title: SOX9 CTCF_Sox2 mus rep4

city: Illkirch

first: Tom

last: Sexton

address: 1 rue Laurent Fries

country: France

institute: IGBMC (Institute of Genetics and Molecular and Cellular Biology)

description: 4C-seq with hSOX9 bait

data_processing:
Library strategy: 4C-seq
Image analysis and base calling were performed using RTA 2.7.7 and bcl2fastq
Individual 4C-seq experiments were demultiplexed according to bait primer sequence using sabre, with two mismatches permitted (-m 2)
Reads were aligned using Bowtie (v1.0.0) with the settings -a -m 1 --best --strata
Mapped reads were filtered and mapped to DpnII restriction fragments with the 4See utilities described in Ben Zouari et al. 4See: a flexible browser to explore 4C data. Front. Genet. 10: 1372.
Genome_build: mm10
Supplementary_files_format_and_content: Cis files, containing a header and two columns (restriction fragment midpoint coordinate, counts), the input format for 4See (Ben Zouari et al. 4See: a flexible browser to explore 4C data. Front. Genet. 10: 1372.)

organism: Mus musculus musculus x Mus musculus castaneus

source_name: Embryonic stem cells

growth_protocol: Mouse F1 ESCs (M. musculus129 × M. castaneus, female cells obtained from Barbara Panning) and derivatives were cultured on 0.1% gelatin-coated plates in ES medium (DMEM containing 15% FBS, 0.1 mM MEM nonessential amino-acids, 1 mM sodium pyruvate, 2 mM GlutaMAX, 0.1 mM 2-mercaptoethanol, 1000 U/mL LIF, 3 µM CHIR99021 [GSK3β inhibitor; Biovision], and 1 µM PD0325901 [MEK inhibitor; Invitrogen]), which maintains ESCs in a pluripotent state in the absence of a feeder layer.

extract_protocol:
Cells were fixed with 2% paraformaldehyde in 10% FBS in PBS for 10 min at 23°C. The fixation was quenched with cold glycine at a final concentration of 125 mM, then cells were washed with PBS and permeabilized on ice for 1 h with 10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.1% NP-40, and protease inhibitors. Nuclei were resuspended in DpnII restriction buffer at 10 million nuclei/mL concentration, and 5 million nuclei aliquots were further permeabilized by treatment for 1 h with 0.4% SDS at 37°C, then incubating for a further 1 h with 3.33% Triton X-100 at 37°C. Nuclei were digested overnight with 1500 U DpnII at 37°C, then washed twice by centrifuging and resuspending in T4 DNA ligase buffer. In situ ligation was performed in 400 μL T4 DNA ligase buffer with 20,000 U T4 DNA ligase overnight at 23°C. DNA was purified by reverse cross-linking with an overnight incubation at 65°C with proteinase K, followed by RNase A digestion, phenol/chloroform extraction, and isopropanol precipitation. The DNA was digested with 5 U/μg Csp6I at 37°C overnight, then re-purified by phenol/chloroform extraction and isopropanol precipitation. The DNA was then circularized by ligation with 200 U/μg T4 DNA ligase under dilute conditions (3 ng/μL DNA), and purified by phenol/chloroform extraction and isopropanol precipitation. For allele-specific 4C from the SCR-proximal bait, samples of the DNA were digested with BveI or Alw26I, cutting specifically at the region between the DpnII site and the 4C reading primer annealing site on the 129 or castaneus allele, respectively. For 129-specific 4C from the SCR bait, the material was digested with AvaIII, cutting specifically at the region between the Csp6I site and the 4C non-reading primer annealing site on the castaneus allele. There were no SNPs allowing castaneus-specific 4C from this bait. No digestion was required for allele-specific 4C from the human insertion sequence, since this was only present on the 129 allele. 100 ng aliquots of treated DNA were then used as template for PCR with bait-specific primers containing Illumina adapter termini. PCR reactions were pooled, primers removed by washing with 1.8× AMPure XP beads, then quantified on a Bioanalyzer (Agilent) before sequencing with a HiSeq 4000 (Illumina).
4C PCR products are quantified in a Bioanalyzer, and pooled in equimolar quantities for direct cluster generation on an Illumina HiSeq4000

