T5, STM2457, rep4

description: male


title: shcontrol_forints6_3h_r1


title: Astrocyte IP, male, stroke, 4 hours (495_IP)

value: Astrocyte


title: R9A10_D6


title: mESC cells, T5, DMSO, rep2

description: male


title: R9A10_D10


title: shcontrol_forints6_15min_r1


title: mESC cells, T5, STM2457, rep2

description: male


title: mESC cells, T5, DMSO, rep1

description: male


title: shcontrol_forints6_0min_r2


title: Astrocyte IP, female, stroke, 4 hours (490_IP)

value: Female

value: Aldh1l1-creERT2 hemi; Ribotag homo


title: R9A10_C8


title: shcontrol_forints6_0min_r1

description: shGFP


title: mESC cells, T4, STM2457, rep3

description: male


title: smrna_hela_shints11_cytoplasm_r2

molecule: cytoplasmic RNA

growth_protocol: HeLa cell lines containing stable shRNA were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. DMEM medium was supplemented with 10% FBS and puromycin (2 μg/ml). Knockdowns were induced by adding of 1 μg/ml doxycycline (Dox) into the culture medium daily for 3 days.

extract_protocol:
Cells were fractionated into nucleus and cytoplasm according to Bhatt et al., Cell 2012, https://doi.org/10.1016/j.cell.2012.05.043. Nuclear RNA was extracted using Trizol reagent  (Thermo Fisher Scientific, #15596026), cytoplasmic RNA using Trizol LS reagent (Thermo Fisher Scientific #10296028) according to the manufacturer’s instructions. Genomic DNA was removed by Turbo DNAse treatment
Small RNA libraries were prepared using the SMARTer smRNA-seq kit (Takara, #635030) with 700 ng of total RNA; 5% (35 ng) of Drosophila RNA were added as spike-in

value: Tet-pLKO_shINTS11 stable cells


title: R9A10_C7


title: mESC cells, T4, STM2457, rep2

description: male


title: Astrocyte IP, female, stroke, 4 hours (484_IP)

value: IP


title: Astrocyte IP, male, stroke, 4 hours (483_IP)


title: R9A10_C5


title: mESC cells, T4, STM2457, rep1

description: male


title: smrna_hela_shints11_cytoplasm_r1


title: R9A10_C4


title: Astrocyte IP, male, stroke, 4 hours (481_IP)


title: Astrocyte IP, female, sham, 4 hours (491_IP)

value: Sham


title: mESC cells, T4, DMSO, rep4

description: male


title: R9A10_C3


title: Astrocyte IP, male, sham, 4 hours (480_IP)


title: Astrocyte IP, female, sham, 4 hours (478_IP)


title: mESC cells, T4, DMSO, rep3

description: male


title: R9A10_C9


title: smrna_hela_shints11_nuclear_r2

molecule: nuclear RNA


title: smrna_hela_shints11_nuclear_r1


title: Astrocyte IP, male, stroke, 4 hours (378_IP)


title: R9A10_C2


title: mESC cells, T4, DMSO, rep2

description: male


title: smrna_hela_shints6_cytoplasm_r2

value: Tet-pLKO_shINTS6 stable cells


title: R9A10_C11

value: iHPCs


title: smrna_hela_shints6_cytoplasm_r1


title: mESC cells, T3, STM2457, rep4

description: male


title: smrna_hela_shints6_nuclear_r2


title: Astrocyte IP, male, stroke, 3 days (371_IP)

value: Brain


title: mESC cells, T4, DMSO, rep1

description: male


title: smrna_hela_shints6_nuclear_r1


title: Astrocyte IP, male, sham, 3 days (368_IP)


title: mESC cells, T3, STM2457, rep2

description: male


title: mESC cells, T3, STM2457, rep3

description: male


title: R9A10_B8


title: smrna_hela_shcontrol_cytoplasm_r2

description: shGFP

value: Tet-pLKO_shGFP stable cells


title: smrna_hela_shcontrol_cytoplasm_r1

description: shGFP


title: R9A10_B6


title: smrna_hela_shcontrol_nuclear_r2

description: shGFP


title: mESC cells, T3, STM2457, rep1

description: male


title: R9A10_B7


title: Astrocyte IP, male, sham, 4 hours (363_IP)

value: 4 hours


title: Astrocyte IP, female, stroke, 4 hours (364_IP)

value: Stroke


title: Astrocyte whole brain input, male, sham, 4 hours (363_Input)

value: Input


title: Astrocyte IP, male, stroke, 3 days (373_IP)


title: smrna_hela_shcontrol_nuclear_r1

description: shGFP


title: mESC cells, T3, DMSO, rep4

description: male

value: mESC

value: mouse embryonic stem cells (mESC)

value: DMSO


title: R9A10_C10


title: R9A10_B4


title: mESC cells, T3, DMSO, rep3

description: male


title: mESC cells, T3, DMSO, rep2

description: male


title: R9A10_B9


title: R9A10_B5


title: Astrocyte IP, female, stroke, 4 hours (355_IP)


title: R9A10_B3


title: Astrocyte IP, female, stroke, 4 hours (357_IP)


