title: ROI2_WT_plasmid_techrep2

city: Austin

first: Can

last: Cenik

state: TX

address: Moffett Molecular Biology Building 2.122 2500 Speedway

department: Molecular Biosciences

institute: The University of Texas at Austin

library_source: other

data_processing:
Variant calling: Variants were called with satmut_utils v1.0.3-dev001 (https://github.com/ijhoskins/satmut_utils/releases/tag/v1.0.3-dev001). The satmut_utils  parameters were as follows: `satmut_utils -r pDEST_HC_Rec_Bxb_v2_UTR5_Flag_COMT_moxGFP_LPS.fa -p pDEST_HC_Rec_Bxb_v2_UTR5_Flag_COMT_moxGFP_LPS_ROI_primers_v2.bed call -k pDEST_HC_Rec_Bxb_v2_UTR5_Flag_COMT_moxGFP_LPS.fa -g pDEST_HC_Rec_Bxb_v2_UTR5_Flag_COMT_moxGFP_LPS.gff -n 2 -m 1 --r1_fiveprime_adapters TACACGACGCTCTTCCGATCT --r1_threeprime_adapters AGATCGGAAGAGCACACGTCT --r2_fiveprime_adapters AGACGTGTGCTCTTCCGATCT --r2_threeprime_adapters AGATCGGAAGAGCGTCGTGTA`.  Ribosome profiling analysis: Analysis was conducted with RiboFlow (umi_devel branch) and RiboR (Ozadam et al., 2020). The reference files were modified to include a Flag tag upstream of the COMT coding sequence to enable mapping of footprints arising from the stably-expressed COMT transgene.
Assembly: Data were aligned to custom a reference FASTA file. Reference files are available at https://github.com/ijhoskins/integrated_MAVEs/tree/main/references
Supplementary files format and content: satmut_utils summary.txt files contain VCF fields with each INFO tag value in a unique column. See https://github.com/ijhoskins/satmut_utils/blob/satmut_utils_dev/docs/satmut_utils_manual.md for details on each field. RiboFlow .ribo file documentation is available at https://ribopy.readthedocs.io/en/latest/ribo_file_format.html.

molecule: other

growth_protocol: HEK293T LLP iCasp9 Blast cells (Matreyek et al., 2020) were periodically tested and confirmed free of Mycoplasma, and were cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, 11995065) with 10% fetal bovine serum (Gibco), and 1% penicillin-streptomycin (Gibco, 15140122).

extract_protocol:
gDNA extraction: gDNA was extracted from approximately 3-4 million cells with the Cell and Tissue DNA Isolation Kit (Norgen Biotek Corp, 24700), including RNaseA treatment at 37 ˚C for 15 min, and eluting in 200 µL warm elution buffer.  RNA extraction: Approximately 3-4 million cells were solubilized with 1 mL QIAzol (Qiagen, 79306) and 0.2 mL chloroform in 5PRIME Phase-Lock Gel heavy tubes (QuantaBio, 2302830), according to the manufacturer’s recommendations. RNA was precipitated at -20 ˚C following the addition of 2 µL GlycoBlue (Thermo Fisher, AM9515) and 2.5 volumes of cold absolute ethanol. RNA was washed once with cold 70% ethanol then resuspended in 30 µL water. 10 µg total RNA or 15 µL of polysomal RNA (half of 30 µL eluate, volumetric inputs) was treated with DNaseI (NEB, M0303) in a 100 µL reaction at 37 ˚C for 15 min, then re-purified by the RNA Clean and Concentrator Kit (Zymo Research, R1015) and eluted in 20 µL water.  Polysome fractionation and polysomal RNA purification: For COMT library stable cell lines, cells at 80-95% confluency (one 15 cm dish per gradient) were incubated with 100 µg/mL cycloheximide (Sigma Aldrich, C4859) in media for 10 min at 37 ˚C, then resuspended in ice cold PBS with 100 µg/mL cycloheximide (PBS-CHX), collected at 4 ˚C by pipetting, and pelleted at 300 x g for 7 min at 4 ˚C. Pellets were flash frozen in liquid nitrogen, then lysed on ice for 10 min with 300-400 µL lysis buffer (20 mM Tris-HCl pH 7.5; 150 mM NaCl; 5 mM MgCl2, 1 mM fresh DTT, 20 mM ribonucleoside vanadyl complex (Fisher Scientific, 50-812-650), 1X protease inhibitor cocktail EDTA-free (Fisher Scientific, 539196), and 100 μg/ml cycloheximide. Lysates were clarified by centrifugation at 1,300 x g for 10 min at 4 ˚C, and 1/10th of the lysate was saved for total RNA extraction. 20-50% sucrose gradients were made with the BioComp Gradient Maker in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 100 μg/ml cycloheximide using Beckman Coulter UC tubes 9/16 x 3-1/2 (Fisher Scientific, NC9194790), and equilibrating overnight at 4 ˚C. Clarified lysates were loaded onto sucrose gradients, and fractionated by ultracentrifugation at 38,000 rpm for 2.5 h at 4 ˚C in a Beckman Coulter SW-41 Ti rotor. Approximately 500 µL polysome fractions were collected using the Biocomp Piston Gradient Fractionator (v8.04) with a Triax flow cell (BioComp model FC-1) and Gilson Fraction Collector using a piston speed of 0.2 mm/s. RNA was precipitated from raw polysome fractions by addition of sodium acetate to 300 mM pH 5.2, 2 µL Glycoblue, and 2x volumes cold ethanol, stored overnight at -20 ˚C. RNA was resuspended in 50-80 µL ultrapure water, and these raw fractions were pooled into metafractions and extracted by phenol-chloroform (5:1) and ethanol precipitation overnight at -20 ˚C. After a single 70% ethanol wash, polysomal RNA was resuspended in 30 µL ultrapure water. Lastly, DNaseI treatment and a column purification was performed prior to library preparation.  Ribosome profiling: Monosomes were pelleted through a sucrose cushion (1 M sucrose, 20 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1x protease inhibitor cocktail EDTA-free) by spinning at 38,000 rpm at 4 ˚C in a SW-41 Ti rotor (Beckman Coulter). Monosome pellets were resuspended in 400 µL lysis buffer and rocked overnight. RNA was extracted with 1.2 mL phenol/chloroform/isoamyl alcohol and precipitated with 300 mM sodium acetate pH 5.2, 1.5 µL Glycoblue, and 2.5 volumes of cold absolute ethanol overnight at -20 ˚C. RNA was pelleted at 21,000 x g for 1 h, washed once with cold 70% ethanol, and resuspended with 30 µL water. 15 µL of RNA was run on a 15% TBE-urea polyacrylamide gel at 150 V for 1.5 h. The gel was stained with 1x SYBR Gold and footprints between 21 nt and 34 nt were cut and isolated by crush and soak in RNA extraction buffer (300 mM sodium acetate pH 5.5, 1 mM EDTA, 0.25% v/v SDS) with overnight rotation. The eluate was passed through a Spin X Centrifuge Tube Filter with a cellulose acetate membrane (Thermo Fisher Scientific, 07-200-385), and the RNA was precipitated with 300 mM sodium acetate pH 5.2, 1.5 µL Glycoblue, and 2.5 volumes of cold absolute ethanol overnight at -20 ˚C. RNA was pelleted at 21,000 x g for 1 h, washed once with cold 70% ethanol, and resuspended in 12 µL water.
cDNA synthesis: 10 µL DNaseI-treated total RNA (<10 µg) or DNaseI-treated polysomal RNA (200 ng - 5 µg) was denatured at 65 ˚C for 5 min followed by RT primer annealing at 4 ˚C for 2 min, using 2 pmol pDEST_HC_Rec_Bxb_v2_R primer specific for the landing pad (see Supplementary Table 4) and 2.5 mM random hexamers. Primed total RNA was included in 20 µL SuperScript IV cDNA synthesis reaction (Thermo Fisher, 18090010) with SUPERase-In RNase inhibitor (Thermo Fisher, AM2696), and first-strand cDNA was synthesized by incubating at 23 ˚C for 10 min, 55 ˚C for 1 h, followed by RT inactivation at 80 ˚C for 10 min. RNA was digested with addition of 5 U RNaseH (NEB, M0297) to the first strand cDNA synthesis reaction and incubation at 37 ˚C for 20 min.  Landing pad amplification (PCR1): Nucleic acids (gDNA, 1st strand cDNA) were amplified with Q5 polymerase (NEB, M0491) in 50 µL PCR reactions for 16 cycles with 500 nM landing-pad-specific primers (pDEST_HC_Rec_Bxb_v2_F/R) flanking the entire COMT insert (~1.7 kb). For negative control (plasmid) samples, 10 ng was used as template. For total RNA and polysome RNA samples, 15 µL of unpurified 1st strand cDNA synthesis reaction was used as template. For total gDNA samples, 4 µg template was used in each of three PCR reactions. For flow-sorted gDNA samples, either 1.75 µg template was used in three PCR reactions or 2.5 µg was input into two reactions, with 14 cycles instead of 16. The cycling parameters were: initial denaturation at 98 ˚C for 30 s; 3-step cycling with denaturation at 98 ˚C for 10 s, anneal at 65 ˚C for 30 s, extension at 72 ˚C for 1 min; final extension at 72 ˚C for 2 min. Products from total RNA and polysome RNA samples were purified with the QIAquick PCR Purification Kit (Qiagen, 28106) and eluted in 30 µL elution buffer. Products for total gDNA samples were pooled, resolved on a 0.8% agarose/TAE gel, stained with 1x SYBR Gold, and extracted using the Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) or Zymoclean Gel DNA Recovery Kit (Zymo Research, D4007) with 20 µL 70 ˚C elution buffer. At this point, PCR1 products for gDNA samples cannot typically be seen in the gel, and the expected band size is cut using comparison to a high-molecular weight ladder.  Coding sequence amplification (PCR2): Purified PCR1 products (landing pad insert) were amplified for each of COMT target regions in a 50 µL NEB Q5 reaction (NEB, M0491) for 8 cycles, following the same cycling parameters as for PCR1 and 500 nM tailed primers (see Supplementary Table 4). 15 µL PCR1 product was used as template (50% of eluate for total RNA and polysome RNA samples; 75% of eluate for gel-extracted gDNA samples). Products were purified with the Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) with 1:5 buffer NTI dilution and 25 µL 70 ˚C elution buffer, or with the QIAquick PCR Purification Kit (Qiagen, 28106) with 30 µL elution buffer.  Illumina adapter addition (PCR3): A final Q5 PCR reaction was carried out for 8 cycles with the same formulation as PCR2 but using NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 (NEB, E7600S) according to manufacturer’s recommendations (65 ˚C annealing). 25 µL/30 µL eluate was used as template for total gDNA, total RNA, and polysome RNA samples. For flow cytometry gDNA samples, 15 µL/25 µL eluate was used as template. PCR3 products (final library) were purified with the Nucleospin Gel and PCR Cleanup kit (Takara, 740609) and eluted in 25 µL 70 ˚C buffer.  Ribosome profiling: 25 ng of RNA in 8 µL water was input to the D-Plex Small RNA kit (Diagenode, C0503000) and libraries were prepared according to the manufacturer recommendations with the exception that 9 cycles of PCR were carried out. The library was purified with 1.25x AMPure beads (Beckman Coulter, A63880) and quantified by the High Sensitivity DNA Kit (Agilent, 5067-4626).  Quantification and size-selection of final libraries: Final libraries were quantified using the KAPA Library Quantification Kit for Illumina (Roche, KK4873) according to the manufacturer’s recommendations, using 1:10,000 dilution of libraries and size-correction with an average fragment size of 300 nt (region 1) and 285 nt (region 2). qPCR was performed on the Applied Biosystems ViiA 7 instrument and values were used to pool individual libraries at equimolar ratios to target ~5 M read pairs per library. Final library pools were size-selected use PAGE purification on a 15% TBE gel followed by a crush-and-soak method (Sambrook and Russell, 2006), or with BluePippin 2% gel (Sage Science, BDF2010) to select fragments from 250-340 bp. Following size-selection, final pools were quantified by High Sensitivity DNA Kit (Agilent, 5067-4626) prior to sequencing.
A targeted library preparation method using three sequential PCRs was used (see library construction protocol).

treatment_protocol: For each region 1 and region 2 biological replicate cell line, 20 µg of the library along with an equal mass of Bxb1 recombinase (pCAG-NLS-HA-Bxb1) was transfected into a 15 cm dish of HEK293T LLP iCasp9 Blast cells using Lipofectamine 3000 (Thermo Fisher Scientific, L3000008), with volumes scaled based on 3.75 µL reagent per 6-well. For single mutants, 1.5 µg was transfected for each of the donor and Bxb1 recombinase plasmids. After 48 h, at near full confluency, 2 µg/mL Doxycycline and 10 nM AP1903 (MedChemExpress, HY-16046), both solubilized in DMSO, were added for negative selection of non-recombined cells. The next day, dead cells were removed and recombined cells were grown out for an additional two days with fresh media containing Doxycycline and AP1903. Cells were recovered for two days by growth in media without Doxycycline and AP1903. Before functional assays, transcription was induced with 2 µg/mL Doxycycline for 24 h (total RNA, polysome RNA readouts) or 21 h (flow cytometry readouts), and cells were stimulated with fresh media for 1 h 15 min prior to harvest. Importantly, because the COMT transgene is membrane localized and faces the extracellular space, cells are collected by pipetting in cold PBS, and not by trypsinization.  Flow cytometry sorting: Following induction with doxycycline and media stimulation, cells were washed once with cold PBS, resuspended with PBS, and assayed in a 5 mL Falcon polystyrene round bottom tube with a cell-strainer cap (Fisher Scientific, 08-771-23). Analysis of point mutants and deletion cell lines was conducted on a LSRFortessa SORP instrument (BD) with FACSDiva v6.1.3, and flow cytometry was conducted on a FACSAria Fusion SORP instrument (BD) at the Center for Biomedical Research Support Microscopy and Imaging Facility at UT Austin (RRID# SCR_021756). Gating was performed with FlowJo. A gate for cells versus debris was defined based on FSC-H versus SSC-H, and another gate for single cells versus doublets was defined based on FSC-A versus FSC-H. Fluorescence gating was based on autofluorescence of 239T LLP iCasp9 Blast cells without recombination or of a stable cell line expressing the template transgene but without doxycycline induction. After flow cytometry sorting, each population was expanded up to a confluent T-25 flask, washed, trypsinized, pelleted, and flash frozen prior to gDNA extraction.  Ribosome profiling: A 15 cm dish of cells with stable expression of the 5′ UTR-containing COMT transgene at 95% confluency was washed twice with cold PBS containing 100 µg/mL cycloheximide, then lysed on the plate with 1.5 mL lysis buffer (20 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/ml cycloheximide, 100 µg/ml anisomycin, 1% Triton-X, and 1x protease inhibitor cocktail EDTA-free). Lysate was partitioned into two tubes and 7 µL RNaseI (Invitrogen, AM2249) was added and incubated for 1 h at 4 ˚C. Digestion was stopped by addition of ribonucleoside-vanadyl complex to 20 mM final.