tag: hybrid generation

value: Sox2-Venus; FRT-hSOX9-2CTCF(Sox2)-F3/+

tag: restriction enzyme

value: DpnII - Csp6I

value: Embryonic stem cells


title: SOX9 CTCF_Sox2 mus rep2

status: Public on Feb 04 2022

type: SRA

description: 4C-seq with hSOX9 bait

library_source: genomic

molecule: genomic DNA

tag: genotype

tag: cell type


title: SOX9 CTCF_Sox2 mus rep3

description: 4C-seq with hSOX9 bait


title: Input_siH2A_DRIP_R1

city: Leicester

first: Michael

last: Tellier

state: Select State

address: Lancaster Road

department: Department of Molecular and Cell Biology

country: United Kingdom

institute: University of Leicester

data_processing:
Library strategy: DRIP-seq
Adapters were trimmed with Cutadapt v1.13
Mapping to the hg38 genome was performed with STAR v2.6.1d.
Extraction of properly paired and properly mapped reads and Bam files sorting and indexing were done with SAMtools v1.3.1
Mapped reads were de-duplicated with Picard (ChIP-seq and DRIP-seq)
Bigwig files were generated with the Deeptools2 v2.5.0.1 bamCoverage tool
Genome_build: hg38
Supplementary_files_format_and_content: bigwig files: genome-coverage files for all samples

organism: Homo sapiens

growth_protocol: HeLa cells were maintained in DMEM with 10% fetal bovine serum

extract_protocol:
RNA were purified from the chromatin fraction. DNA were purified from chromatin IP products.
For Chromatin RNA-seq, libraries were prepared using Illumina's TruSeq Stranded Total RNA library Preparation Kit according to Illumina's instructions. For ChIP-seq and DRIP-seq, libraries were prepared using Illumina's TruSeq ChIP library Preparation Kit according to Illumina's instructions.

treatment_protocol: siRNAs were transfected for 72 hours according to Lipofectamine 2000 reagent protocol.

tag: cell line

value: HeLa

tag: antibody

tag: sirna

value: RNaseH2A siRNA


title: SOX9 CTCF_Sox2 mus rep1

description: 4C-seq with hSOX9 bait


title: AN5-S37-d0-1

city: Edinburgh

first: Tilo

last: Kunath

address: 5 LIttle France Drive

department: Centre for Regenerative Medicine

institute: University of Edinburgh

library_source: transcriptomic

data_processing:
QIAGEN GeneGlobe RNA-seq bioinformatics tool
Genome_build: GRCh38
Supplementary_files_format_and_content: raw counts
Supplementary_files_format_and_content: normalised counts

molecule: total RNA

source_name: hESCs

growth_protocol: hESCs were cultured on Laminin-521 (BioLamina) in StemMACS iPS-Brew XF self-renewal media (Miltenyi).

extract_protocol:
Total RNA was extracted with the QIAGEN RNeasy kit
QIAseq UPX 3' Transcriptome Kit

treatment_protocol: hESCs were differentiated into cortical neuronal progenitor cells for 25 days using a dual-Smad inhibition protocol

tag: differentiation


title: AN5-S8-d0-1


title: Input_RNaseH2A_R2

data_processing:
Adapters were trimmed with Cutadapt v1.13
Mapping to the hg38 genome was performed with STAR v2.6.1d.
Extraction of properly paired and properly mapped reads and Bam files sorting and indexing were done with SAMtools v1.3.1
Mapped reads were de-duplicated with Picard (ChIP-seq and DRIP-seq)
Bigwig files were generated with the Deeptools2 v2.5.0.1 bamCoverage tool
Genome_build: hg38
Supplementary_files_format_and_content: bigwig files: genome-coverage files for all samples