title: smrna_hela_eq_dox_r2_30mio

extract_protocol:
Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, #15596026) according to the manufacturer’s instructions. Genomic DNA was removed by Turbo DNAse treatment (Invitrogen, #AM1907).
Small RNA libraries were prepared using the SMARTer smRNA-seq kit (Takara, #635030) with 800 ng of total RNA

value: cervical adenocarcinoma


title: smrna_hela_eq_dox_r1_30mio


title: R9A10_B11


title: Astrocyte IP, male, stroke, 4 hours (350_IP)

value: Male

value: Astrocyte


title: smrna_hela_wt_dox_r2_30mio

description: WT


title: mESC cells, T3, DMSO, rep1

description: male


title: Astrocyte IP, female, stroke, 4 hours (338_IP)

value: Female

value: Aldh1l1-creERT2 hemi; Ribotag homo


title: R9A10_B10


title: smrna_hela_wt_dox_r1_30mio

description: WT


title: mESC cells, T2, STM2457, rep3

description: male


title: R9A10_A9


title: Astrocyte IP, male, stroke, 4 hours (331_IP)

value: IP


title: R9A10_A8


title: smrna_hela_shints11_r2_30mio

value: Tet-pLKO_shINTS11 stable cells


title: mESC cells, T2, STM2457, rep2

description: male


title: Astrocyte whole brain input, male, stroke, 4 hours (331_Input)


title: mESC cells, T2, STM2457, rep1

description: male


title: mESC cells, T2, DMSO, rep4

description: male


title: R9A10_A7


title: Astrocyte IP, female, sham, 3 days (317_IP)

value: Sham

value: 3 days


title: smrna_hela_shints11_r1_30mio


title: smrna_hela_shints6_r2_30mio

value: Tet-pLKO_shINTS6 stable cells


title: smrna_hela_shints6_r1_30mio


title: Astrocyte IP, female, sham, 3 days (311_IP)

value: Brain


title: Astrocyte IP, male, sham, 4 hours (326_IP)

value: 4 hours


title: R9A10_A5

value: iHPCs


title: mESC cells, T2, DMSO, rep3

description: male


title: R9A10_A6


title: R9A10_A4


title: mESC cells, T2, STM2457, rep4

description: male


title: smrna_hela_shcontrol_r2_30mio

description: shGFP

value: Tet-pLKO_shGFP stable cells


title: Astrocyte IP, male, stroke, 3 days (306_IP)

value: Stroke


title: mESC cells, T2, DMSO, rep1

description: male

value: mESC

value: mouse embryonic stem cells (mESC)


title: mESC cells, T2, DMSO, rep2

description: male

value: DMSO


title: Astrocyte IP, male, stroke, 3 days (305_IP)

value: Male


title: mESC cells, T1, STM2457, rep4

description: male


title: R9A10_A11


title: Astrocyte whole brain input, male, stroke, 3 days (305_Input)

value: Astrocyte

value: Input


title: Astrocyte IP, female, sham, 3 days (300_IP)

value: Female

value: Aldh1l1-creERT2 hemi; Ribotag homo


title: mESC cells, T1, STM2457, rep3

description: male


title: R9A10_A10


title: mESC cells, T1, STM2457, rep2

description: male


title: iN_44_cre_d43_rep2

city: Heidelberg

first: Bryce

address: In Neuenheimer Feld 280

institute: DKFZ

data_processing:
Sequenced reads were trimmed for adaptor sequence and masked for low-complexity or low-quality sequence
Filtered reads were mapped to hg38 with STAR to generate raw counts files
Differential expression and normalised counts were determined using DESeq2 (R package version 1.28.1)
Genome_build: hg38
Supplementary_files_format_and_content: Tab-delimited text file with raw counts for all samples
Supplementary_files_format_and_content: Tab-delimited text file with DESeq2 output for all conditions (sample-collapsed)
Supplementary_files_format_and_content: Tab-delimited text file with normalised counts for all samples

source_name: NGN2-induced neurons

growth_protocol: H1 (male) human ES cells (WiCell) were maintained feeder-free in mTeSR Plus medium (Stem Cell Technologies) and one allele of human MYT1L (ENSG00000186487) was targeted by  homologous recombination to flank exon 7 with loxP sites. Cre-mediated recombination deleted exon 7 resulting in a frameshift starting in exon 8 at amino acid (aa) 19 and terminating in a premature STOP at aa 69 in exon 9 (p.V19AfsX51). Heterozygous targeting was confirmed by Sanger sequencing that showed insertion of loxP sites within one allele for each clone (+/fl) and MYT1L protein depletion was confirmed by Western blot directly in induced human neurons.