title: LC_D4_M

status: Public on Nov 06 2023

type: SRA

city: Palo Alto

first: Avnesh

last: Thakor

state: California

address: 3155 Porter Dr

department: Radiology

country: USA

institute: Stanford Medicine

library_source: transcriptomic

data_processing:
Sequenced reads were trimmed for adaptor sequence,
Then mapped to rat genome Rattus Norvegicus using HISTAT
HTSeq was used to generate the raw read counts
DESeq2 was used for differential gene expression analysis
Assembly: Rnor_6.0
Supplementary files format and content: tab-delimited text file include FPKM values for all samples
Supplementary files format and content: tab-delimited text file include raw counts for all samples

molecule: total RNA

organism: Rattus norvegicus

source_name: Colon

extract_protocol:
total RNA was extracted from each replicate using the RNAeasy minikit (qiagen)
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: tissue

value: Colon

tag: genotype

value: SD

tag: treatment

value: DSS


title: ST_48_1

city: Tucson

first: Megha

last: Padi

state: AZ

address: 1071 East Lowell St.

department: Molecular and Cellular Biology

institute: University of Arizona

description: normalized_counts_cpm1filter_nooutliers.txt

data_processing:
Reads were mapped to a transcriptome index generated from the hg19 human reference genome, using Tophat 2.0.4 and Bowtie1 with all default parameters except for the following: --no-novel-juncs --transcriptome-only -p 6.
Counts were quantified using HTSeq with default bam file parameters, except the following: -s no.
Samples with low read counts were removed from further analysis.  Additionally, outliers were identified using principle component analysis and were subsequenty removed from further analysis.  Samples that were excluded are: TLT_8_1 (low read count), TLT_8_2 (low read count), TLT_40_1 (low read count), TLT_20_1,  and GFP_4_1 (outlier).
Raw counts were then normalized and log-transformed using edgeR.
Genes with average counts-per-million less than 1 were excluded from further analysis.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file with normalized counts-per-million for included genes and samples

organism: Homo sapiens

source_name: IMR90 human lung embryonic fibroblast

growth_protocol: IMR90 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Cellgro) supplemented with 1% Pen-Strep (Gibco), 1% Glutamax (Gibco), 1% non-essential amino acids (Gibco) and 15% fetal bovine serum (FBS) (Sigma).

extract_protocol:
Total RNA was purified using RNeasy Plus Mini Kit (Qiagen).
Sequencing libraries were prepared with NEBNext mRNA library Prep Master Mix Set for Illumina (New England BioLabs), passed Qubit, Bioanalyzer and qPCR QC analyses, and were subsequently sequenced on HiSeq 2000 system (Illumina).

treatment_protocol: Dox-inducible IMR90 lines expressing ST, ER, TLT or GFP were seeded in 6 cm dishes 24 hours before initiation of timecourse with normal media (no dox). At time zero, cells were refed with dox-containing media.  Time zero cells did not receive dox treatment.  Cells were harvested every 4 hours for the first 24 hours, then every 8 for the next 24 hours.  Cells were not refed after the initial addition of dox-containing media.

tag: cell type

value: lung embryonic fibroblast

tag: vector

value: ST

tag: time point (hours)

tag: replicate


title: ST_40_3


title: ROI2_fractionated_RNA_biorep2_techrep1_F4

source_name: HEK293T LLP iCasp9 Blast

tag: cell line

value: HEK293T LLP iCasp9 Blast

value: Induction of transcription from landing pad with addition of 2 µg/mL Doxycycline


title: ST_40_2


title: ROI1_WT_plasmid_techrep3


title: TLT_0_1

value: TLT


title: ROI2_fractionated_RNA_biorep2_techrep1_F1


title: RC_D4_M


title: ROI2_fractionated_RNA_biorep2_techrep1_F2


title: ROI2_fractionated_RNA_biorep2_techrep1_F3


title: IL_D4_M


title: ROI2_fractionated_RNA_biorep1_techrep1_F4


title: ST_40_1


title: LC_D3_M


title: ST_32_3


title: Young_OA_Male_A

city: Rochester

first: Jessica

last: Hohenstein

state: MN

address: 200 1st St SW

institute: Mayo Clinic

data_processing:
MAP-RSeqversion 3.1.4 and theSTARv2.6.1c tool were usedto align the raw RNA seq reads to the reference human genome build hg38
QC was performed by FastQC pre- and post-read trimming
The RNA paired end reads were aligned using the STAR aligner 2.6.1c
Gene counts were generated using subread featureCounts
edgeR v3.40.2 for defining differentially expressed genes
Assembly: hg38
Supplementary files format and content: tab-separated tsv file containing raw counts

source_name: knee articular cartilage

growth_protocol: Chondrocytes were grown in DMEM/F-12 media supplemented with 10% fetal bovine serum and 1 % Penicillin/streptomycin.

extract_protocol:
RNA isolation: Two step RNA isolation protocols were followed for total RNA extraction: Organic phase isolation was performed with TriReagent®/ bromo–3–chloropropane (BCP) (Thermo Fisher Scientific/Molecular Research Center, Inc., Cincinnati, OH, USA) on the samples and then used for total RNA isolation using a RNeasy Plus Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. RNA concentration was measured using a nanophotometer® (Implen, Inc., Westlake Village, CA, USA). RNA purity was A260/280 and A260/230 ˃ 2.0.
Illumina Stranded mRNA library prep with Illlumina NextSeq2000 Sequencing: RNA libraries were prepared using 200 ng of total RNA in 20 µl according to the manufacturer’s instructions for the Illumina Stranded mRNA Ligation Sample Prep Kit (Illumina, San Diego, CA).  The concentration and size distribution of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). Libraries were sequenced using Illumina’s standard protocol for the NovaSeq 6000. The NovaSeq SP PE100 flow cell was sequenced as 100 X 2 paired end reads using NovaSeq Control Software v1.8.0. Base-calling is performed using Illumina’s RTA version 3.4.4.

treatment_protocol: Chonndrocytes that were passage 1-3 designated as young cells. Chondrocytes later were subjected to serial passaging until they reached to passage 15-18. Late passages were designated as old cells.

value: knee articular cartilage


title: ROI2_fractionated_RNA_biorep1_techrep1_F2


title: ROI2_fractionated_RNA_biorep1_techrep1_F1


title: ROI2_fractionated_RNA_biorep2_techrep2_F4


title: ROI2_fractionated_RNA_biorep2_techrep2_F3


title: ST_32_2


title: IL_D3_M


title: RC_D3_M


title: ROI2_fractionated_RNA_biorep2_techrep2_F1


title: ROI2_fractionated_RNA_biorep2_techrep2_F2


title: ST_32_1


title: ROI2_fractionated_RNA_biorep1_techrep2_F4


title: GFP_4_1

description: removed from analysis

value: GFP


title: ROI2_fractionated_RNA_biorep1_techrep1_F3


title: ROI2_fractionated_RNA_biorep1_techrep2_F3


title: ST_24_3


title: LC_D2_M


title: IL_D2_M


title: ROI2_fractionated_RNA_biorep1_techrep2_F1


title: ST_24_2


title: LC_D1_M


title: ROI1_fractionated_RNA_biorep4_techrep1_F3


title: ST_24_1


title: ROI1_fractionated_RNA_biorep4_techrep1_F1


title: ROI1_fractionated_RNA_biorep4_techrep1_F4


title: ST_20_3


title: ROI1_fractionated_RNA_biorep3_techrep1_F4


title: ROI1_fractionated_RNA_biorep3_techrep1_F2


title: ROI1_fractionated_RNA_biorep3_techrep1_F3


title: IL_D1_M


title: ST_20_2


title: ROI1_fractionated_RNA_biorep3_techrep1_F1


title: ROI1_genomic_DNA_FC_biorep3_techrep2_input


title: ROI2_fractionated_RNA_biorep1_techrep2_F2


title: RNA-Seq of rat hippocampus after TBI (Controlled Corticol Impact):  Thymus  dosage. rep 5 [90B_S15]

city: Kannapolis

first: Robert

last: Reid

state: NC

address: 150 Research Campus Drive, Suite 3333

department: Bioinformatics and Genomics

institute: UNC Charlotte

description:
Hippocampus_TD-CCI_versus_Control.deseq2_output.tsv
featurecount_all.tsv

data_processing:
Alignment done using STAR
Gene counting done using Salmon (genecounts.txt)
Differential expression table done using DESEq2
Assembly: Rat genome RatBN7.2 / GCF_015227675.2_mRatBN7.2_genomic.fna
Supplementary files format and content: (Tab delimited file of gene counts for all experiments generated by FeatureCount)
Supplementary files format and content: Hippocampus_TNF-CCI_versus_Control.deseq2_output.tsv
Supplementary files format and content: Hippocampus_TCF-CCI_versus_Control.deseq2_output.tsv
Supplementary files format and content: Hippocampus_TD-CCI_versus_Control.deseq2_output.tsv
Supplementary files format and content: Hippocampus_Vehicle-CCI_versus_Control.deseq2_output.tsv

molecule: polyA RNA

source_name: Hippocampus TD-CCI

extract_protocol:
RNeasy Fibrous Tissue Mini Kit
the Universal Plus mRNA-Seq kit (TECAN), following manufacturer's instructions. Completed libraries were quantitated using qPCR and their sizes were visualized on the BioAnalyzer (Agilent). All libraries passed QC, showing no presence of adapter dimers contamination and excess amplification, and an average size peak around 400-500bp.
Universal Plus mRNA-Seq kit (TECAN)

value: hippocampus

tag: strain

value: Sprague-Dawley

tag: age

value: adult; 6-7 weeks

tag: Sex

value: male


title: ST_20_1


title: ROI2_genomic_DNA_FC_biorep1_techrep2_input


title: WT IL, replicate 2, scRNAseq

city: Pittsburgh

first: Shanshan

last: Song

state: PA

address: 3501 Fifth Ave

institute: University of Pittsburgh

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
Cell Ranger (10x Genomics) analysis pipeline was used to perform alignment, filtering and counting of barcodes, and unique molecular identifiers. The sequencing data was aligned to the mouse genome mm10. Count matrices generated by Cell Ranger were imported into Partek Flow 8.0 for further analysis.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

organism: Mus musculus

source_name: brain white matter (ipsilateral to stroke)

extract_protocol:
Single-cell RNA sequencing and bioinformatic analysis were performed in white matter tissues dissected from the WT or Nhe1 cKO brains at 3 days post-tMCAO. Briefly, mice were euthanized with an overdose of CO2 and transcardially perfused with ice-cold saline, as described before (10). The brains were dissected and cut into 4 coronal slices of 2 mm thickness in a brain slicer matrix. Cortices were removed and white matter tissues (corpus callosum and external capsules) were separated from the contralateral and ipsilateral hemispheres and dissociated into single-cell suspensions using a Neural Tissue Dissociation Kit with the gentleMAC Octo Dissociator (Miltenyi Biotech Inc., Germany). Myelin was removed using the 30/70 Percoll gradient method as described (10). Single-cell suspension was prepared as we described before 13 and used for droplet-based scRNAseq (10x Genomics).
The quality of the single-cell suspension was confirmed prior to the start of library preparation with an acridine-orange-propidium iodide stain (Nexcelcom Biosciences) and a Cellometer Auto 2000 Viability Counter (Nexcelcom Biosciences). Library Preparation (Chromium Next GEM Single Cell 3’ Reagent kit v3.1) was conducted as per the manufacturer’s protocol. The quality of the cDNA libraries was verified using a High Sensitivity D5000 ScreenTape and 4200 Tapestation system (Agilent Technologies).

treatment_protocol: Both genotypes of mice (male or female) at postnatal day 30-40 (P30-40) received tamoxifen (Tam, Sigma) (75 mg/kg body weight/day at a concentration of 20 mg/ml in corn oil, intraperitoneally) for 5 consecutive days. As Cx3cr1 is expressed by both brain resident microglia and peripheral infiltrating bone marrow-derived myeloid cells (BMDM), a 30-day post-injection waiting period was given for clearance of Tam 3-5 and for replenishing of Cx3cr1+ monocytes 6 prior to induction of ischemic stroke.