title: Input_siH2A_DRIP_R2


title: SOX9 CTCF_SCR mus rep4

description: 4C-seq with hSOX9 bait

value: Sox2-Venus; FRT-hSOX9-2CTCF(SCR)-F3/+


title: SOX9 CTCF_SCR mus rep2

description: 4C-seq with hSOX9 bait


title: SOX9 CTCF_SCR mus rep3

description: 4C-seq with hSOX9 bait


title: Input_RNaseH2A_R1


title: SOX9 CTCF_SCR mus rep1

description: 4C-seq with hSOX9 bait


title: AN4-S37-d0-1


title: AN6-S37-d0-2


title: SOX9 hSOX9 mus rep3

description: 4C-seq with hSOX9 bait

value: Sox2-Venus; FRT-hSOX9-F3/+


title: SOX9 hSOX9 mus rep4

description: 4C-seq with hSOX9 bait


title: Input_PolII


title: OUH_miR_117

type: RNA

city: Odense

first: Ines

last: Block

address: J.B. Winsløws Vej 4

department: Department of Clinical Genetics

country: Denmark

institute: Odense University Hospital

description: Total RNA extracted from fresh frozen primary breast tumour of a female patient with relapse

hyb_protocol: Hybridization, washing, and scanning (Agilent G2565CA Microarray scanner) were performed according to the recommendations provided by Exiqon.

scan_protocol: Scanned images were imported into GenePixPro6.0 software (Molecular Devices) for quality control and raw data extraction.

data_processing: Signals from 3 spots were compiled, only human microRNAs were selected, the R-package limma was used for LOESS normalization and quantile normalization of raw signal intensities, and the ComBat function embedded in the sva R-package was used for adjustment of the normalized intensities and to eliminate potential batch effects

source_name: Primary breast tumour of patient with relapse

label_protocol: The miRCURY Power labeling kit (Exiqon), miRCURY LNA Array hybridization buffer (Exiqon) and miRCURY LNA Array Washing buffer kit (Exiqon) were used for sample labelling, hybridization and washing, respectively.

extract_protocol: Total RNA was isolated from tumor samples with Trizol (Invitrogen) and further purified with the RNeasy micro kit (Qiagen), including DNAse treatment. NanoDrop Spectrophotometer (NanoDrop Technologies) was used for RNA quantification. The quality of extracted RNA was assessed with Bioanalyzer 2100 (Agilent Technologies) using the RNA 6000 Nano Kit (Agilent Technologies).

treatment_protocol: After surgical removal, tumor samples were snap-frozen and stored at -80°C.

tag: pair no.

tag: metastasis (1= yes; 0=no)

tag: tumour type

value: Mucinous carcinoma

tag: esr1 expression (1=yes; 0=no)

tag: lymph node status (1=positive; 0=negative)

tag: status at last follow up (1=dead, 0=alive)

source_name: common reference pool of all samples

tag: tissue

value: human breast tumour


title: SOX9 hSOX9 mus rep2

description: 4C-seq with hSOX9 bait


title: AN4-S8-d0-1


title: DRIP_siLuc_RNaseH

value: S9.6 mouse monoclonal antibody; Gromak lab; RRID:AB_2810829

value: Scramble siRNA


title: AN6-Shef4-d0-1


title: SCR CTCF_Sox2 mus rep4

description: Musculus-specific 4C-seq, combining SCR bait with pre-digestion with AvaIII


title: DRIP_siLuc_R2


title: AN5-S9-d0-1


title: SCR CTCF_Sox2 mus rep3


title: SCR CTCF_Sox2 mus rep2


title: SCR CTCF_Sox2 mus rep1


title: DRIP_siLuc_R1


title: AN4-S34-d0-2


title: SCR CTCF_SCR mus rep4


title: SCR CTCF_SCR mus rep3


title: DRIP_siH2A_RNaseH


title: DRIP_siH2A_R2


title: AN4-S9-d0-1


title: SCR CTCF_SCR mus rep2


title: SCR CTCF_SCR mus rep1


title: Donor16_TNFa_low

city: San Francisco

first: Zachary

last: Steinhart

state: CA

address: 1650 Owens St

department: Gladstone-UCSF Institute of Genomic Immunology

country: USA

institute: J. David Gladstone Institutes

description: other (CRISPR sgRNA capture)