extract_protocol:
For human neurons pure cultures of induced human neurons at day 7 or induced human neurons co-cultured with primary mouse glia at day 43 were harvested by addition of TRIzol to the cultures (Invitrogen) and RNA was isolated using the RNA Miniprep kit (Zymo Research).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Lentivirus encoding doxycycline-inducible Ngn2 and either cre-recombinase or an inactive truncated delta-cre-recombinase, both under control of the ubiquitin promoter, were used to transduce conditional MYT1L knockout cells (+/fl). On day 0, Doxycycline (2 mg/ml, Sigma) was added to induce Ngn2 expression and retained in the medium until day 14. On day 1, cells were treated with Accutase (Gibco) and plated on Matrigel (Corning)-coated 6-well plates in DMEM/F12 containing N2-supplement, non-essential amino acids (all from Invitrogen), human BDNF (10 ng/ml, PeproTech), human NT-3 (10 ng/ml, PeproTech), and mouse Laminin-1 (0.2 mg/ml, Invitrogen) containing 10 µM Y-27632 (Axon Medchem) and puromycin (2 µg/ml c.f.) was added for 3 days to select infected cells. On day 4, iN cells were treated with Accutase and replated in Neurobasal-A medium supplemented with B27/glutamax (Invitrogen) containing BDNF, NT3, and Laminin-1, and Ara-C (2 µM, Sigma). For day 7 harvest, iN cells were seeded on Matrigel-coated 12-well plates without addition of glia. For functional maturation, iN cells were seeded on Matrigel-coated coverslips together with primary mouse glia with media changes every 2 days. After 1 week media was gradually exchanged with MEM supplemented with 0.5% glucose, 0.02% NaHCO3), 2 µM Ara-C (all Sigma), Transferrin (100 µg/ml c.f.; Merck), 5% FBS, B27, 0.5 mM L-glutamine (all Invitrogen), following media changes every 3–4 days for the duration of the experiment. All cells were grown at 37 °C and 5% CO2.

tag: donor cell line

value: neuron

tag: harvest day

value: cre

tag: clone

value: clone 2


title: iN_44_cre_d43_rep1


title: Astrocyte IP, male, stroke, 4 hours (289_IP)

tag: tissue

tag: genotype

value: IP


title: iN_44_dcre_d43_rep2

value: dcre


title: Astrocyte IP, male, stroke, 4 hours (288_IP)

value: Brain

tag: time

value: 4 hours


title: 513_WT_rep1

description:
mouse_bulk_deseq2_table.tsv
mouse_bulk_norm_counts.tsv
mouse_bulk_raw_counts.tsv

data_processing:
Sequenced reads were trimmed for adaptor sequence and masked for low-complexity or low-quality sequence
Filtered reads were mapped to mm10 with STAR to generate raw counts files
Differential expression and normalised counts were determined using DESeq2 (R package version 1.28.1) with sequencing batch ID and sex as covariates
Assembly: mm10
Supplementary files format and content: Tab-delimited text file with raw counts for all samples
Supplementary files format and content: Tab-delimited text file with DESeq2 output for all conditions (sample-collapsed)
Supplementary files format and content: Tab-delimited text file with normalised counts for all samples

source_name: mouse cortex

growth_protocol: The cortices of P0 Myt1l mutant or control mouse pups were isolated and cultured on Matrigel-coated coverslips in MEM with 2% B27, 0.04% glucose, 2mM L-glutamine and 5% FBS for one day and then in Neurobasal-A medium with 2% B27, 2 mM L-glutamine and 5% FBS. Three days after plating, the medium was supplemented with 2 µM Ara-C.

extract_protocol:
Cells were placed into TRIzol (Invitrogen) followed by RNA harvest using RNA Miniprep kit (Zymo Research).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Myt1l frameshift mutation was induced using CRISPR/Cas9 ribonucleoprotein (RNP) electroporation into mouse zygotes that caused a 7 bp deletion in Myt1l (ENSG00000186487) exon 6, resulting in a frameshift at amino acid (aa) 11 and terminating in a premature STOP at aa 78 in exon 9 (p.R11GfsX68). Myt1l mutant mice showed no nonsense-mediated decay of Myt1l transcript, but Western blot analysis showed expected depletion of full-length MYT1L, suggesting that this mouse model mimicked MYT1L deficiency reported in a patient with a premature STOP mutation at aa 75 (R75X).

value: cortex

tag: age

value: WT

value: male


title: mESC cells, T1, STM2457, rep1

description: male

value: mESC

tag: cell type

value: mouse embryonic stem cells (mESC)


title: iN_44_dcre_d43_rep1


title: mESC cells, T1, DMSO, rep4

description: male

value: DMSO


title: R9A10_A3

value: iHPCs


title: iN_44_cre_d7_rep1


title: Astrocyte IP, female, stroke, 4 hours (277_IP)

value: Astrocyte

value: Stroke


title: smrna_hela_shcontrol_r1_30mio

description: shGFP

value: cervical adenocarcinoma

tag: cell source

tag: modification

value: Tet-pLKO_shGFP stable cells


title: mESC cells, T1, DMSO, rep3

description: male


title: iN_44_dcre_d7_rep1

value: neuron


title: 511_WT_rep1


title: 511_WT_rep2


title: Astrocyte IP, male, sham, 4 hours (230_IP)

value: Male

value: Aldh1l1-creERT2 hemi; Ribotag homo

value: Sham


title: 510_WT_rep2


title: mESC cells, T1, DMSO, rep1

description: male


title: iN_44_dcre_d7_rep2

value: dcre

value: clone 2


title: 510_WT_rep1

value: cortex


title: iN_30_cre_d43_rep2

value: cre

value: clone 1


title: 513_WT_rep2

value: WT

value: male


title: Astrocyte IP, female, stroke, 4 hours (223_IP)