value: tamoxifen


title: RNA-Seq of rat hippocampus after TBI (Controlled Corticol Impact):  Thymus Nuclear Factor dosage . rep 5 [88B_S25]

description:
Hippocampus_TNF-CCI_versus_Control.deseq2_output.tsv
featurecount_all.tsv

source_name: Hippocampus TNF-CCI


title: RC_D2_M


title: ST_16_3


title: ROI1_fractionated_RNA_biorep4_techrep1_F2


title: ROI2_genomic_DNA_FC_biorep2_techrep2_P6

library_source: genomic

molecule: genomic DNA


title: RNA-Seq of rat hippocampus after TBI (Controlled Corticol Impact):  Thymus Nuclear Factor dosage . rep 4 [80B_S24]


title: WT IL, replicate 1, scRNAseq

description: 10x Genomics

value: brain white matter (ipsilateral to stroke)


title: ST_16_2


title: RC_D1_M


title: ST_16_1


title: ROI2_genomic_DNA_FC_biorep2_techrep2_P4


title: ROI2_genomic_DNA_FC_biorep2_techrep2_P5


title: WT CL, replicate 2, scRNAseq

description: 10x Genomics

source_name: brain white matter (contralateral to stroke)


title: RNA-Seq of rat hippocampus after TBI (Controlled Corticol Impact):  Thymus  dosage. rep 4 [70B_S14]


title: WT CL, replicate 1, scRNAseq

description: 10x Genomics

value: brain white matter (contralateral to stroke)


title: ROI2_genomic_DNA_FC_biorep2_techrep2_P3


title: KO IL, replicate 2, scRNAseq

description: 10x Genomics


title: RNA-Seq of rat hippocampus after TBI (Controlled Corticol Impact):  Thymus  dosage. rep 3 [6B_S11]


title: GFP_0_3


title: ROI1_genomic_DNA_FC_biorep2_techrep2_P4


title: ST_12_3


title: ROI2_genomic_DNA_FC_biorep4_techrep1_P6


title: ST_12_2


title: RNA-Seq of rat hippocampus after TBI:  Vehicle (Controlled Corticol Impact). rep 5 [60B_S10]

description:
Hippocampus_Vehicle-CCI_versus_Control.deseq2_output.tsv
featurecount_all.tsv

source_name: Hippocampus Vehicle-CCI

value: Vehicle (Controlled Corticol Impact)


title: KO CL, replicate 2, scRNAseq

description: 10x Genomics


title: RNA-Seq of rat hippocampus after TBI:  Vehicle sham. rep 5 [62B_S5]

source_name: Hippocampus Vehicle-sham

value: Vehicle sham


title: ST_8_3


title: RNA-Seq of rat hippocampus after TBI:  Thymus Cytosolic factor dosage. rep 5 [66B_S20]

description:
Hippocampus_TCF-CCI_versus_Control.deseq2_output.tsv
featurecount_all.tsv

source_name: Hippocampus TCF-CCI

value: Thymus Cytosolic factor dosage


title: KO CL, replicate 1, scRNAseq

description: 10x Genomics


title: ROI2_genomic_DNA_FC_biorep4_techrep1_P3


title: RNA-Seq of rat hippocampus after TBI (Controlled Corticol Impact):  Thymus Nuclear Factor dosage. rep 3 [72B_S23]


title: ST_8_1

value: lung embryonic fibroblast


title: ROI2_genomic_DNA_FC_biorep4_techrep1_input


title: Slc35d3 adipose-specific knockin mouse IngWAT 1, SAKI group

city: Beijing

first: Yibo

last: Wang

state: Beijin

address: 167 Beilishi Road, Xicheng District, Beijing

country: China

institute: Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College

description:
column name: SAKI-1
FPKMs_allsamples.txt

data_processing:
Hisat2（version:2.0.4）
Adaptor sequences contained in reads were removed using trimadap, bases with a mass Q below 20 at the 3 'end were removed by seqtk trimfq (version: 1.0), i.e., the base error rate was less than 0.01, (Q = -10 logerrorratio).
Reads less than 25 in length were removed using lengthsort.pl. Ribosome RNA reads of the species to which they belonged were removed by bowtie2 (version: 2.2.6).
Genome mapping of pre-processed reads was performed applying the spliced mapping algorithm of Hisat2 (version: 2.0.4).
Stringtie (version: 1.3.0) was applied to count the number of Fragments per gene after Hisat2 alignment, then the TMM (trimmed mean of M values) method was used to normalize, and finally the FPKM value of each gene was calculated using a perl script.
Assembly: GRCm38
Supplementary files format and content: tab-delimited text file includes FPKM values for each sample.
Supplementary files format and content: tab-delimited text files include counts for each sample.

source_name: IngWAT (inguinal white adipose tissue)

growth_protocol: All mice were housed under a 12-hour light/dark cycle at a temperature of 23 ± 1 °C and relative humidity of 50%-60%, with free access to water.

extract_protocol:
Total RNA was extracted using the MJzol Animal RNA Isolation Kit (Majorivd) and purified using RNAClean XP Kit (Beckman Coulter) and RNase-Free DNase Set (QIAGEN) according to standard operating procedures provided by the manufacturer.
The mRNA was enriched using VAHTS ® mRNA Capure Beads (Human/Mouse/Rat), and mRNA sequencing libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina ® (Vazyme) according to standard operating procedures provided by the manufacturer.

treatment_protocol: Male 6-week-old Slc35d3 adipose-specific knockin mice (SAKI group) and their control mice (wild-type mice, WT group) were fed a normal standard chow diet (NCD, 10 kal% fat, D09100304, Research Diets, Inc) for 10 weeks.

value: IngWAT (inguinal white adipose tissue)

value: Slc35d3 adipose-specific knockin

value: PBS perfusion


title: ROI2_genomic_DNA_FC_biorep4_techrep1_P4

value: HEK293T LLP iCasp9 Blast

value: Induction of transcription from landing pad with addition of 2 µg/mL Doxycycline


title: RNA-Seq of rat hippocampus after TBI:  Vehicle sham. rep 4 [42B_S4]


title: RNA-Seq of rat hippocampus after TBI:  Thymus Cytosolic factor dosage. rep 4 [56B_S19]


title: KO IL, replicate 1, scRNAseq

description: 10x Genomics


title: ST_8_2

value: ST


title: Control mouse IngWAT 3, WT group

description:
column name: WT-3
FPKMs_allsamples.txt

value: wild type


title: ROI1_genomic_DNA_FC_biorep4_techrep1_P6


title: Slc35d3 adipose-specific knockin mouse IngWAT 2, SAKI group

description:
column name: SAKI-2
FPKMs_allsamples.txt


title: RNA-Seq of rat hippocampus after TBI:  Vehicle (Controlled Corticol Impact). rep 3 [40B_S8]

value: hippocampus

value: Sprague-Dawley


title: ST_4_3


title: ROI2_genomic_DNA_FC_biorep4_techrep1_P5


title: ST_4_2


title: ST_12_1


title: ROI1_genomic_DNA_FC_biorep4_techrep1_P5


title: RNA-Seq of rat hippocampus after TBI:  Thymus Cytosolic factor dosage. rep 3 [36B_S18]

value: adult; 6-7 weeks

value: male


title: Control mouse IngWAT 2, WT group

description:
column name: WT-2
FPKMs_allsamples.txt


title: ST_4_1


title: ROI1_genomic_DNA_FC_biorep4_techrep1_P4


title: Slc35d3 adipose-specific knockin mouse IngWAT 3, SAKI group

description:
column name: SAKI-3
FPKMs_allsamples.txt


title: Control mouse IngWAT 1, WT group

description:
column name: WT-1
FPKMs_allsamples.txt


title: RNA-Seq of rat hippocampus after TBI:  Vehicle (Controlled Corticol Impact). rep 2 [34B_S7]


title: RNA-Seq of rat hippocampus after TBI:  Vehicle (Controlled Corticol Impact). rep 4 [52B_S9]


title: ROI1_genomic_DNA_FC_biorep4_techrep1_P3


title: RNA-Seq of rat hippocampus after TBI:  Vehicle sham. rep 3 [2B_S1]


title: ST_0_3


title: Sublibraries 1 to 8 (excludes #6)

city: Irvine

first: Jie

last: Wu

state: CA

address: Sprague Hall 332

department: Biological Chemistry

institute: University of California, Irvine

data_processing:
Illumina NovaSeq 6000 RTA (v3.4.4) software was used to perform basecalling, and FASTQ files were generated using the bcl2fastq Conversion software (v2.20) to convert BCL base call files outputted by the sequencing instrument into the FASTQ format
The data preprocessing and alignment was performed using the SPLiT-seq pipeline (https://github.com/yjzhang/split-seq-pipeline) from Parse Biosciences (split-pipe v0.9.6p) to convert raw FASTQ files into gene expression matrices.
The obtained count matrices for each sample were merged and filtered for nuclei with <500 genes, or >5–10% mitochondrial genes. Prior to normalization, two mouse transcripts, AY036118 and Gm42418, were removed and disregarded in the downstream analysis due to potential rRNA contamination caused by sequence overlap with Rn45s.
Downstream analyses with Seurat (v3.1.5) include normalization, dimension reduction by principal component analysis (‘RunPCA’), uniform manifold approximation and projection (‘RunUMAP’), Shared Nearest Neighbor (SNN) Graph construction (‘FindNeighbors’), and finally Louvain clustering (‘FindClusters’) using the first 30 principal components and a resolution of 0.05 to determine cluster assignment.
To identify cell type markers and differentially expressed genes between different conditions, Wilcoxon rank sum test was used (‘FindMarkers’)
Assembly: mm10
Supplementary files format and content: csv, gene counts

source_name: striatum

extract_protocol:
Mice were euthanized by pentobarbital overdose and perfused with 0.01 M PBS. Striatum was dissected out of the left hemisphere and flash-frozen. Single nuclei were isolated from flash frozen ½ hemisphere full striatal in Nuclei EZ Lysis buffer (Cat#NUC101-1KT, Sigma-Aldrich) and incubated for 5min. Samples were passed through a 70 μm filter and incubated in additional lysis buffer for 5min and centrifuged at 500 × g for 5min at 4 °C before two washes in Nuclei Wash and Resuspension buffer (1×PBS, 1% BSA, 0.2 U/μl RNase inhibitor). Nuclei were fixed using a Parse Biosciences WF100 kit and the manufacturers protocol (Parse Biosciences, Seattle, WA).
Single-nucleus suspensions were fixed and frozen for 8 mouse brain tissue samples following the Parse Bioscience Fixation User Manual V1.3.0 (Parse Biosciences). Single-nucleus RNA-seq libraries were generated following the Parse Biosciences Single Cell Whole Transcriptome Kit User Manual V1.3.0 (Parse Biosciences). Quantification of cDNA libraries was performed using Qubit dsDNA HS Assay Kit (Life Technologies Q32851) and high-sensitivity DNA chips (Agilent. 5067-4626). Quantification of final cDNA libraries was performed using KAPA qPCR (Kapa Biosystems KK4824). Sequencing of single-cell libraries was performed on the Illumina NovaSeq 6000 platform to target an average of ~50,000 reads per cell.

value: striatum


title: RNA-Seq of rat hippocampus after TBI:  Thymus Cytosolic factor dosage. rep 2 [28B_S17]


title: RNA-Seq of rat hippocampus after TBI (Controlled Corticol Impact):  Thymus  dosage. rep 2 [26B_S13]


title: ROI2_genomic_DNA_FC_biorep3_techrep1_P6


title: GFP_0_2

value: GFP


title: ROI2_genomic_DNA_FC_biorep3_techrep1_P5


title: ST_0_1


title: D21_P_2

city: Durham

first: Zachary

last: Hartman

address: Research Dr MSRBI

department: Surgery

institute: Duke University

description:
D21_P_2
readcount_cells.txt
fpkm_sample_cells.txt

data_processing:
Data processing was performed by Novogene according to their standard protocol.
The procedure of raw reads filtering is as follows: (1) Remove reads containing adapters; (2) Remove reads containing N > 10% (N represents base that could not be determined); (3) Remove low quality reads: The Qscore (Quality value) of over 50% bases of the read is <= 5.
STAR software is used to accomplish the mapping.
Assembly: mm10
Supplementary files format and content: tab-delimited text file containing raw counts for each sample
Supplementary files format and content: tab-delimited text file containing FPKM values for each sample

source_name: in vitro cells

extract_protocol:
Individual tumors were snap frozen and stored in RNAlater-ICE (Invitrogen) until RNA extraction. Total RNA was extracted using PureLink RNA mini Kits (Invitrogen) and DNA was removed via DNA-free DNA Removal kit (Invitrogen). RNA quality and concentration was determined using an Agilent 2100 Bioanalyzer.
Library construction was performed by Novogene according to their standard protocol.