data_processing:
Library strategy: CRISPR sgRNA amplicons
Fastq files were aligned to Calabrese (Addgene #92379, #92380) libraries using MAGeCK version 0.5.9.2 count command using --trim-5 22,23,24,25,26,28,29,30 argument
Genome_build: NA
Supplementary_files_format_and_content: Raw counts of sgRNAs

source_name: Primary CD4+ T cells

growth_protocol: Primary human CD4+ T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641). Primary T cell activation was done by anti-humanCD3/CD28 CTS dynabeads (Fisher Scientific cat 40203D) at a 1:1 cell to bead ratio at 1e6 cells/ml.

extract_protocol:
After sorting, cells were washed with PBS, counted, pelleted, and resuspending at up to 5E6 cells per 400 µl of lysis buffer (1% SDS, 50 mM Tris, pH 8, 10 mM EDTA). The remaining protocol reflects additives/procedures performed per each 400 µl of sample. 16µl of NaCl (5M) was added, and the sample was incubated on a heat block overnight at 66°C. The next morning, 8µl of RNAse A (10mg/ml, resuspended in ddH2O) (Zymo, Cat #E1008) was added, and the sample was vortexed briefly, and incubated at 37°C for 1 hour. Next, 8µl of Proteinase K (20mg/ml) (Zymo, Cat #D3001) was added, the sample was vortexed briefly, and incubated at 55°C for 1 hour. A phase lock tube (Quantabio, Cat #2302820) was prepared for each sample by spinning down the gel to the bottom of the tube at 20,000g for 1 minute and then 400µl of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) was added to each tube. 400µl of the sample was then added to the phase lock tube and the tube was shaken vigorously. The sample was centrifuged at maximum speed at room temperature for 5 minutes. The aqueous phase was transferred to a low-binding eppendorf tube (Eppendorf, Cat #022431021) and then 40µl of Sodium Acetate (3M), 1µl GlycoBlue (Invitrogen, Cat # AM9515), and 600µl of room temperature isopropanol was added. The sample was then vortexed and stored at -80°C for 30 minutes or until the sample had frozen solid. Next the sample was centrifuged at maximum speed at 4°C for 30 minutes, the pellet was washed with fresh 70% room temperature Ethanol, and allowed to air dry for 15 minutes. Pellets were then resuspended in Zymo DNA elution buffer (Zymo, Cat No: D3004-4-10), and placed on the heat block at 65°C for 1 hour to completely dissolve the genomic DNA.
sgRNA barcodes were amplified from genomic DNA using Takara Ex-Taq polymerase (Takara RR001C), using 40ug of isolated genomic dna as template per sample, following this protocol, https://media.addgene.org/cms/filer_public/61/16/611619f4-0926-4a07-b5c7-e286a8ecf7f5/broadgpp-sequencing-protocol.pdf. PCR amplicons were agarose gel purified using nucleoSpin Gel and PCR Clean‑up Mini kit (Machery-Nagel cat 740609.50)

treatment_protocol: One day after activation, T cells were infected with dCas9-VP64 (CRISPRa) lentivirus. Two days after activation, T cells were infected with Calabrese Set A (addgene 92379) and Calabrese Set B (addgene 92380) lentivirus (mixed 50/50) at an MOI of 0.5. The following day 2ug/ml puromycin was added to the culture medium and cells were passaged every two days maintaining a minimum concentration of 0.3e6cells/ml. Eight days after initial activation, cells were harvested, spun down and resuspended at 2e6 cells/ml X-VIVO 15 without supplements. Next day, cells were restimulated with anti-CD3/CD28/CD2 immunocult and treated with Brefeldin A for 9 hours, and fixed/stained for FACS. Cells were sorted into IL-2 low and, IFN-γ low and high, or TNF-a low and high bins.