value: Brain

value: Female

value: 4 hours

value: IP


title: mESC cells, T0, STM2457, rep4

description: male

value: mESC

value: mouse embryonic stem cells (mESC)


title: mESC cells, T0, STM2457, rep3

description: male


title: Astrocyte IP, male, stroke, 4 hours (287_IP)

value: Astrocyte

value: Stroke


title: 508_HET_rep2

value: HET


title: Astrocyte whole brain input, female, stroke, 4 hours (223_Input)

value: Aldh1l1-creERT2 hemi; Ribotag homo

value: Input


title: 508_HET_rep1


title: mESC cells, T0, STM2457, rep2

description: male


title: iN_30_cre_d43_rep1

value: neuron


title: iN_30_dcre_d43_rep1

value: dcre


title: 506_HET_rep2

value: cortex


title: iN_44_cre_d7_rep2

value: cre

value: clone 2


title: Astrocyte IP, female, sham, 4 hours (222_IP)

value: Sham


title: iN_30_dcre_d43_rep2

value: clone 1


title: mESC cells, T1, DMSO, rep2

description: male

value: DMSO


title: 506_HET_rep1

value: male


title: mESC cells, T0, STM2457, rep1

description: male


title: 505_HET_rep2


title: mESC cells, T0, DMSO, rep4

description: male


title: iN_30_cre_d7_rep1


title: mESC cells, T0, DMSO, rep3

description: male


title: Astrocyte IP, female, stroke, 3 days (203_IP)

value: Brain

value: Female

value: 3 days

value: IP


title: Astrocyte whole brain input, female, sham, 4 hours (222_Input)

value: Astrocyte

value: 4 hours


title: Astrocyte IP, male, sham, 4 hours (228_IP)

value: Male

value: Aldh1l1-creERT2 hemi; Ribotag homo


title: 505_HET_rep1

value: HET


title: mESC cells, T0, DMSO, rep2

description: male

value: mESC

value: mouse embryonic stem cells (mESC)


title: iN_30_dcre_d7_rep2

value: neuron

value: dcre


title: mouse_P0_3_Myt1l

data_processing:
Sequenced reads were trimmed for adaptor sequence and masked for low-complexity or low-quality sequence
Filtered reads were mapped to mm10 and peaks called based upon coverage using nf-core CUT&RUN pipeline v1.0.0
For each timepoint, peaks that were present in two or more replicates were considered consensus peaks.
Assembly: mm10
Supplementary files format and content: Tab-delimited text file with peak information for adult (bed format)
Supplementary files format and content: Tab-delimited text file with peak information for E18.5 (bed format)
Supplementary files format and content: Tab-delimited text file with peak information for P0 (bed format)
Library strategy: CUT&RUN

extract_protocol:
The cortex of E18.5 or the prefrontal cortex of P0, and adult (3-month-old) mice was dissected and dissociated into single cells for CUT&RUN.
CUT&RUN libraries were prepared for sequencing using standard Illumina protocols

value: cortex

value: WT

value: unknown


title: Astrocyte IP, female, stroke, 3 days (198_IP)

value: Stroke


title: Astrocyte IP, female, sham, 3 days (196_IP)

value: Brain

value: Sham


title: mouse_P0_3_IgG


title: iN_29_cre_d43_rep2

value: cre

value: clone 2


title: mouse_P0_2_Myt1l


title: Astrocyte whole brain input, female, sham, 3 days (196_Input)

value: Female

value: Astrocyte

value: 3 days

value: Input


title: mESC cells, no 4SU, STM2457, rep3

description: male


title: mouse_P0_2_IgG


title: mESC cells, no 4SU, STM2457, rep2

description: male


title: iN_30_cre_d7_rep2

value: clone 1


title: Astrocyte IP, male, stroke, 3 days (191_IP)

value: Male

value: Aldh1l1-creERT2 hemi; Ribotag homo

value: IP


title: mESC cells, no 4SU, STM2457, rep4

description: male

value: mESC

value: mouse embryonic stem cells (mESC)


title: iN_29_dcre_d43_rep2

value: neuron

value: dcre


title: Astrocyte IP, male, sham, 3 days (193_IP)


title: Astrocyte IP, female, sham, 3 days (202_IP)


title: mouse_P0_1_Myt1l

value: cortex

value: WT

value: unknown


title: mESC cells, no 4SU, DMSO, rep4

description: male

value: DMSO


title: Astrocyte IP, male, sham, 3 days (184_IP)

value: Brain

value: Sham


title: mESC cells, no 4SU, STM2457, rep1

description: male


title: iN_29_cre_d43_rep1

value: cre

value: clone 2


title: iN_29_dcre_d43_rep1


title: mouse_P0_1_IgG


title: iN_29_cre_d7_rep1


title: Astrocyte IP, male, sham, 3 days (183_IP)

value: Astrocyte

value: 3 days


title: mouse_E18_3_Myt1l


title: iN_30_dcre_d7_rep1

value: clone 1


title: mESC cells, no 4SU, DMSO, rep3

description: male


title: mESC cells, T0, DMSO, rep1

description: male


title: iN_29_cre_d7_rep2


title: mouse_E18_3_IgG


title: Astrocyte whole brain input, male, sham, 3 days (182_Input)