value: in vitro cells


title: D21_P_1

description:
D21_P_1
readcount_cells.txt
fpkm_sample_cells.txt


title: ROI1_genomic_DNA_FC_biorep4_techrep1_input


title: RNA-Seq of rat hippocampus after TBI (Controlled Corticol Impact):  Thymus Nuclear Factor dosage. rep 2 [20B_S22]


title: D2A_P_3

description:
D2A_P_3
readcount_cells.txt
fpkm_sample_cells.txt


title: ROI2_genomic_DNA_FC_biorep3_techrep1_P3


title: RNA-Seq of rat hippocampus after TBI:  Vehicle sham. rep 2 [24B_S3]

value: Vehicle sham


title: GFP_48_3


title: D2A_P_2

description:
D2A_P_2
readcount_cells.txt
fpkm_sample_cells.txt


title: ROI2_genomic_DNA_FC_biorep3_techrep1_P4


title: RNA-Seq of rat hippocampus after TBI:  Thymus Cytosolic factor dosage. rep 1 [18B_S16]

value: Thymus Cytosolic factor dosage


title: D2A_P_1

description:
D2A_P_1
readcount_cells.txt
fpkm_sample_cells.txt


title: GFP_48_1

value: lung embryonic fibroblast


title: RNA-Seq of rat hippocampus after TBI (Controlled Corticol Impact):  Thymus  dosage. rep 1 [16B_S12]


title: ROI1_genomic_DNA_FC_biorep3_techrep1_P6


title: ROI2_genomic_DNA_FC_biorep3_techrep1_input


title: GFP_40_3


title: ST_0_2


title: D2A1_SCID_5

description:
D2A586SC
readcount_tumor.txt
fpkm_sample_tumor.txt

source_name: Tumor

value: Tumor

value: SCID beige

value: Female


title: GFP_40_2


title: RNA-Seq of rat hippocampus after TBI:  Vehicle sham. rep 1 [14B_S2]


title: ROI1_genomic_DNA_FC_biorep3_techrep1_P5

value: HEK293T LLP iCasp9 Blast


title: D21_P_3

description:
D21_P_3
readcount_cells.txt
fpkm_sample_cells.txt


title: RNA-Seq of rat hippocampus after TBI:  Vehicle (Controlled Corticol Impact). rep 1 [12B_S6]

value: Vehicle (Controlled Corticol Impact)


title: D2A1_SCID_4

description:
D2A585SC
readcount_tumor.txt
fpkm_sample_tumor.txt


title: D2A1_SCID_3

description:
D2A584SC
readcount_tumor.txt
fpkm_sample_tumor.txt


title: RNA-Seq of rat hippocampus after TBI (Controlled Corticol Impact):  Thymus Nuclear Factor dosage. rep 1 [10B_S21]


title: GFP_48_2

description: normalized_counts_cpm1filter_nooutliers.txt


title: GFP_32_3


title: GFP_32_2


title: ROI1_genomic_DNA_FC_biorep3_techrep1_input

value: Induction of transcription from landing pad with addition of 2 µg/mL Doxycycline


title: ROI1_genomic_DNA_FC_biorep3_techrep1_P4


title: ROI1_genomic_DNA_FC_biorep3_techrep1_P3


title: D2A1_SCID_2

description:
D2A583SC
readcount_tumor.txt
fpkm_sample_tumor.txt


title: GFP_32_1


title: BZR1-YFP, bio rep 1

city: seoul

first: So-Hee

last: Kim

address: Wangsimni-ro

country: South Korea

institute: Hanyang univ.

description: 2.Col_0_1-Col_0_2.vs.R1_101_C_1-R1_101_C_2.xlsx

data_processing:
At first, reads for each sample were mapped to the reference genome by Tophat (v2.0.13).
The aligned results were added to Cuffdiff(v2.2.0) to report differentially expressed genes.
For library normalization and dispersion estimation, geometric and pooled (“blind” when each condition has single replicates, or “pooled” when multiple replicates are available) methods were applied.
Cuffdiff data were added to CummeRbund(v2.8.2) to visualize RNA-Seq data.
Assembly: TAIR10
Supplementary files format and content: tab-delimited xlsx files include differentially expressed genes data

organism: Arabidopsis thaliana

source_name: whole plant

growth_protocol: Plants on 1/2 Murashige and Skoog (MS; Duchefa, Harrlem, Netherlands) medium were grown in a growth chamber at 23 ℃, under long day condition (16 h light and 8 h dark) for 12 days.

extract_protocol:
Total RNA was extracted from Arabidopsis seedlings using plant total RNA extraction kit (Macherey-Nagel).
mRNA sequencing libraries were prepared according to the manufacturer’s instructions (Illumina Truseq stranded mRNA library prep kit).

treatment_protocol: 12-d-old Col-0 seedlings treated with mock or 100 nM BL for 6 h, and Col-0, BEH2-YFP, BZR1-YFP are non-treatment.

value: whole plant

tag: ecotype

value: BZR1 overexpression in Columbia-0

value: non-treatment


title: BZR1-YFP, bio rep 2


title: GFP_40_1


title: BEH2-YFP, bio rep 2

description: 1.Col_0_1-Col_0_2.vs.H2_101_C_1-H2_101_C_2.xlsx

value: BEH2 overexpression in Columbia-0


title: D2A1_SCID_1

description:
D2A582SC
readcount_tumor.txt
fpkm_sample_tumor.txt


title: GFP_0_1


title: D2A1_Balb_4

description:
D2A581Ba
readcount_tumor.txt
fpkm_sample_tumor.txt

value: Balb/c


title: ROI2_genomic_DNA_biorep3_techrep1


title: D2A1_Balb_3

description:
D2A580Ba
readcount_tumor.txt
fpkm_sample_tumor.txt


title: BEH2-YFP, bio rep 1


title: GFP_24_3


title: Col-0, bio rep 2

description:
1.Col_0_1-Col_0_2.vs.H2_101_C_1-H2_101_C_2.xlsx
2.Col_0_1-Col_0_2.vs.R1_101_C_1-R1_101_C_2.xlsx

value: wild-type


title: D2A1_Balb_2

description:
D2A579Ba
readcount_tumor.txt
fpkm_sample_tumor.txt


title: ROI2_genomic_DNA_biorep2_techrep1


title: GFP_24_2

value: GFP


title: Col-0, bio rep 1


title: D2A1_Balb_1

description:
D2A578Ba
readcount_tumor.txt
fpkm_sample_tumor.txt


title: Col-0, treatment BL, bio rep 2

description: 1.Col-0_NBL-1,Col-0_NBL-2.vs.Col-0_PBL-1,Col-0_PBL-2.xlsx

value: treatment brassinosteroid


title: D21_SCID_5

description:
D219SCID
readcount_tumor.txt
fpkm_sample_tumor.txt


title: D21_SCID_4

description:
D218SCID
readcount_tumor.txt
fpkm_sample_tumor.txt


title: ROI2_genomic_DNA_biorep4_techrep1


title: GFP_20_2


title: GFP_20_3


title: Col-0, treatment BL, bio rep 1


title: ROI1_genomic_DNA_biorep3_techrep1


title: ROI1_genomic_DNA_biorep1_techrep2


title: D21_SCID_2

description:
D216SCID
readcount_tumor.txt
fpkm_sample_tumor.txt


title: ROI1_genomic_DNA_biorep2_techrep3


title: Col-0, non-treatment BL, bio rep 1


title: D21_SCID_3

description:
D217SCID
readcount_tumor.txt
fpkm_sample_tumor.txt


title: GFP_16_3


title: ROI2_library_plasmid_techrep1


title: ROI2_genomic_DNA_biorep1_techrep1


title: Col-0, non-treatment BL, bio rep 2


title: ROI1_library_plasmid_techrep2


title: GFP_16_2


title: GFP_24_1


title: D21_SCID_1

description:
D215SCID
readcount_tumor.txt
fpkm_sample_tumor.txt


title: ROI1_genomic_DNA_biorep4_techrep1


title: GFP_16_1


title: D21_Balb_3

description:
D213Balb
readcount_tumor.txt
fpkm_sample_tumor.txt


title: H1K1 cells, RIC-KO, Rep2

city: London

first: Li

last: Tong

address: Du Cane Road

department: Metabolism, Digestion & Reproduction

country: United Kingdom

institute: Imperial College London

data_processing:
Raw fastq format data were trimmed by Fastp v0.23.2 to remove sequencing adaptors and low-quality bases (< Q15).
Trimmed sequence reads were aligned to  GRCh38 release 104 using STAR v2.7.10a. The expression of genes was quantified by RSEM v1.3.3
Differential expression and statistical analysis were performed by DESeq2 v1.34.0 in R v4.1.2
Assembly: GRCh38 release 104
Supplementary files format and content: Text files include raw counts, TPM, and FPKM values for each sample.

source_name: H1 hESCS

growth_protocol: H1 hESCs were cultured in mouse embryonic fibroblast conditioned medium (MEF-CM) which contains knockout serum replacement (KSR) medium (Knockout DMEM containing 20% KSR, 1 mM L-glutamine, 1% non-essential amino acid, 0.1 M β-mercaptoethanol, 1% Penicillin-Streptomycin, 4 ng/ml bFGF) supplemented with 10 ng/ml bFGF and refreshed daily.

extract_protocol:
Total RNA was extracted from cells using TRIzolTM Plus RNA Purification Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. 500 ng of total RNA was used for constructing sequencing libraries.
Strand-specific RNA libraries were prepared using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® following manufacturer's instructions.

value: H1 hESCS

value: human embryonic stem cells

value: RICTOR-KO

value: routine MEF-CM culture


title: ROI2_WT_plasmid_techrep1


title: GFP_20_1

description: normalized_counts_cpm1filter_nooutliers.txt

value: lung embryonic fibroblast


title: H1K5 cells, RIC-KO, Rep1


title: ER_48_3

value: ER


title: ER_48_2


title: ROI1_WT_plasmid_techrep2


title: D21_Balb_2

description:
D212Balb
readcount_tumor.txt
fpkm_sample_tumor.txt

value: Tumor

value: Female


title: H1K5 cells, RIC-KO, Rep2


title: H1K1 cells, RIC-KO, Rep1


title: D21_Balb_4

description:
D214Balb
readcount_tumor.txt
fpkm_sample_tumor.txt


title: D21_Balb_1

description:
D211Balb
readcount_tumor.txt
fpkm_sample_tumor.txt


title: H1C3 cells, RIC-WT, Rep2

value: WT


title: ROI2_total_RNA_biorep4_techrep2

value: HEK293T LLP iCasp9 Blast

value: Induction of transcription from landing pad with addition of 2 µg/mL Doxycycline


title: ROI2_total_RNA_biorep3_techrep2


title: H1C3 cells, RIC-WT, Rep1


title: ER_48_1


title: Mouse replicate 1 Nur77GFP LO ADT/HTO/FBT library [scRNASeq]

city: Minneapolis

first: Tyler

last: Bold

address: 2101 6th St SE

institute: University of Minnesota

description:
10x Genomics
Bold_004_S4_barcodes.tsv.gz
Bold_004_S4_features.tsv.gz
Bold_004_S4_matrix.mtx.gz

data_processing:
Fastq files from gene expression (GEX) and ADT libraries were processed with 10X cellranger using a custom reference genome consisting of the Mus musculis genome mm10 with the addition of the EGFP coding sequence to enable detection of cells expressing GFP reporter gene. TCR libraries were processed using Cellranger VDJ
ADT
TotalSeq™-C0157 anti-mouse CD45.2 Antibody
Barcode sequence
CACCGTCATTCAACC
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Lung

extract_protocol:
Mycobacterium tuberculosis-infected Nur77-GFP mice were euthanized with CO2 and lungs were suspended in a digestion buffer containing DMEM, FBS, Collagenase D, DNase, Heparin, CaCl 2 , and MgCl 2 . Lungs were incubated at 37˚C for 30 minutes and dissociated with a gentleMACS dissociator (Miltenyi Biotec), according to the manufacturer’s protocol. To isolate cells, lung suspensions were filtered through a 70 μm cell strainer. Sorting for the tetramer-enriched scRNA-Seq was performed using a CD4 + T cell isolation kit (Miltenyi Biotec) followed by tetramer staining and enrichment of tetramer-specific cells with anti-PE microbeads (Miltenyi Biotec).
Cells FACS sorted (either GFP-HI or -LO) into DMEM with 40% FBS were used for scRNA-Seq sample preparation. After sorting, cell number was confirmed with the Luna II (Logos Biosystems). The appropriate number of cells necessary to achieve a target cell input of 2-10,000 cells was resuspended and used to generate GEMs (Gel Bead-In Emulsion). The Chromium Next GEM Single-cell 5’ Gel beads v2 kit (10X Genomics) was used and manufacturer instructions were followed for GEM generation through cDNA synthesis. A Chromium Control was used for GEM generation and barcoding then a thermocycler was used for cDNA synthesis. cDNA was submitted to the University of Minnesota Genomics Center for sequencing on an Illumina Novaseq. Tetramer-enriched scRNA-seq data pooled from three biological replicates.

value: Lung

value: CD4 T cells

value: Mycobacterium tuberculosis infection

tag: antibody

value: TotalSeq™-C0157 anti-mouse CD45.2 Antibody.