tag: donor

value: donor 16

tag: group


title: SOX9 hSOX9 mus rep1

description: 4C-seq with hSOX9 bait

tag: hybrid generation


title: DRIP_siH2A_R1


title: SCR hSOX9 mus rep2

tag: restriction enzyme


title: SCR hSOX9 mus rep3

tag: genotype


title: SCR FRT/F3 mus rep1

value: Sox2-Venus; FRT-purotk-F3/+

tag: cell type


title: SCR hSOX9 mus rep1


title: Donor16_IL2_low

description: other (CRISPR sgRNA capture)


title: SCR Venus mus rep1


title: SCR F1 mus rep1

value: Wild type


title: Donor16_IL2_high

description: other (CRISPR sgRNA capture)


title: Infected (Wild Type) - 4dpi - Rep3

city: Cantoblanco

first: Juan

middle: Carlos

last: Oliveros

state: Madrid

address: Darwin 3

country: Spain

institute: CNB, CSIC

data_processing:
Filtering: Strand-specific, single-end reads longer than 18 nt provided by BGI sequencing company were quality-checked with FASTQCjects/fastqc/). No additional filters were necessary.
Read alignment: Short reads were aligned against mouse genome sequence (GRCm38) or SARS-CoV genome, with BWA (Li and Durbin, 2009) allowing up to 1 mismatch and no gaps (bwa aln -n 1 -k 1 -o 0). Only unique hits were considered for posterior steps. SAM alignment files generated by BWA were compressed, sorted and indexed using “view”, “sort” and “index” functions of Samtools package (Li et al., 2009).
Small RNA loci definition: A unified list of candidate regions (small RNA loci) was created by collapsing coverage data of all 16 samples on a single combined coverage track where, for each nucleotide, the maximum individual coverage was recorded. In this track, continuous genomic regions with a combined coverage of 2 or more nucleotides and with a summit of at least 10 nt were considered. By this way, a set of 5208 genomic features was defined with an average size of 36 nt. Features overlapping with known mmu-miRNAs from miRBase v.20 (Kozomara and Griffiths-Jones, 2014) were annotated. By this way, 593 out of 2035 (29%) of all miRBase v.20 mmu-miRNAs were detected.
Genome_build: GRCm38
Supplementary_files_format_and_content: tab-delimited text file include raw read count values for all samples

organism: Mus musculus

source_name: Infected (Wild Type)_4dpi_lung

growth_protocol: Specific-pathogen-free 8 week-old BALB/c OlaHsd female mice were purchased from Harlan. Mice were maintained for 8 additional weeks in the animal care facility at the National Center of Biotechnology (CNB-CSIC, Madrid).

extract_protocol: Small RNAs isolated (mirVana miRNA Isolation Kit, Ambion) from mouse lungs were analyzed using the Bioanalyzer 2100 expert_Eukaryote Total RNA to determine their integrity. 1 μg of the small RNA fraction was used for library construction (BGI Genomics, Hong-Kong).

treatment_protocol: 16-week-old mice were inoculated with rSARS-CoV wt or rSARS-CoV-ΔE in 50 μl of DMEM containing 2% fetal bovine serum (FBS, Biowhittaker). Mice were sacrificed at days 2 and 4 post infection and lung and serum samples were collected.

tag: strain

value: BALB/c OlaHsd

tag: age

tag: gender

value: female

tag: infected with

value: rSARS-CoV wild type

tag: time point

value: lung

tag: molecule subtype

value: small RNAs


title: Donor16_IFNG_low_Rep2

description: other (CRISPR sgRNA capture)


title: nearSCR ΔSRR85-95,SRR107-dCTCF/+ cast rep1

description: Castaneus-specific 4C-seq, combining nearSCR bait with pre-digestion with BveI

value: DpnII - Csp6I

value: Embryonic stem cells


title: Infected (Wild Type) - 4dpi - Rep1


title: Donor16_IFNG_low_Rep1

description: other (CRISPR sgRNA capture)