value: Male

value: Aldh1l1-creERT2 hemi; Ribotag homo

value: Input


title: mouse_E18_2_Myt1l


title: iN_29_dcre_d7_rep2


title: iN_29_dcre_d7_rep1


title: Astrocyte IP, male, sham, 3 days (161_IP)

value: IP


title: Astrocyte IP, female, stroke, 3 days (159_IP)

value: Female

value: Stroke


title: female mESC, single clone39, DMSO

data_processing:
Quality control: FastQC (v0.11.8)
Trimming: Cutadapt (v1.18) with non-default parameters: cutadapt -a file:NexteraPE-PE.fa --minimum-length 50 (additional parameter used for C643, HEK293T, RPE1, male mESC samples: --cut 1)
Mapping: STAR (v2.7.3a) with non-default parameters: STAR --runMode alignReads --outFilterMismatchNoverLmax 0.04 --outFilterMismatchNmax 999 --outStd SAM --outSAMattributes NH HI AS nM MD --outSJfilterReads Unique --outSAMunmapped Within --runThreadN 4 --readFilesCommand zcat --sjdbOverhang READLENGTH-1
Filter/remove secondary hits: Samtools (v1.9) with parameters: samtools view -F 256
Counts per gene: Subread (v2.0.0) with non-default parameters: featureCounts --donotsort -s2 -t exon (default used for -g gene_id)
Assembly: human: GRCh38.p12 (Gencode release 31) (+ ERCC spike-ins in C643, HEK293T, RPE1)
Assembly: mouse: GRCm38.p6 (Gencode release M23) (+ ERCC spike-ins in male mESC)
Supplementary files format and content: tab-delimited text (.tsv) files include absolute read counts per gene for each sample

growth_protocol: For C643 and HEK293T: Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 1% L-glutamine (Life Technologies) and 1% penicillin/streptomycin (P/S Life Technologies). For RPE1: Cells were cultured in DMEM/F12 (Life Technologies) supplemented with 10% foetal bovine serum (FBS, Life Technologies), 1% penicillin/streptomycin (P/S, Life Technologies), 1% L-Glutamin and 0.04% Hygromycin B (Life Technologies). Cells were grown at 37◦C with 5% CO2. For male and female mESC: Female mouse embryonic stem cells (mESC) were taken from a previous publication (PMID: 24506884). Single clones were picked and cultured until confluency. Male mESC were taken from a previous publication (PMID: 25569111).  Cells were cultured in 2i/LIF medium. Briefly, 500 ml medium was generated by adding 235 ml of each DMEM/F12 and neurobasal (Gibco; 21331020, 21103049), 7.5 ml BSA solution (7.5%, ThermoFisher; 11500496), 5 ml penicillin-streptomycin (Gibco; 15140-122), 2 mM L-Glutamin (ThermoSci; 25030024), 100 µM β-mercaptoethanol (Gibco; 21985023), 5 ml mM nonessential amino acids (Gibco; 11140050), 2.5 ml N2 supplement (Gibco; 17502048), 5 ml B27 supplement (Gibco; 17504044), 3 µM CHIR99021 (Sigma; SML1046), 1 µM PD 0325901 (Biomol; 13034-1), 10 ng/ml LIF (IMB Protein Production core facility). Cells were cultured at 37°C  with 5% CO2. Cell culture dishes were coated using 0.1% gelatin (Sigma; ES-006-B). The medium was exchanged every day and cells were passaged every second day.

extract_protocol:
For RNA extraction, cells were washed twice with ice cold 1x PBS and collected on ice. For total RNA isolation the RNeasy Plus Mini Kit (Qiagen) was used following the manufacturer’s instructions.
RNA-seq: All NGS library preps were performed with Illumina's Stranded mRNA Prep Ligation Kit following Stranded mRNA Prep Ligation Reference Guide (June 2020) (Document # 1000000124518 v00). For RNA-seq in HEK293T and C643 cells: Libraries were prepared with a starting amount of 350 ng for C643 samples and 1000 ng for HEK293T samples and amplified in 11 PCR cycles and 10 cycles respectively. For normalization, 2μl of a 1:300 dilution of ERCC spike-ins (Ambion) were added to the starting material for C643 samples and for HEK293T samples 2μl of a 1:100 dilution. Libraries were profiled in a DNA 1000 chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All samples were pooled in equimolar ratio and sequenced on 1 NextSeq500 Highoutput FC, SR for 1x 80 cycles plus 10 cycles for the index read plus 1 dark cycle upfront Read 1. For RNAseq in male mESC cells upon 24 h of STM2457 treatment: Libraries were prepared with a starting amount of 1000 ng and amplified in 10 PCR cycles. For normalization, 2μl of a 1:100 dilution of ERCC spike-ins (Ambion) were added to the starting material. Libraries were profiled in a DNA 1000 chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All samples were pooled in equimolar ratio and sequenced on 1 NextSeq500 Highoutput FC, SR for 1x 155 cycles plus 10 cycles for the index read and 1 dark cycle upfront Read 1. For RNAseq in RPE1 cells: Libraries were prepared with a starting amount of 1000 ng and amplified in 10 PCR cycles. For normalization, 2μl of a 1:100 dilution of ERCC spike-ins (Ambion) were added to the starting material. Libraries were profiled in a HS DNA chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All samples were pooled in equimolar ratio and sequenced on 1 NextSeq500 Highoutput FC, SR for 1x 80 cycles plus 10 cycles for the index read and 1 dark cycle upfront Read 1. For RNAseq in male mESC upon STM2457 timecourse 3h-12 h: Libraries were prepared with a starting amount of 1000 ng and amplified in 10 cycles. For normalization, 2μl of a 1:100 dilution of ERCC spike-ins (Ambion) were added to the starting material. Libraries were profiled in a DNA 1000 chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All samples were pooled in equimolar ratio and sequenced on 2 NextSeq500 Highoutput FC, SR for 1x 80 cycles plus 10 cycles for the index read plus 1 dark cycle upfront Read 1.  For RNAseq in female mESC single clones: Libraries were prepared with a starting amount of 500 ng and amplified in 10 PCR cycles. Two post PCR purification steps were performed to exclude residual primer and adapter dimers. Libraries were profiled in a DNA 1000 chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All 12 samples were pooled in equimolar ratio and sequenced on 1 NextSeq500 Highoutput FC, SR for 1x 79 cycles plus 10 cycles for the index read and 2 dark cycles upfront Read 1.