title: ROI1_total_RNA_biorep4_techrep2


title: H1C1 cells, RIC-WT, Rep2


title: Mouse replicate 1 Nur77GFP HI ADT/HTO/FBT library [scRNASeq]

description:
10x Genomics
Bold_004_S3_barcodes.tsv.gz
Bold_004_S3_features.tsv.gz
Bold_004_S3_matrix.mtx.gz

value: TotalSeq™-C0157 anti-mouse CD45.2 Antibody


title: H1C1 cells, RIC-WT, Rep1


title: ROI1_total_RNA_biorep3_techrep2


title: Mouse replicate 1 Nur77GFP HI TCR library [scRNASeq]

description: 10x Genomics


title: ER_40_1

source_name: IMR90 human lung embryonic fibroblast


title: ER_32_3


title: VEXASLIKE2

city: Paris

first: Mickael

middle: Mathieu

last: Menager

address: 24 Boulevard du Montparnasse

country: France

institute: Institut Imagine

data_processing:
Sequencing reads were demultiplexed and aligned to the human reference genome (GRCh38-2020-A directly download from 10x), using the CellRanger Pipeline v5.0.1
Unfiltered RNA UMI counts were loaded into Seurat v4.0.3 (Stuart et al., 2019) for quality control, data integration and downstream analyses. Apoptotic cells and empty sequencing capsules were excluded by filtering out cells with fewer than 500 features or a mitochondrial content higher than 20%.
Data from each sample were SCT normalisedand scaled, before batch correction using Seurat’s FindIntegratedAnchors.
On this integrated dataset, we computed the principal component analysis on the 2000 most variable genes. UMAP was carried out using the 20 most significant PCs, and community detection was performed using the graph-based modularity-optimization Louvain algorithm from Seurat’s FindClusters function with a 0.8 resolution.
Cell types labels were assigned to resulting clusters based on a manually curated list of marker genes as well as previously defined signatures of the well-known PBMC subtypes (Monaco et al., 2019).  63630 cells were kept for further analysis.
Assembly: GRCh38-2020-A
Supplementary files format and content: h5 files contain the raw count matrix for each sample
Supplementary files format and content: the rds file contains the Seurat object of all integrated samples

source_name: pbmc

extract_protocol: RNA libraries were prepared for sequencing using standard Illumina protocols

value: pbmc

tag: group

value: Patient

tag: disease

value: autoinflammatory disease

tag: mutation

value: no


title: GFP_12_1

value: GFP


title: Mouse replicate 1 Nur77GFP LO TCR library [scRNASeq]

description: 10x Genomics


title: ROI2_total_RNA_biorep2_techrep2

source_name: HEK293T LLP iCasp9 Blast


title: Mouse replicate 4 Nur77GFP LO [scRNASeq]

description: 10x Genomics


title: ROI2_total_RNA_biorep1_techrep2


title: VEXASLIKE1


title: Mouse replicate 4 Nur77GFP HI [scRNASeq]

description: 10x Genomics


title: ER_40_3

value: lung embryonic fibroblast


title: ER_32_2

value: ER


title: VEXAS2

value: Vexas syndrome


title: VEXAS1


title: ER_32_1


title: Mouse replicate 3 Nur77GFP LO [scRNASeq]

description: 10x Genomics


title: ROI2_total_RNA_biorep3_techrep1


title: ER_24_3


title: ROI2_total_RNA_biorep2_techrep1


title: MDS2

value: low-risk myelodysplastic syndromes


title: Mouse replicate 3 Nur77GFP HI [scRNASeq]

description: 10x Genomics


title: ER_24_2


title: ROI2_total_RNA_biorep1_techrep1


title: MDS1


title: ER_24_1


title: Mouse replicate 2 Nur77GFP LO [scRNASeq]

description: 10x Genomics


title: ROI1_total_RNA_biorep4_techrep1


title: CONTROL2

value: Control

value: Healthy controls


title: Mouse replicate 2 Nur77GFP HI [scRNASeq]

description: 10x Genomics


title: ROI1_total_RNA_biorep3_techrep1

value: HEK293T LLP iCasp9 Blast

value: Induction of transcription from landing pad with addition of 2 µg/mL Doxycycline


title: Mouse replicate 1 Nur77GFP LO [scRNASeq]

value: Lung

value: CD4 T cells


title: ER_20_2


title: ER_20_3


title: TIER_PMN_Blood

city: Woluwe Waint Lambert

first: Pierre

last: van der Bruggen

address: Avenue Hippocrate 75

country: Belgium

institute: de Duve institute

description:
normal density neutrophil from blood of healthy donor
Dataset_2_matrix_ub_fcounts.txt

data_processing:
Quality control of reads using FastQC before and after trimming with Trimmomatic version 0.38 (headcrop:5 ; Leading:20; slidingwindow:5:20; Minlen:50)
Reads were aligned on genome Grch38 with hisat2 program using default options
Reads counts table was generated from .bam files through FeatureCounts program (Subread package) with automatic settings
Assembly: hg38
Supplementary files format and content: 1 matrix tab-delimited text files including raw count values for all samples (one per column)

source_name: Blood

growth_protocol: 5 000 to 2 500 neutrophils sorted from blood and tumors of healthy donors or cancer patients were centrifuged and resuspended in 200ul of PBS. The suspension was then mixed with 900ul of Qiazol lysis solution (Qiagen, 79305), transfered to RNAse-free 1.5ml low-binding Eppendorfs (Ambion, AM12450) and then vortexed for 1min. Lysates were kept at -80°C until RNA extraction

extract_protocol:
RNA was extracted with miRNeasy kit (Qiagen, 217084) according to manufacturer protocol. RNA was eluted in 15ul of RNase-free water and precipitated with 0,1 volume of sodium acetate 3M (Themofisher Scientific, AM9740), 3 volumes of ice-cold ethanol 100% (Anafor-Normapur, 20821.310) and 1ug glycogen (Thermo Fisher Scientific, R0551). After overnight incubation at -20°C, samples were centrifuged at 14000g 30min 4°C and washed 2 times with ice-cold ethanol 75%. Supernatant was removed and samples were left to dry at RT°C before being resuspended in 4.5ul RNase-free water
RNA-seq libraries were constructed using the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096) in combination with Nextera XT Index Kit v2 Set D (Illumina, FC-131-2004). The construction was unstranded and poly-A-based. RNA libraries were sent for ultra-low input RNA-sequencing to Genewiz

value: Blood

tag: patient id

value: PMN

value: Healthy


title: CONTROL1

value: pbmc


title: ROI1_total_RNA_biorep2_techrep3 (COMT_95)


title: ROI2_total_RNA_biorep4_techrep1


title: ROI1_total_RNA_biorep1_techrep2 (COMT_94)


title: Mouse replicate 1 Nur77GFP HI [scRNASeq]

tag: tissue

value: Mycobacterium tuberculosis infection


title: Mouse replicate 4 Nur77GFP HI

data_processing:
RNA was submitted to GeneWiz from Azenta Life Sciences for RNA sequencing and differential gene expression analysis using DeSeq2.
Assembly: mm10
Supplementary files format and content: Tab delimited csv file contains read counts for each sample

extract_protocol:
Mycobacterium tuberculosis-infected Nur77-GFP mice were euthanized with CO2 and lungs were suspended in a digestion buffer containing DMEM, FBS, Collagenase D, DNase, Heparin, CaCl 2 , and MgCl 2 . Lungs were incubated at 37˚C for 30 minutes and dissociated with a gentleMACS dissociator (Miltenyi Biotec), according to the manufacturer’s protocol. To isolate cells, lung suspensions were filtered through a 70 μm cell strainer. Sorting for CD44hi, CD4+, GFP HI and LO cells was performed with a Sony MA900 cell sorter.
Cells FACS sorted (either GFP-HI or -LO) into DMEM with 40% FBS were used for scRNA-Seq sample preparation. After sorting, cell number was confirmed with the Luna II (Logos Biosystems). Approximately 100,000 cells from each population were sorted into DMEM-40 before resuspension in Tri reagent. Samples in TRI Reagent were centrifuged at 1000 x g for 10 minutes. Samples were resuspended in chloroform and incubated for 3 minutes then centrifuged at 12,000 x g for 15 minutes. The supernatant was collected, isopropyl alcohol added, incubated for 10 minutes, then centrifuged at 7,500 x g for 5 minutes. The supernatant was discarded then samples were air dried before resuspension in RNase-free water.

tag: genotype


title: ER_20_1


title: PEFR_PMN_Blood


title: ER_16_2


title: ROI2_fractionated_RNA_biorep4_techrep1_F3

tag: treatment


title: ROI2_fractionated_RNA_biorep4_techrep1_F4


title: ER_16_3

tag: cell type


title: SLAY_PMN_Blood


title: Mouse replicate 3 Nur77GFP HI


title: PEFR_LDN_CD45high_Blood

description:
low density neutrophil CD45hi from blood of healthy donor
Dataset_2_matrix_ub_fcounts.txt


title: Mouse replicate 1 Nur77GFP HI


title: PB026_PMN_Blood

description:
normal density neutrophil from blood of lung cancer patient
Dataset_2_matrix_ub_fcounts.txt

value: Lung Cancer


title: ROI2_fractionated_RNA_biorep4_techrep1_F2


title: ROI2_fractionated_RNA_biorep4_techrep1_F1


title: PB026_LDN_Tumor

description:
low density neutrophil CD45hi from tumor of lung cancer patient
Dataset_2_matrix_ub_fcounts.txt

value: Tumor


title: Mouse replicate 3 Nur77GFP LO


title: PB026_LDN_CD45low_Blood

description:
low density neutrophil CD45low from blood of lung cancer patient
Dataset_2_matrix_ub_fcounts.txt


title: ROI2_fractionated_RNA_biorep3_techrep1_F4


title: Mouse replicate 2 Nur77GFP LO


title: ROI2_fractionated_RNA_biorep3_techrep1_F3


title: ROI1_genomic_DNA_biorep2_techrep2


title: PB026_LDN_CD45high_Blood

description:
low density neutrophil CD45hi from blood of lung cancer patient
Dataset_2_matrix_ub_fcounts.txt


title: Mouse replicate 1 Nur77GFP LO


title: ROI1_genomic_DNA_biorep2_techrep1


title: ROI1_genomic_DNA_biorep1_techrep1


title: ROI2_fractionated_RNA_biorep3_techrep1_F1


title: ROI2_fractionated_RNA_biorep3_techrep1_F2


title: ROI1_fractionated_RNA_biorep2_techrep3_F'5


title: PB013_PMN_Blood


title: Mouse replicate 2 Nur77GFP HI


title: Mouse replicate 3

description: 10x Genomics

data_processing:
Fastq files were processed with 10X cellranger using a custom reference genome consisting of the Mus musculis genome mm10 with the addition of the EGFP coding sequence to enable detection of cells expressing GFP reporter gene.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol:
Mice were euthanized with CO2 and lungs were suspended in a digestion buffer containing DMEM, FBS, Collagenase D, DNase, Heparin, CaCl 2 , and MgCl 2 . Lungs were incubated at 37˚C for 30 minutes and dissociated with a gentleMACS dissociator (Miltenyi Biotec), according to the manufacturer’s protocol. To isolate cells, lung suspensions were filtered through a 70 μm cell strainer. Sorting for the tetramer-enriched scRNA-Seq was performed using a CD4 + T cell isolation kit (Miltenyi Biotec) followed by tetramer staining and enrichment of tetramer-specific cells with anti-PE microbeads (Miltenyi Biotec).
Cells sorted into DMEM with 40% FBS were used for scRNA-Seq sample preparation. After sorting, cell number was confirmed with the Luna II (Logos Biosystems). The appropriate number of cells necessary to achieve a target cell input of 2-10,000 cells was resuspended and used to generate GEMs (Gel Bead-In Emulsion). The Chromium Next GEM Single-cell 5’ Gel beads v2 kit (10X Genomics) was used and manufacturer instructions were followed for GEM generation through cDNA synthesis. A Chromium Control was used for GEM generation and barcoding then a thermocycler was used for cDNA synthesis. cDNA was submitted to the University of Minnesota Genomics Center for sequencing on an Illumina Novaseq. Tetramer-enriched scRNA-seq data pooled from three biological replicates.

value: Multiple


title: ROI1_library_plasmid_techrep1


title: ROI1_fractionated_RNA_biorep2_techrep1_F4


title: PB013_LDN_CD45low_Blood


title: PB013_LDN_CD45high_Blood


title: ROI1_WT_plasmid_techrep1


title: Mouse replicate 4 Nur77GFP LO


title: Mouse replicate 2

description: 10x Genomics


title: PB007B_PMN_Blood


title: Mouse replicate 1

description: 10x Genomics


title: ROI1_fractionated_RNA_biorep2_techrep1_F3


title: ROI1_fractionated_RNA_biorep2_techrep1_F2


title: ROI1_fractionated_RNA_biorep2_techrep3_F'3


title: ROI1_fractionated_RNA_biorep2_techrep3_F'4


title: PB007B_LDN_CD45low_Blood


title: 4T1, Primary Tumor, 1583-SO-45

city: Ann Arbor

first: Sophia

last: Orbach

state: MI

address: 1600 Huron Parkway

institute: University of Michigan

data_processing:
Sequence reads were trimmed using Trim_Galore (v 0.5.0)
Trimmed sequence reads were aligned to GECm38 from ENSEMBL using STAR (v 2.6.0)
UMI collapsed using Lexogen's supplied collapse_UMI_bam binary
Counted using Bioconductor R package Rsubread and featureCounts function
Assembly: GECm38
Supplementary files format and content: tab-delimited text file including raw counts of each sample

source_name: Primary Tumor

growth_protocol: 67NR, 4T07, and 4T1 triple negative breast cancer cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, and 1 mM mixed non-essential amino acids in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
RNA was harvested using RNeasy Mini Kit (Qiagen). 50 uL of total RNA was submitted for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using the Lexogen QuantSeq 3' mRNA FWD with the UMI add-on kit.
NovaSeq (S1) 100 Cycle

treatment_protocol:
Tumor-bearing mice received an orthotopic inoculation of 2 million cancer cells (67NR, 4T07, or 4T1) resusupended in 50 uL PBS
Tumors were allowed to progress for 14 days before tissue extraction

value: Primary Tumor

value: Balb/c

value: female

tag: time

value: Day 14


title: ROI1_fractionated_RNA_biorep2_techrep1_F1

data_processing:
Variant calling: Variants were called with satmut_utils v1.0.3-dev001 (https://github.com/ijhoskins/satmut_utils/releases/tag/v1.0.3-dev001). The satmut_utils  parameters were as follows: `satmut_utils -r pDEST_HC_Rec_Bxb_v2_UTR5_Flag_COMT_moxGFP_LPS.fa -p pDEST_HC_Rec_Bxb_v2_UTR5_Flag_COMT_moxGFP_LPS_ROI_primers_v2.bed call -k pDEST_HC_Rec_Bxb_v2_UTR5_Flag_COMT_moxGFP_LPS.fa -g pDEST_HC_Rec_Bxb_v2_UTR5_Flag_COMT_moxGFP_LPS.gff -n 2 -m 1 --r1_fiveprime_adapters TACACGACGCTCTTCCGATCT --r1_threeprime_adapters AGATCGGAAGAGCACACGTCT --r2_fiveprime_adapters AGACGTGTGCTCTTCCGATCT --r2_threeprime_adapters AGATCGGAAGAGCGTCGTGTA`.  Ribosome profiling analysis: Analysis was conducted with RiboFlow (umi_devel branch) and RiboR (Ozadam et al., 2020). The reference files were modified to include a Flag tag upstream of the COMT coding sequence to enable mapping of footprints arising from the stably-expressed COMT transgene.
Assembly: Data were aligned to custom a reference FASTA file. Reference files are available at https://github.com/ijhoskins/integrated_MAVEs/tree/main/references
Supplementary files format and content: satmut_utils summary.txt files contain VCF fields with each INFO tag value in a unique column. See https://github.com/ijhoskins/satmut_utils/blob/satmut_utils_dev/docs/satmut_utils_manual.md for details on each field. RiboFlow .ribo file documentation is available at https://ribopy.readthedocs.io/en/latest/ribo_file_format.html.