title: nearSCR ΔSRR85-95,SRR107-dCTCF/+ cast rep2


title: SCR hSOX9 mus rep4


title: nearSCR ΔSRR85-95,SRR107-dCTCF/+ mus rep1

description: Musculus-specific 4C-seq, combining nearSCR bait with pre-digestion with Alw26I


title: Infected (Wild Type) - 2dpi - Rep3

source_name: Infected (Wild Type)_2dpi_lung

value: small RNA


title: Infected (Wild Type) - 2dpi - Rep2


title: Infected (Wild Type) - 2dpi - Rep1


title: nearSCR ΔSRR85-95,SCR-dCTCF/+ mus rep2


title: Donor16_TNFa_high

description: other (CRISPR sgRNA capture)


title: nearSCR ΔSRR85-95,SCR-dCTCF/+ cast rep1


title: nearSCR ΔSRR85-95,SRR107-dCTCF/+ mus rep2


title: nearSCR ΔSRR85-95,SCR-dCTCF/+ mus rep1


title: Infected (Wild Type) - 4dpi - Rep2


title: nearSCR ΔSCR-dCTCF/+ cast rep2

value: delSCR-dCTCF/+


title: Donor15_TNFa_low

description: other (CRISPR sgRNA capture)

value: donor 15


title: nearSCR ΔSCR-dCTCF/+ cast rep1


title: Mock - 4dpi - Rep2

value: mock


title: nearSCR ΔSRR85-95,SCR-dCTCF/+ cast rep2


title: Donor16_IFNG_high_Rep1

description: other (CRISPR sgRNA capture)


title: Resident, MID25, 1A

city: Aachen

first: Oliver

last: Pabst

address: Pauwelsstrasse 30

country: Germany

institute: RWTH Aachen University

data_processing:
Library strategy: TCR-Seq
UMI demultiplexing: migec Checkout with “oute” parameters follow by Histogram command
UMI consensus sequences: migec Assemble with “-m” parameter as found by Histogram step
Alignment: mixcr analyze amplicon --library imgt --species MusMusculus --starting-material rna --5-end v-primers –3-end j-primers --adapters adapters-present --receptor-type TRA --region-of-interest CDR3 --only-productive --align "-OreadsLayout=Collinear" --align "-OsaveOriginalReads=true" --assemble "-OseparateByV=true" --assemble "-OseparateByJ=true" --assemble "-ObadQualityThreshold=20" --assemble "-OmaxBadPointsPercent=0" --assemble "-OcloneClusteringParameters=null" S0_R1.fastq S0_R2.fastq output_directory
Joined all clonotype tables across samples into a matrix (mids_counts.csv)
Genome_build: IMGT 202122 mouse reference
Supplementary_files_format_and_content: MiXCR clonotype table, tab-separated files with the following columns:
Supplementary_files_format_and_content: '- cloneCount: number of unique V-CDR3-J clones identified by MiXCR
Supplementary_files_format_and_content: '- nSeqCDR3: CDR3 nucleotide sequence
Supplementary_files_format_and_content: '- aaSeqCDR3: CDR3 aminoacid sequence
Supplementary_files_format_and_content: '- bestVHit: V gene with highest score
Supplementary_files_format_and_content: '- bestJHit: J gene with highest score
Supplementary_files_format_and_content: '- bestDHit: D gene with highest score
Supplementary_files_format_and_content: '- cloneFraction: relative abundance within this sample

source_name: Mesenteric lymph node

extract_protocol:
RNA was isolated using RNeasy Micro Plus Kit  (Qiagen) according to manufacturer's instructions
Library preparation was performed using the template switch technique and UMI-adaptor incorporation during the TCR-specific cDNA synthesis (Mint-2 cDNA synthesis kit, Evrogen).  This was followed by two nested PCRs and a third amplification for the Illumina adaptor incorporation. DNA was purified using MinElute PCR Purification Kit (Qiagen)
RNA-Seq (RACE)