treatment_protocol: Cells were cultured in respective medium supplemented with Mettl3 inhibitor STM2457 or DMSO as negative control for 3h and up to 24h.

value: female mouse embryonic stem cells (mESC)

tag: gender

value: female


title: iN_51_cre_d7_rep1

value: neuron


title: iN_51_cre_d7_rep2


title: female mESC, single clone39, STM2457

tag: cell line


title: mouse_E18_1_Myt1l

value: cortex

value: WT

value: unknown


title: Astrocyte whole brain input, female, stroke, 3 days (157_Input)

value: Brain


title: mouse_adult_HET_3_Myt3l

description: mouse_adult_HET_Myt1l.cat.sort.merge.flat.bed

value: adult

value: HET


title: Astrocyte IP, female, stroke, 3 days (157_IP)

value: Astrocyte

value: 3 days


title: Astrocyte IP, male, stroke, 3 days (133_IP)

value: Male


title: iN_51_dcre_d7_rep1

value: dcre

value: clone 1


title: female mESC, single clone38, STM2457


title: female mESC, single clone38, DMSO

value: DMSO


title: mouse_E18_1_IgG


title: Astrocyte IP, male, sham, 3 days (182_IP)

value: Aldh1l1-creERT2 hemi; Ribotag homo

value: Sham

value: IP


title: mouse_adult_HET_3_IgG


title: iN_51_dcre_d7_rep2


title: iN_43_cre_d43_rep2

value: cre


title: female mESC, single clone35, DMSO

value: female mouse embryonic stem cells (mESC)

value: female


title: female mESC, single clone35, STM2457


title: mESC cells, no 4SU, DMSO, rep1

description: male

value: mESC

value: mouse embryonic stem cells (mESC)


title: Astrocyte IP, female, stroke, 3 days (114_IP)

value: Brain

value: Female

value: Stroke


title: Astrocyte IP, female, stroke, 3 days (111_IP)

value: Astrocyte


title: mouse_adult_HET_2_IgG

value: cortex

value: adult

value: HET

value: unknown


title: female mESC, single clone7, DMSO

value: DMSO


title: mouse_E18_2_IgG

value: WT


title: Astrocyte IP, female, sham, 3 days (110_IP)

value: Aldh1l1-creERT2 hemi; Ribotag homo

value: Sham

value: 3 days


title: iN_43_dcre_d43_rep1

value: neuron

value: dcre

value: clone 1


title: mouse_adult_HET_1_Myt1l


title: female mESC, single clone7, STM2457

value: female mouse embryonic stem cells (mESC)

value: female


title: mouse_adult_HET_1_IgG


title: mouse_adult_WT_3_Myt3l

description: mouse_adult_WT_Myt1l.cat.sort.merge.flat.bed


title: mouse_adult_HET_2_Myt2l


title: iN_43_dcre_d43_rep2


title: mouse_adult_WT_2_Myt2l


title: iN_43_dcre_d7_rep2


title: mouse_adult_WT_3_IgG


title: female mESC, single clone4, STM2457


title: female mESC, single clone3, DMSO


title: iN_43_cre_d7_rep2

value: cre


title: NFAT2_CARIT - Replicate 2

city: New Haven

first: Joe

last: Craft

state: CT

address: 300 Cedar St

department: Internal Medicine

institute: Yale University

data_processing:
Sequences were trimmed, processed, mapped, aligned and normalized using Partek Flow software v10
Assembly: mm10
Supplementary files format and content: tab limited text files
Supplementary files format and content: normalized counts in CPM, differential expression using DESeq2

source_name: CD4 Cells, Transduced with NFAT2_CARIT vector

growth_protocol: Primary T cells were incubated in RPMI-1640 supplemented with 10%FBS