extract_protocol:
gDNA extraction: gDNA was extracted from approximately 3-4 million cells with the Cell and Tissue DNA Isolation Kit (Norgen Biotek Corp, 24700), including RNaseA treatment at 37 ˚C for 15 min, and eluting in 200 µL warm elution buffer.  RNA extraction: Approximately 3-4 million cells were solubilized with 1 mL QIAzol (Qiagen, 79306) and 0.2 mL chloroform in 5PRIME Phase-Lock Gel heavy tubes (QuantaBio, 2302830), according to the manufacturer’s recommendations. RNA was precipitated at -20 ˚C following the addition of 2 µL GlycoBlue (Thermo Fisher, AM9515) and 2.5 volumes of cold absolute ethanol. RNA was washed once with cold 70% ethanol then resuspended in 30 µL water. 10 µg total RNA or 15 µL of polysomal RNA (half of 30 µL eluate, volumetric inputs) was treated with DNaseI (NEB, M0303) in a 100 µL reaction at 37 ˚C for 15 min, then re-purified by the RNA Clean and Concentrator Kit (Zymo Research, R1015) and eluted in 20 µL water.  Polysome fractionation and polysomal RNA purification: For COMT library stable cell lines, cells at 80-95% confluency (one 15 cm dish per gradient) were incubated with 100 µg/mL cycloheximide (Sigma Aldrich, C4859) in media for 10 min at 37 ˚C, then resuspended in ice cold PBS with 100 µg/mL cycloheximide (PBS-CHX), collected at 4 ˚C by pipetting, and pelleted at 300 x g for 7 min at 4 ˚C. Pellets were flash frozen in liquid nitrogen, then lysed on ice for 10 min with 300-400 µL lysis buffer (20 mM Tris-HCl pH 7.5; 150 mM NaCl; 5 mM MgCl2, 1 mM fresh DTT, 20 mM ribonucleoside vanadyl complex (Fisher Scientific, 50-812-650), 1X protease inhibitor cocktail EDTA-free (Fisher Scientific, 539196), and 100 μg/ml cycloheximide. Lysates were clarified by centrifugation at 1,300 x g for 10 min at 4 ˚C, and 1/10th of the lysate was saved for total RNA extraction. 20-50% sucrose gradients were made with the BioComp Gradient Maker in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 100 μg/ml cycloheximide using Beckman Coulter UC tubes 9/16 x 3-1/2 (Fisher Scientific, NC9194790), and equilibrating overnight at 4 ˚C. Clarified lysates were loaded onto sucrose gradients, and fractionated by ultracentrifugation at 38,000 rpm for 2.5 h at 4 ˚C in a Beckman Coulter SW-41 Ti rotor. Approximately 500 µL polysome fractions were collected using the Biocomp Piston Gradient Fractionator (v8.04) with a Triax flow cell (BioComp model FC-1) and Gilson Fraction Collector using a piston speed of 0.2 mm/s. RNA was precipitated from raw polysome fractions by addition of sodium acetate to 300 mM pH 5.2, 2 µL Glycoblue, and 2x volumes cold ethanol, stored overnight at -20 ˚C. RNA was resuspended in 50-80 µL ultrapure water, and these raw fractions were pooled into metafractions and extracted by phenol-chloroform (5:1) and ethanol precipitation overnight at -20 ˚C. After a single 70% ethanol wash, polysomal RNA was resuspended in 30 µL ultrapure water. Lastly, DNaseI treatment and a column purification was performed prior to library preparation.  Ribosome profiling: Monosomes were pelleted through a sucrose cushion (1 M sucrose, 20 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1x protease inhibitor cocktail EDTA-free) by spinning at 38,000 rpm at 4 ˚C in a SW-41 Ti rotor (Beckman Coulter). Monosome pellets were resuspended in 400 µL lysis buffer and rocked overnight. RNA was extracted with 1.2 mL phenol/chloroform/isoamyl alcohol and precipitated with 300 mM sodium acetate pH 5.2, 1.5 µL Glycoblue, and 2.5 volumes of cold absolute ethanol overnight at -20 ˚C. RNA was pelleted at 21,000 x g for 1 h, washed once with cold 70% ethanol, and resuspended with 30 µL water. 15 µL of RNA was run on a 15% TBE-urea polyacrylamide gel at 150 V for 1.5 h. The gel was stained with 1x SYBR Gold and footprints between 21 nt and 34 nt were cut and isolated by crush and soak in RNA extraction buffer (300 mM sodium acetate pH 5.5, 1 mM EDTA, 0.25% v/v SDS) with overnight rotation. The eluate was passed through a Spin X Centrifuge Tube Filter with a cellulose acetate membrane (Thermo Fisher Scientific, 07-200-385), and the RNA was precipitated with 300 mM sodium acetate pH 5.2, 1.5 µL Glycoblue, and 2.5 volumes of cold absolute ethanol overnight at -20 ˚C. RNA was pelleted at 21,000 x g for 1 h, washed once with cold 70% ethanol, and resuspended in 12 µL water.
cDNA synthesis: 10 µL DNaseI-treated total RNA (<10 µg) or DNaseI-treated polysomal RNA (200 ng - 5 µg) was denatured at 65 ˚C for 5 min followed by RT primer annealing at 4 ˚C for 2 min, using 2 pmol pDEST_HC_Rec_Bxb_v2_R primer specific for the landing pad (see Supplementary Table 4) and 2.5 mM random hexamers. Primed total RNA was included in 20 µL SuperScript IV cDNA synthesis reaction (Thermo Fisher, 18090010) with SUPERase-In RNase inhibitor (Thermo Fisher, AM2696), and first-strand cDNA was synthesized by incubating at 23 ˚C for 10 min, 55 ˚C for 1 h, followed by RT inactivation at 80 ˚C for 10 min. RNA was digested with addition of 5 U RNaseH (NEB, M0297) to the first strand cDNA synthesis reaction and incubation at 37 ˚C for 20 min.  Landing pad amplification (PCR1): Nucleic acids (gDNA, 1st strand cDNA) were amplified with Q5 polymerase (NEB, M0491) in 50 µL PCR reactions for 16 cycles with 500 nM landing-pad-specific primers (pDEST_HC_Rec_Bxb_v2_F/R) flanking the entire COMT insert (~1.7 kb). For negative control (plasmid) samples, 10 ng was used as template. For total RNA and polysome RNA samples, 15 µL of unpurified 1st strand cDNA synthesis reaction was used as template. For total gDNA samples, 4 µg template was used in each of three PCR reactions. For flow-sorted gDNA samples, either 1.75 µg template was used in three PCR reactions or 2.5 µg was input into two reactions, with 14 cycles instead of 16. The cycling parameters were: initial denaturation at 98 ˚C for 30 s; 3-step cycling with denaturation at 98 ˚C for 10 s, anneal at 65 ˚C for 30 s, extension at 72 ˚C for 1 min; final extension at 72 ˚C for 2 min. Products from total RNA and polysome RNA samples were purified with the QIAquick PCR Purification Kit (Qiagen, 28106) and eluted in 30 µL elution buffer. Products for total gDNA samples were pooled, resolved on a 0.8% agarose/TAE gel, stained with 1x SYBR Gold, and extracted using the Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) or Zymoclean Gel DNA Recovery Kit (Zymo Research, D4007) with 20 µL 70 ˚C elution buffer. At this point, PCR1 products for gDNA samples cannot typically be seen in the gel, and the expected band size is cut using comparison to a high-molecular weight ladder.  Coding sequence amplification (PCR2): Purified PCR1 products (landing pad insert) were amplified for each of COMT target regions in a 50 µL NEB Q5 reaction (NEB, M0491) for 8 cycles, following the same cycling parameters as for PCR1 and 500 nM tailed primers (see Supplementary Table 4). 15 µL PCR1 product was used as template (50% of eluate for total RNA and polysome RNA samples; 75% of eluate for gel-extracted gDNA samples). Products were purified with the Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) with 1:5 buffer NTI dilution and 25 µL 70 ˚C elution buffer, or with the QIAquick PCR Purification Kit (Qiagen, 28106) with 30 µL elution buffer.  Illumina adapter addition (PCR3): A final Q5 PCR reaction was carried out for 8 cycles with the same formulation as PCR2 but using NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 (NEB, E7600S) according to manufacturer’s recommendations (65 ˚C annealing). 25 µL/30 µL eluate was used as template for total gDNA, total RNA, and polysome RNA samples. For flow cytometry gDNA samples, 15 µL/25 µL eluate was used as template. PCR3 products (final library) were purified with the Nucleospin Gel and PCR Cleanup kit (Takara, 740609) and eluted in 25 µL 70 ˚C buffer.  Ribosome profiling: 25 ng of RNA in 8 µL water was input to the D-Plex Small RNA kit (Diagenode, C0503000) and libraries were prepared according to the manufacturer recommendations with the exception that 9 cycles of PCR were carried out. The library was purified with 1.25x AMPure beads (Beckman Coulter, A63880) and quantified by the High Sensitivity DNA Kit (Agilent, 5067-4626).  Quantification and size-selection of final libraries: Final libraries were quantified using the KAPA Library Quantification Kit for Illumina (Roche, KK4873) according to the manufacturer’s recommendations, using 1:10,000 dilution of libraries and size-correction with an average fragment size of 300 nt (region 1) and 285 nt (region 2). qPCR was performed on the Applied Biosystems ViiA 7 instrument and values were used to pool individual libraries at equimolar ratios to target ~5 M read pairs per library. Final library pools were size-selected use PAGE purification on a 15% TBE gel followed by a crush-and-soak method (Sambrook and Russell, 2006), or with BluePippin 2% gel (Sage Science, BDF2010) to select fragments from 250-340 bp. Following size-selection, final pools were quantified by High Sensitivity DNA Kit (Agilent, 5067-4626) prior to sequencing.
A targeted library preparation method using three sequential PCRs was used (see library construction protocol).


title: 4T1, Primary Tumor, 1583-SO-44


title: ROI1_fractionated_RNA_biorep1_techrep1_F4


title: 4T1, Primary Tumor, 1583-SO-43


title: ROI1_fractionated_RNA_biorep2_techrep3_F'1


title: PB007B_LDN_CD45high_Blood


title: ROI1_fractionated_RNA_biorep2_techrep2_F'5


title: PB007A_PMN_Blood


title: 4T1, Spleen, 1583-SO-42

source_name: Spleen

value: Spleen


title: ROI1_fractionated_RNA_biorep2_techrep2_F'4

value: HEK293T LLP iCasp9 Blast


title: ROI1_fractionated_RNA_biorep1_techrep1_F3

value: Induction of transcription from landing pad with addition of 2 µg/mL Doxycycline


title: PB007A_LDN_CD45low_Blood


title: ROI1_fractionated_RNA_biorep1_techrep1_F2


title: 4T1, Spleen, 1583-SO-40


title: PB007A_LDN_CD45high_Blood


title: 4T1, Spleen, 1583-SO-41


title: ROI1_fractionated_RNA_biorep2_techrep2_F'3


title: ROI2_genomic_DNA_FC_biorep1_techrep1_P6


title: PA100_PMN_Blood


title: 4T1, Lung, 1583-SO-39

value: Lung


title: PA100_LDN_Tumor


title: ROI1_fractionated_RNA_biorep2_techrep3_F'2


title: 4T1, Lung, 1583-SO-38


title: ROI1_fractionated_RNA_biorep2_techrep2_F'2


title: PA100_LDN_CD45low_Blood


title: ROI2_genomic_DNA_FC_biorep1_techrep1_P5


title: 4T1, Lung, 1583-SO-37


title: ROI1_fractionated_RNA_biorep2_techrep1_F'5


title: PA100_LDN_CD45high_Blood


title: 4T1, Scaffold, 1583-SO-36

source_name: Scaffold

value: Scaffold


title: ROI2_genomic_DNA_FC_biorep1_techrep1_P4


title: PA070_PMN_Blood

value: Blood


title: ROI2_genomic_DNA_FC_biorep1_techrep1_P3


title: ROI1_fractionated_RNA_biorep2_techrep1_F'3


title: ROI2_genomic_DNA_FC_biorep1_techrep1_input


title: ROI1_fractionated_RNA_biorep1_techrep1_F1


title: 4T1, Scaffold, 1583-SO-35


title: PA064_PMN_Blood

value: PMN


title: ROI1_fractionated_RNA_biorep2_techrep1_F'1


title: ROI1_genomic_DNA_FC_biorep1_techrep1_P6


title: ROI1_fractionated_RNA_biorep2_techrep2_F'1


title: PA017D_PMN_Blood


title: 4T1, Scaffold, 1583-SO-34


title: ROI1_fractionated_RNA_biorep2_techrep1_F'2


title: ROI1_genomic_DNA_FC_biorep1_techrep1_P5


title: 4T07, Primary Tumor, 1583-SO-33


title: PA017D_LDN_CD45low_Blood


title: ROI1_genomic_DNA_FC_biorep1_techrep1_P4


title: ROI1_fractionated_RNA_biorep1_techrep3_F'5


title: ROI1_fractionated_RNA_biorep1_techrep3_F'4


title: 4T07, Primary Tumor, 1583-SO-32


title: ROI1_fractionated_RNA_biorep2_techrep1_F'4


title: ROI1_genomic_DNA_FC_biorep1_techrep1_P3


title: 4T07, Primary Tumor, 1583-SO-31


title: ROI1_genomic_DNA_FC_biorep1_techrep1_input


title: PA017C_PMN_Blood


title: ROI1_fractionated_RNA_biorep1_techrep3_F'2


title: MYKA_PMN_Blood

description:
normal density neutrophil from blood of ovarian cancer patient
Dataset_2_matrix_ub_fcounts.txt

value: MYKA

value: Ovarian Cancer


title: 4T07, Spleen, 1583-SO-29


title: ROI2_genomic_DNA_FC_biorep2_techrep1_P6


title: ROI2_genomic_DNA_FC_biorep2_techrep1_P5


title: 4T07, Spleen, 1583-SO-28


title: ROI1_fractionated_RNA_biorep1_techrep3_F'1


title: LISE_PMN_Blood

value: Healthy


title: ROI2_genomic_DNA_FC_biorep2_techrep1_P4


title: ROI1_fractionated_RNA_biorep1_techrep2_F'4


title: LISE_LDN_CD45high_Blood


title: 4T07, Lung, 1583-SO-27


title: ROI1_fractionated_RNA_biorep1_techrep2_F'5


title: ROI1_fractionated_RNA_biorep1_techrep2_F'3


title: ROI2_genomic_DNA_FC_biorep2_techrep1_P3


title: 4T07, Lung, 1583-SO-26


title: PA017D_LDN_CD45high_Blood

value: Lung Cancer


title: LIRI_PMN_Blood


title: ROI1_fractionated_RNA_biorep1_techrep3_F'3


title: ROI2_genomic_DNA_FC_biorep2_techrep1_input


title: LIRI_LDN_CD45high_Blood


title: ROI1_genomic_DNA_FC_biorep2_techrep1_P6


title: ROI1_fractionated_RNA_biorep1_techrep2_F'2


title: ROI1_fractionated_RNA_biorep1_techrep2_F'1


title: DIDI_PMN_Blood


title: ROI1_genomic_DNA_FC_biorep2_techrep1_P5


title: ROI1_fractionated_RNA_biorep1_techrep1_F'5


title: 4T07, Scaffold, 1583-SO-24


title: CTRL_PEFR_PMN

value: PMN CTRL


title: 4T07, Spleen, 1583-SO-30


title: ROI1_genomic_DNA_FC_biorep2_techrep1_P4


title: LOYE_PMN_Blood


title: 4T07, Scaffold, 1583-SO-22


title: CTRL_LIRI_PMN


title: 67NR, Primary Tumor, 1583-SO-21


title: P70_gMDSC_CD45low_Blood

description:
low density neutrophil CD45low from blood of lung cancer patient
Dataset_1_matrix_ub_fcounts.txt

growth_protocol: At least 10 000 neutrophils sorted from blood and tumors of healthy donors or cancer patients were centrifuged and resuspended in 200ul of PBS. The suspension was then mixed with 900ul of Qiazol lysis solution (Qiagen, 79305), transfered to RNAse-free 1.5ml low-binding Eppendorfs (Ambion, AM12450) and then vortexed for 1min. Lysates were kept at -80°C until RNA extraction

value: gMDSC CD45 Low


title: ROI1_genomic_DNA_FC_biorep2_techrep1_P3


title: 67NR, Primary Tumor, 1583-SO-20

value: Primary Tumor


title: ROI1_fractionated_RNA_biorep1_techrep1_F'4


title: ROI1_fractionated_RNA_biorep1_techrep1_F'2


title: P70_gMDSC_CD45hi_Blood

description:
low density neutrophil CD45hi from blood of lung cancer patient
Dataset_1_matrix_ub_fcounts.txt

value: gMDSC CD45 High


title: ROI1_fractionated_RNA_biorep1_techrep1_F'3


title: 4T07, Lung, 1583-SO-25

value: Balb/c

value: female


title: P64_gMDSC_CD45low_Blood


title: CSF cell specimen

description: 10x Genomics

data_processing:
Human scRNA-Seq raw expression data were mapped with Cell Ranger (version 7.0.0) for alignment to a custom reference comprised of the human genome with segments of an Ugandan HIV pro-viral genome (GenBank: AB098330.1), relevant to the geographic region from which the specimens were originally isolated
Assembly: hg38 with hybrid HIV reference genome (GenBank: AB098330.1)
Supplementary files format and content: Tab-separated values files and matrix files

source_name: CSF

extract_protocol:
All sorted samples were pre-gated on CD3+, CD4+ or CD8+, live, single-cells. 5,000 sorted cells from each CD4+ and CD8+ population were pooled and used for scRNA-Seq analysis as below
Cells FACS sorted into DMEM with 40% FBS were used for scRNA-Seq sample preparation. After sorting, cell number was confirmed with the Luna II (Logos Biosystems). The appropriate number of cells necessary to achieve a target cell input of 5,000 cells was resuspended and used to generate GEMs (Gel Bead-In Emulsion). The Chromium Next GEM Single-cell 5’ Gel beads v2 kit (10X Genomics) was used and manufacturer instructions were followed for GEM generation through cDNA synthesis. A Chromium Controller was used for GEM generation and barcoding then a thermocycler was used for cDNA synthesis. cDNA was submitted to the University of Minnesota Genomics Center for sequencing on an Illumina Novaseq. Tetramer-enriched scRNA-seq data pooled from three biological replicates.

value: CSF

value: CD4 and CD8 T cells

value: Human

value: Mycobacterium tuberculosis meningitis infection


title: 67NR, Primary Tumor, 1583-SO-19

value: Day 14


title: 67NR, Spleen, 1583-SO-18

value: Spleen


title: ROI1_fractionated_RNA_biorep1_techrep1_F'1

value: HEK293T LLP iCasp9 Blast

value: Induction of transcription from landing pad with addition of 2 µg/mL Doxycycline


title: ROI1_total_RNA_biorep2_techrep3


title: BD-220-5

city: Boston

first: Katharina

last: Meyer

state: MA

address: 201 Brookline Ave

department: Synthetic Biology

institute: Wyss Institute

data_processing:
reads were aligned to GRCh38 with Ensembl GRCh38.92 gene models using STAR version 2.5.4a with options --alignSJDBoverhangMin 1 --alignSJoverhangMin 8 --outFilterMultimapNmax 20 --outFilterType BySJout --alignIntronMin 20 --alignIntronMax 5000000 --alignMatesGapMax 5000000. Expression levels of genes were quantified as gene counts using STAR version 2.5.4a with option --twopassMode Basic.
Gene annotation and gene sets used for functional gene classification into biological process (BP), molecular function (MF), and cellular component (CC) were from the GRCh38.p12 database downloaded from Ensembl Biomart on March 4, 2018.
ENCODE ChIP-seq transcription factor target gene sets were from the ENCODE_TF_ChIP-seq_2015 database downloaded from http://amp.pharm.mssm.edu/Enrichr/ on March 8, 2018
REST target genes. The REST RE1 motif position-specific weight matrix MA0138.2 was obtained from JASPAR. FIMO was used with the Homo sapiens genome sequence GRCh38 to predict REST binding sites. A gene was defined as a REST target if it had a RE1 motif with motif pvalue < 1e-7 that was +/-10kb from the transcription start site of any transcript of the gene in the Ensembl GRCh38.86 gene models. This procedure identified 2632 REST target genes before filtering for expressed genes in each data set during gene set enrichment analysis.
Assembly: hg38
Supplementary files format and content: gene_counts.tsv

source_name: forebrain organoid

growth_protocol: Cerebral organoids were differentiated using an approach adapted from a previously described protocol by Lancaster et al with modifications. Briefly, 9000 hiPSCs were seeded in each well in a Corning® 96-well clear round bottom ultra-low attachment microplate (Corning #7007) to form embryoid bodies (EBs) in the presence of 50µM ROCK inhibitor (Sigma #Y-27632). When EBs reached 600 µm in diameter, medium was replaced with neural induction medium for 4 days before embedding EBs in Matrigel (Corning #354234). Embedded EBs were grown in ultra-low attachment plates in differentiation medium without vitamin A for 5 days without shaking. Differentiation medium with vitamin A was used for long term maintenance with shaking on an orbital shaker (90rpm). Medium was changed every 2-3 day.

extract_protocol:
RNA was extracted using Qiagen RNAeasy kit
RNA quality control, library preparation and sequencing were facilitated by Novogene Corporation.

value: forebrain organoid

value: iPSC

value: bipolar disorder


title: 4T07, Scaffold, 1583-SO-23

value: Scaffold


title: 67NR, Spleen, 1583-SO-17


title: 67NR, Spleen, 1583-SO-16


title: P17C_gMDSC_CD45low_Blood

value: Blood

value: gMDSC CD45 Low

value: Lung Cancer


title: P17C_gMDSC_Tumor

description:
low density neutrophi CD45hi from tumor of lung cancer patient
Dataset_1_matrix_ub_fcounts.txt

value: Tumor

value: gMDSC Tumor


title: BD-193-3


title: BD-25-3


title: ROI1_genomic_DNA_FC_biorep2_techrep1_input


title: BD-12-33


title: 67NR, Lung, 1583-SO-15

value: Lung

value: Balb/c

value: female


title: Conventional_RiboSeq_UTR5_Flag_COMT_moxGFP


title: ROI1_total_RNA_biorep1_techrep2


title: 67NR, Lung, 1583-SO-14


title: P17C_gMDSC_CD45hi_Blood

value: gMDSC CD45 High


title: 67NR, Lung, 1583-SO-13

value: Day 14


title: MYKA_gMDSC_Tumor

description:
low density neutrophi CD45hi from tumor of ovarian cancer patient
Dataset_1_matrix_ub_fcounts.txt

value: MYKA

value: Ovarian Cancer


title: BD-PSC-01-003


title: MYKA_gMDSC_CD45low_Blood

description:
low density neutrophil CD45low from blood of ovarian cancer patient
Dataset_1_matrix_ub_fcounts.txt


title: ROI1_total_RNA_biorep1_techrep3

value: HEK293T LLP iCasp9 Blast

value: Induction of transcription from landing pad with addition of 2 µg/mL Doxycycline


title: ROI1_total_RNA_biorep1_techrep1


title: 67NR, Scaffold, 1583-SO-12


title: BD-PSC-01-002

value: forebrain organoid

value: iPSC


title: GSTZ1-KO3

city: chongqing

first: tang

last: ni

address: yixueyuan Road NO.1

institute: Chongqing Medical University

data_processing:
Reads filtering under criteria removing reads with 20% of the base quality lower than 13
HISAT2.1.0 mapping to reference genome
count counting
Genome_build: GRCh38
Supplementary_files_format_and_content: TXT file contain counts for each sample

source_name: HepG2 cells

growth_protocol: GSTZ1-KO HepG2 cells and parental cells were cultured under hypoxic condition for 12h

extract_protocol:
Total RNA was extracted from cell  by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Bioanalyzer 2200 (Aligent) and kept at -80℃.The RNA with RIN >8.0 is right for cDNA library construction.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: HepG2 cells

value: liver


title: P64_gMDSC_CD45hi_Blood

value: Blood


title: ROI1_total_RNA_biorep2_techrep2

city: Austin

first: Can

last: Cenik

address: Moffett Molecular Biology Building 2.122 2500 Speedway

institute: The University of Texas at Austin


title: MYKA_gMDSC_CD45hi_Blood

description:
low density neutrophil CD45hi from blood of ovarian cancer patient
Dataset_1_matrix_ub_fcounts.txt


title: PSC-01-026

value: healthy


title: GSTZ1-KO2


title: PSC-01-020


title: 67NR, Scaffold, 1583-SO-11

value: Scaffold


title: ATAC-seq_CN3-day-100

city: New York

first: Hyein

middle: Sophia

last: Cho

state: NY

address: 430 E 67th St

department: Developmental Biology

institute: Memorial Sloan Kettering Cancer Center

data_processing:
Genome alignment by bowtie2 2.3.4.1
Peak calling by macs2 2.1.1.20160309
Peak filter for reproducibility by IDR 2.0.3
Differential accessibility by DESeq2 1.25.1
Genome_build: hg19
Supplementary_files_format_and_content: Coverage signal in bigWig

source_name: hPSC-derived neurons, day 100, replicate 2

growth_protocol: WA09 (H9) hPSC line was cultured in Essential 8 on Vitronectin coated dishes and expanded by dissociation with 0.5mM EDTA. Neural induction was completed using the dual SMAD inhibition protocol and WNT inhibition on Matrigel coated dishes. hPSC for differentiation and neuronal progenitors were dissociated using Accutase. Neurons were maintained on poly-L-ornithine and Laminin/ Fibronectin coated plates in Neurobasal medium supplemented with B27, L-glutamine, BDNF, GDNF, cAMP, AA. DAPT was added to the neuronal maintenance medium from day20 to day30 of differentiation.