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen).
RNA sample was submitted to Yale Center for Genoma Analysis where library construction and paired-end sequencing was performed.

treatment_protocol: Primary T cells were transduced following the retroviral transduction protocol outlined in the manuscript

value: Primary Murine CD4+ T Cells

value: Isolated from C57BL/6 Mice

value: Transduced with NFAT2_CARIT vector


title: NFAT2_CARIT - Replicate 3


title: NFAT2_CA - Replicate 3

source_name: CD4 Cells, Transduced with NFAT2_CA vector

value: Transduced with NFAT2_CA vector


title: iN_43_dcre_d7_rep1


title: female mESC, single clone3, STM2457


title: NFAT2_CA - Replicate 2


title: mESC cells, no 4SU, DMSO, rep2

description: male

value: mESC

value: mouse embryonic stem cells (mESC)


title: mouse_adult_WT_2_IgG

value: cortex

value: adult

value: unknown


title: mESC, STM2457, 12h, rep4

value: male


title: iN_34_cre_d43_rep1


title: NFAT2_CA - Replicate 1


title: iN_43_cre_d7_rep1


title: female mESC, single clone4, DMSO

value: DMSO


title: mouse_adult_WT_1_Myt1l

value: WT


title: Control - Replicate 3

source_name: CD4 Cells, Transduced with CTRL vector

value: Transduced with CTRL vector


title: mESC, STM2457, 12h, rep3


title: NFAT2_CARIT - Replicate 1


title: mouse_adult_WT_1_IgG


title: Control - Replicate 2


title: iN_34_dcre_d43_rep2

value: neuron

value: dcre

value: clone 1


title: mESC, STM2457, 12h, rep2


title: iN_34_dcre_d43_rep1


title: Mrps5 fl/fl; MCM mouse heart tissue, Mrps5 cKO for 14 weeks, AAV9 klf15 infection for 7 weeks, rep 4

city: Hang Zhou

first: Feng

last: Gao

state: Zhejiang

address: Kaixuan road 268

department: Department of Cardiology

institute: Second Afliated Hospital, Zhejiang University School of Medicine

data_processing:
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated.
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed.
Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias wascorrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies (http://www.genome.jp/kegg/). We used clusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathways.
Assembly: mm9
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: Heart

growth_protocol: Mrps5 fl/fl; MCM mouse were generated and grown up to 8 weeks old in SPF animal labratory

extract_protocol:
RNA was harvested using Trizol (Life). RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) . RNA concentration was measured using Qubit® RNA Assay Kit in Qubit®2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently  performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

treatment_protocol: 8 weeks old Mrps5 fl/fl; MCM mouse were treated with tamoxifen through intraperitoneal injection at the dosage of 45 mg/kg/day for 5 continuous days to activate Cre recombinase and delete Mrps5 gene in genome. 7 weeks after Mrps5 gene deletion, infected Mrps5 fl/fl; MCM mouse heart with AAV9 luci virus or klf15 virus, and heart samples were harvested at 7 weeks after AAV9 luci or klf15 virus infection.

value: Heart

value: Mrps5 fl/fl; MCM

value: Tamoxifen injection

value: AAV9 klf15 injection

value: 14 weeks after tamoxifen injection (7 weeks after AAV9 injection)


title: Control - Replicate 1

value: Primary Murine CD4+ T Cells

value: Isolated from C57BL/6 Mice


title: iN_43_cre_d43_rep1

value: cre


title: iN_34_cre_d7_rep1


title: mESC, STM2457, 12h, rep1

value: mESC

value: mouse embryonic stem cells (mESC)


title: CD3_CD28_ICOS - Replicate 3

source_name: CD4 Cells, Stimulated with Anti-CD3/Anti-CD28/Anti-ICOS Antibodies

treatment_protocol: Primary T cells were activated as described in the manuscript

value: Activated with soluble Anti-CD3/Anti-CD28/Anti-ICOS for 72 hours


title: Mrps5 fl/fl; MCM mouse heart tissue, Mrps5 cKO for 14 weeks, AAV9 klf15 infection for 7 weeks, rep 3


title: mESC, DMSO, 12h, rep3

value: DMSO

value: male


title: Mrps5 fl/fl; MCM mouse heart tissue, Mrps5 cKO for 14 weeks, AAV9 klf15 infection for 7 weeks, rep 1


title: Mrps5 fl/fl; MCM mouse heart tissue, Mrps5 cKO for 14 weeks, AAV9 klf15 infection for 7 weeks, rep 2


title: mESC, DMSO, 12h, rep4


title: mESC, DMSO, 12h, rep2


title: iN_34_cre_d7_rep2


title: iN_34_dcre_d7_rep2


title: CD3_CD28_ICOS - Replicate 2


title: Mrps5 fl/fl; MCM mouse heart tissue, Mrps5 cKO for 14 weeks, AAV9 luci infection for 7 weeks, rep 3