extract_protocol:
Illumina Nextera DNA library preparation kit
ATAC libraries were prepared for sequencing using standard Illumina protocols

value: Neurons

value: WT


title: GSTZ1-KO1


title: ROI1_total_RNA_biorep2_techrep1


title: 67NR, Scaffold, 1583-SO-10

value: Balb/c


title: Parental3


title: PSC-01-017


title: Control, Spleen, 1583-SO-9

value: Spleen

value: female


title: ATAC-seq_CN3-day-51

source_name: hPSC-derived neurons, day 50, replicate 2


title: MH0185983


title: Control, Spleen, 1583-SO-8

value: Day 14


title: ATAC-seq_CN3-day-25

source_name: hPSC-derived neurons, day 25, replicate 2


title: Parental2


title: Parental1


title: Control, Spleen, 1583-SO-7


title: MH0185863


title: ATAC-seq_CN2-day-100

source_name: hPSC-derived neurons, day 100, replicate 1


title: MH0185865


title: ATAC-seq_CN2-day-75

source_name: hPSC-derived neurons, day 75, replicate 1


title: Control, Lung, 1583-SO-6

value: Lung


title: MCF10A-ha-tfcp2-wt-r3-IgG

city: Heidelberg

first: Claudia

last: Scholl

address: Im Neuenheimer Feld 581

country: Germany

institute: German Cancer Research Center (DKFZ)

description: ACT-Seq of MCF10A cells transduced with wild-type TFCP2 targeting IgG, replicate 3

data_processing:
nf-core ATAC-seq v1.2.1 in broad peak mode for calling histone marks
nf-core ChIP-seq v1.2.1 in narrow peak mode for calling FUS/EWSR1-TFCP2 and wild-type TFCP2/FUS/EWSR1 binding sites
Assembly: hs37d5 + PhiX
Supplementary files format and content: bigWig

source_name: breast

extract_protocol:
MinElute Kit (Qiagen, #28004)
To generate sequencing libraries, real-time PCR was performed in a total volume of 50 µl using 25 µl NEBNext High Fidelity 2x Mix (New England Biolabs, #M0541), 0.5 µl 100x SYBR Green, 19.5 µl tagmented eluate, and 5 µl of Nextera UDI primer (replicates R1, R3, and R4) or 2.5 µl primer T5McP1n and 2.5 µl barcode primer (replicate R2) for each sample with the following program: 72°C, 5 minutes (gap repair); 98 °C, 30 seconds (initial melting); 98°C, 10 seconds; 63°C, 10 seconds; 72°C, 10 seconds (cycling). The reaction was stopped after an increase of five or more fluorescence units. Libraries were purified using AMPure XP beads with a bead-to-DBA ratio of 1.4:1 and 12 µl elution buffer for single-phase purification (anti-HA antibodies and IgG) and with bead-to-DBA ratios of 0.5:1 and 1.4:1 for biphasic purification (anti-H3K27me3 and anti-H3K27ac).
ACT-Seq, paired-end

value: breast

value: mammary gland cell line

value: Control


title: ATAC-seq_CN2-day-51

source_name: hPSC-derived neurons, day 50, replicate 1


title: Control, Lung, 1583-SO-5

source_name: Lung


title: MCF10A-ha-tfcp2-wt-r2-IgG

description: ACT-Seq of MCF10A cells transduced with wild-type TFCP2 targeting IgG, replicate 2


title: MCF10A-ha-fus-tfcp2-r4-IgG

description: ACT-Seq of MCF10A cells transduced with FUS-TFCP2 targeting IgG, replicate 4


title: ATAC-seq_CN3-day-75

source_name: hPSC-derived neurons, day 75, replicate 2


title: Control, Lung, 1583-SO-4


title: MH0185932

value: forebrain organoid


title: MCF10A-ha-fus-tfcp2-r3-IgG

description: ACT-Seq of MCF10A cells transduced with FUS-TFCP2 targeting IgG, replicate 3


title: MH0185880

value: iPSC

value: bipolar disorder


title: MH0185853


title: ATAC-seq_CN2-day-26

source_name: hPSC-derived neurons, day 25, replicate 1


title: Control, Scaffold, 1583-SO-3


title: ATAC-seq_NPC

source_name: hPSC-derived neural progenitor cells

value: Neural progenitor cells


title: MH0185855


title: ATAC-seq_hESC

source_name: hPSC

value: hPSC


title: RNAseq_MDA231_sgITGAV#3

city: duarte

first: LU

last: YANG

address: 1500 duarte rd

institute: city of hope

data_processing:
Illumina RTA was used to create .bcl files, and bcl2fastq v2.20.0.422 software was used for base calling (for samples split between flowcells and later merged per run)
RNA-seq reads aligned to the GRCh38 genome assembly using STAR_2.6.0a, Aligned reads was normalized by edgeR bioconductor package and outputed as rpkm.txt file
Assembly: GRCh38
Supplementary files format and content: raw count table in txt format of every sample in RNAseq project

growth_protocol: Cas9-expressing MDA-MB-231 cells transduced with sgCtrl or sgITGAV and seleted in 1.5 ug/ml puromycine for 3 days.

extract_protocol:
Total RNA was extracted using the RNeasy kit (QIAGEN) for RNAseq.
Eukaryotic mRNA directional library prep were used for RNAseq.

treatment_protocol: sgCtrl and sgITGAV were cloned into ipUSEPR vector and transduced via lentiviral infection.

value: breast cancer cells

value: sgITGAV

value: ITGAV knockout


title: Control, Scaffold, 1583-SO-2

value: Scaffold


title: MCF10A-ha-fus-tfcp2-r2-IgG

description: ACT-Seq of MCF10A cells transduced with FUS-TFCP2 targeting IgG, replicate 2


title: MCF10A-ha-tfcp2-wt-r4-IgG

description: ACT-Seq of MCF10A cells transduced with wild-type TFCP2 targeting IgG, replicate 4


title: CN5_d100

description: ribo-depleted total RNA

data_processing:
Transcriptome alignment by STAR 2.6.0
Gene quantification by HTSeq 0.9.1
Differential expression by DESeq2 1.16.1
Genome_build: hg19
Supplementary_files_format_and_content: txt, gene quantifcation of raw counts

source_name: hPSC-derived neurons, day 100, replicate 3

extract_protocol:
Illumina TruSeq Stranded Total RNA Library Prep Kit
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Neurons

value: WT


title: MCF10A-ha-fus-tfcp2-r1-IgG

description: ACT-Seq of MCF10A cells transduced with FUS-TFCP2 targeting IgG, replicate 1


title: RNAseq_MDA231_sgITGAV#2


title: CN5_d75

description: ribo-depleted total RNA

source_name: hPSC-derived neurons, day 75, replicate 3


title: BS-PD-1_S1

city: Seoul

first: Jeonghwan

address: 369 Sangdo-Ro, Entrepreneurship & Small Business Center 612

institute: Soongsil University

data_processing:
Seqeunces were mapped to mm10 and demultiplexed using Cell Ranger v3.1.0
Assembly: mm10

source_name: Mouse tumor sample

extract_protocol:
B16F10 tumors were collected at 3 days after 2nd administration of anti-PD-1 antibody. The tumor samples were first mechanically dissociated using a scalpel, then enzymatically dissociated in digestion medium (2mg/ml Collagenase P (Sigma Aldrich) and 0.2mg/ml DNAse I (Roche) in DMEM (Thermo Fisher Scientific)). Red blood cells were removed from the cell suspension using red blood cell lysis buffer (Roche), and the cells were filtered using a 40-µm Flowmi tip strainer (VWR). The number of living cells was determined using a LUNA automated cell counter (Logos Biosystems).
We performed single-cell RNA-seq on the single-cell suspension using the Chromium Single Cell 3’ Solution from 10x Genomics according to the manufacturer’s instructions. Up to 5,000 cells were loaded onto a 10x Genomics cartridge for each sample. Cell-barcoded 5′ gene expression libraries were sequenced on an Illumina NextSeq and/or NovaSeq6000 system.


title: RNAseq_MDA231_sgITGAV#1


title: Control, Scaffold, 1583-SO-1

source_name: Scaffold

value: Balb/c

value: female


title: BS-iso_S1

value: IgG


title: MCF10A-ev-r4-IgG

description: ACT-Seq of MCF10A cells transduced with empty vector targeting IgG, replicate 4

value: breast


title: CN5_d50

description: ribo-depleted total RNA

source_name: hPSC-derived neurons, day 50, replicate 3


title: RNAseq_MDA231_sgCtrl#1

value: sgCtrl

value: control


title: BP-PD-1_S1

value: pLKO


title: CN5_d25

description: ribo-depleted total RNA

source_name: hPSC-derived neurons, day 25, replicate 3


title: BP-iso_S1

description: pLKO/IgG


title: MCF10A-ev-r2-IgG

description: ACT-Seq of MCF10A cells transduced with empty vector targeting IgG, replicate 2

value: mammary gland cell line

value: Control


title: CN5_NPC

description: ribo-depleted total RNA

source_name: hPSC-derived neural progenitor cells, replicate 3

value: Neural progenitor cells


title: RNAseq_MDA231_sgCtrl#2


title: WaGa, pyrvinium pamoate, 6h, 3 [WP6h3]

city: Tucson

first: Jiawen

last: Yang

state: Arizona

address: 1515 N Campbell Ave

department: University of Arizona Cancer Center

institute: The University of Arizona

data_processing:
Ran fastqc to check the quality of raw data
Trimmed the adapter sequence from raw sequencing data by using trimmomatic-0.39
Performed data alignment by using STAR 2.7.10a with a hg38 reference genome (“GDC.h38.d1.vd1 STAR2 Index Files (v36)” downloaded from https://gdc.cancer.gov/about-data/gdc-data-processing/gdc-reference-files)
The aligned data were mapped to the same reference genome by using Rsubread package.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include read counts for individual sample
Supplementary files format and content: tab-delimited text files include read count matrix for all samples

source_name: Merkel cell carcinoma

growth_protocol: MKL1 and WaGa cells were cultured in RPMI-1640 medium, containing 10% fetal bovine serum (FBS), 1% glutaMAX, 10 U/ml penicillin, and 10 mg/ml streptomycin, at 37 ℃ under 5% CO2.

extract_protocol:
RNA was harvested using Rneasy mini kit (Qiagen）
Total RNA was sent to Novogene CO.for mRNA library preparation and sequencing.

treatment_protocol: MKL1 and WaGa cells were resuspended in 5 ml RPMI culture medium and seeded in 25 cm^2 flasks. 5 μL DMSO were added for vehicle control group and 5 μL 1 mM pyrvinium pamoate (dissolved in DMSO) were added for drug treated group. The cells were collected at 6 hours and 24h hours after treatment.

value: Merkel cell carcinoma

value: WaGa

value: suspension cells

value: pyrvinium pamoate 1 μM


title: MCF10A-ev-r1-IgG

description: ACT-Seq of MCF10A cells transduced with empty vector targeting IgG, replicate 1


title: MCF10A-ev-r3-IgG

description: ACT-Seq of MCF10A cells transduced with empty vector targeting IgG, replicate 3


title: MCF10A-ha-tfcp2-wt-r3-H3K27ac

description: ACT-Seq of MCF10A cells transduced with wild-type TFCP2 targeting H3K27ac, replicate 3

value: Case


title: CN3_d100

description: ribo-depleted total RNA

source_name: hPSC-derived neurons, day 100, replicate 2

value: Neurons


title: WaGa, pyrvinium pamoate, 6h, 2 [WP6h2]

value: WT


title: CN3_d50

description: ribo-depleted total RNA

source_name: hPSC-derived neurons, day 50, replicate 2


title: Cpeb3_KO_rep4

city: Hamburg

first: Jan Broder

last: Engler

address: Falkenried 94

department: Institut für Neuroimmunologie und Multiple Sklerose

institute: Universitätsklinikum Hamburg-Eppendorf

description:
IWHH_8
norm_counts.tsv

data_processing:
Alignment with STAR (v2.5.2b)
Counting by featureCounts (v1.5.1)
Analysis with DESeq2 (v1.36.0)
Assembly: ensemble.mouse.GRCm38r79
Supplementary files format and content: Rows represent mRNAs, columns represent samples

source_name: Embryonic cortex E15.5

extract_protocol:
Qiagen RNeasy mini kit
Novogene NGS RNA Library Prep Set

value: Embryonic cortex E15.5

value: Neuronal cultures DIV16

tag: mouse strain


title: Wildtype_rep2

description:
IWHH_3
norm_counts.tsv


title: WaGa, pyrvinium pamoate, 24h, 3 [WP24h3]


title: MCF10A-ha-tfcp2-wt-r1-H3K27ac

description: ACT-Seq of MCF10A cells transduced with wild-type TFCP2 targeting H3K27ac, replicate 1

source_name: breast


title: Wildtype_rep1

description:
IWHH_1
norm_counts.tsv


title: CN3_d75

description: ribo-depleted total RNA


title: WaGa, pyrvinium pamoate, 6h, 1 [WP6h1]


title: MCF10A-ha-fus-tfcp2-r4-H3K27ac

description: ACT-Seq of MCF10A cells transduced with FUS-TFCP2 targeting H3K27ac, replicate 4


title: CN3_d25

description: ribo-depleted total RNA

source_name: hPSC-derived neurons, day 25, replicate 2


title: WaGa, pyrvinium pamoate, 24h, 2 [WP24h2]


title: RNAseq_MDA231_sgCtrl#3

value: breast cancer cells


title: Wildtype_rep4

description:
IWHH_7
norm_counts.tsv


title: CN5_ESC

description: ribo-depleted total RNA

source_name: hPSC, replicate 3

value: hPSC


title: Wildtype_rep3

description:
IWHH_6
norm_counts.tsv


title: MCF10A-ha-tfcp2-wt-r2-H3K27ac

description: ACT-Seq of MCF10A cells transduced with wild-type TFCP2 targeting H3K27ac, replicate 2

value: breast


title: Cpeb3_KO_rep3

description:
IWHH_5
norm_counts.tsv


title: WaGa, pyrvinium pamoate, 24h, 1 [WP24h1]


title: CN3_NPC

description: ribo-depleted total RNA

source_name: hPSC-derived neural progenitor cells, replicate 2


title: MCF10A-ha-fus-tfcp2-r2-H3K27ac

description: ACT-Seq of MCF10A cells transduced with FUS-TFCP2 targeting H3K27ac, replicate 2


title: CN3_ESC

description: ribo-depleted total RNA

source_name: hPSC, replicate 2


title: Cpeb3_KO_rep2

description:
IWHH_4
norm_counts.tsv


title: CN1_d100

description: ribo-depleted total RNA

source_name: hPSC-derived neurons, day 100, replicate 1


title: MCF10A-ha-fus-tfcp2-r1-H3K27ac

description: ACT-Seq of MCF10A cells transduced with FUS-TFCP2 targeting H3K27ac, replicate 1