value: AAV9 luci injection


title: CD3_CD28_ICOS - Replicate 1


title: CD3_CD28 - Replicate 3

source_name: CD4 Cells, Stimulated with Anti-CD3/Anti-CD28 Antibodies

value: Activated with plate-bound Anti-CD3/soluble Anti-CD28 for 72 hours


title: iN_34_dcre_d7_rep1

value: neuron

value: dcre


title: mESC, DMSO, 12h, rep1


title: Mrps5 fl/fl; MCM mouse heart tissue, Mrps5 cKO for 14 weeks, AAV9 luci infection for 7 weeks, rep 2

value: Heart


title: CD3_CD28 - Replicate 2


title: mESC, STM2457, 9h, rep4


title: iN_32_cre_d43_rep2

value: clone 1


title: Mrps5 fl/fl; MCM mouse heart tissue, Mrps5 cKO for 14 weeks, AAV9 luci infection for 7 weeks, rep 1

value: Mrps5 fl/fl; MCM

value: Tamoxifen injection


title: mESC, STM2457, 9h, rep3


title: CD3_CD28 - Replicate 1


title: iN_32_cre_d43_rep1


title: iN_32_dcre_d43_rep1


title: Mrps5 fl/fl; MCM mouse heart tissue, cKO, 12 weeks after tamoxifen injection, rep 2

growth_protocol: Mrps5 fl/fl mouse and Mrps5 fl/fl; MCM mouse were generated and grown up to 8 weeks old in SPF animal labratory

treatment_protocol: 8 weeks old Mrps5 fl/fl mouse and Mrps5 fl/fl; MCM mouse were treated with tamoxifen through intraperitoneal injection at the dosage of 45 mg/kg/day for 5 continuous days to activate Cre recombinase and delete Mrps5 gene in genome.

value: 12 weeks after tamoxifen injection


title: CD3 - Replicate 3

source_name: CD4 Cells, Stimulated with Anti-CD3 Antibodies

value: Primary Murine CD4+ T Cells

value: Activated with plate-bound Anti-CD3 for 72 hours


title: mESC, STM2457, 9h, rep2


title: CD3 - Replicate 2

value: Isolated from C57BL/6 Mice


title: iN_32_dcre_d43_rep2


title: Mrps5 fl/fl; MCM mouse heart tissue, cKO, 12 weeks after tamoxifen injection, rep 1


title: mESC, DMSO, 9h, rep4

value: mESC


title: iN_46_cre_d43_rep2

value: cre

value: clone 2


title: CD3 - Replicate 1


title: Mrps5 fl/fl mouse heart tissue, control, 12 weeks after tamoxifen injection, rep 3

value: Mrps5 fl/fl


title: iN_46_cre_d43_rep1


title: mESC, DMSO, 9h, rep3

value: mouse embryonic stem cells (mESC)

value: DMSO

value: male


title: Unstimulated - Replicate 3

source_name: CD4 Cells, Freshly Isolated - No Stimulation

value: Fresh isolation, no activation


title: Unstimulated - Replicate 2


title: iN_46_dcre_d43_rep2

value: neuron

value: dcre


title: mESC, DMSO, 9h, rep2


title: mESC, DMSO, 9h, rep1


title: P0_WT_593_605_file4

description: single-cell RNA

data_processing:
Reads were counted and aggregated with 10x Genomics Cell Ranger (version 4.0.0)
Cells containing fewer than 200 or greater than 4000 features, or with mitochondrial genes making up over 15% of genes, were discarded using Seurat (version 4.0)
We used scArches (v0.3.5) with the scANVI model to calculate latent space of dimension ten, and to assign cell type annotations by mapping our data onto reference data using default parameters
Leiden clustering was performed on latent space (default parameters, k = 15). Two clusters with average annotation probability scores <80% were annotated based on marker gene expression; microglia with Tmem119 and Aif1, and blood cells with Hba-a1 and Hbb-bs. Blood cells were removed for downstream analysis.
Sub-populations of each cell type most affected by Myt1l mutation were identified using MELD (version 1.0) with parameters Beta=31 and KNN=5 which were determined using grid search
Differential expression was performed on these sub-populations using MAST (version 1.16.0) within Seurat’s FindMarkers function (logfc.threshold = 0, min.pct = 0.05, all other parameters default)
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited file with MAST DE analysis (het vs wt)
Supplementary_files_format_and_content: Tab-delimited file with MAST DE analysis (hom vs wt)
Supplementary_files_format_and_content: Tab-delimited file with cell x gene matrix
Supplementary_files_format_and_content: Tab-delimited file with cell x metadata matrix

extract_protocol:
Prefrontal cortices from P0 mice were dissected and dissociated into single cells and fixed in ice-cold 80% methanol and barcoded using the ECCITE-Seq scheme with an amine group at the 5’ end.
We pooled ~20.000 cells from two Myt1l (+/+), (+/-), and (-/-) animals, respectively. Each pool was loaded into an individual lane of the Chromium Controller (10x Genomics).
Chromium Single Cell 5' v1 reagent kit was used to process samples according to the manufacturer’s instructions, with modifications in the cDNA and library preparation steps suggested by the ECCITE-Seq protocol to generate the barcoded oligonucleotide libraries in parallel.
single-cell RNA-seq

value: cortex

value: WT

tag: gem


title: Unstimulated - Replicate 1