treatment_protocol: 7 days after photoconversion of the mesenteric lymph node

value: Mesenteric lymph node

value: Resident T cells

tag: cell subtype

value: Treg

tag: organ

value: siMLN-PC (photoconverted small intestine draining mesenteric lymph node)


title: Resident, MID39, 5C

value: Tcell


title: Donor15_TNFa_high

description: other (CRISPR sgRNA capture)


title: Cre Control Rep3

city: Toon

first: Hiroshi

last: Sakai

state: Ehime

address: Shitsukawa

department: Proteo-Science Center

country: Japan

institute: Ehime University

data_processing: Raw reads were trimmed using TrimGalore (v0.6.6; http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and mapped to the reference genome (GRCm38, PRI) using HISAT2 (v2.2.1) (Kim et al., 2019). The option ‘--trim3 99’ was used to compare reads of different lengths. Expression level was quantified using featureCounts (v2.0.1) (Liao et al., 2014) and annotation (GRCm38.102). The differentially expressed genes were extracted by DESeq2 (Love et al., 2014).

source_name: Skeletal muscle stem cells

extract_protocol: Not provided

value: Hindlimb muscles

value: 9-week old

tag: treatment

value: Tamoxifen


title: Mock - 2dpi - Rep2

value: BALB/c OlaHsd

value: female


title: Donor15_IL2_low

description: other (CRISPR sgRNA capture)


title: Donor16_IFNG_high_Rep2

description: other (CRISPR sgRNA capture)

value: donor 16


title: Cre Control Rep2


title: nearSCR ΔSCR-dCTCF/+ mus rep1

value: DpnII - Csp6I


title: Donor15_IL2_high

description: other (CRISPR sgRNA capture)


title: Cre Control Rep1


title: Mock - 2dpi - Rep1

value: lung

value: small RNAs


title: Mock - 4dpi - Rep1


title: Infected (deltaE) - 4dpi - Rep3

source_name: Infected (deltaE)_4dpi_lung

value: rSARS-CoV-ΔE


title: Donor15_IFNG_low_Rep2

description: other (CRISPR sgRNA capture)

value: donor 15


title: Infected (deltaE) - 4dpi - Rep2


title: nearSCR ΔSCR-dCTCF/+ mus rep2

value: delSCR-dCTCF/+

value: Embryonic stem cells


title: Infected (deltaE) - 2dpi - Rep3

source_name: Infected (deltaE)_2dpi_lung


title: nearSCR ΔSRR107-111/+ mus rep2


title: Donor15_IFNG_low_Rep1

description: other (CRISPR sgRNA capture)


title: Donor15_IFNG_high_Rep2

description: other (CRISPR sgRNA capture)


title: Infected (deltaE) - 4dpi - Rep1


title: Infected (deltaE) - 2dpi - Rep2


title: GFP_S2_RNAseq

city: Cambridge

first: Julia

last: Joung

state: MA

address: 75 Ames St

institute: Broad Institute

data_processing:
bcl2fastq 2.20.0
Bowtie 1.2.3
RSEM 1.3.1
Genome_build: hg38
Supplementary_files_format_and_content: Aggregated TPM determined by RSEM for each condition

molecule: polyA RNA

source_name: A375 melanoma cells

extract_protocol:
RNA was extracted using the RNeasy Plus Mini Kit (Qiagen 74134)
RNA-Seq libraries were prepared using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB E7530S)

value: A375 melanoma cells

tag: perturbation

value: GFP overexpression

tag: perturbation time

value: 7 days


title: nearSCR ΔSRR107-111/+ mus rep1


title: nearSCR ΔSRR107,111/+ cast rep2


title: Infected (deltaE) - 2dpi - Rep1

tag: tissue


title: nearSCR ΔSRR107,111/+ mus rep2


title: nearSCR ΔSRR107,111/+ cast rep1


title: cardiac tissue, clpp, rep 4

city: Cologne

first: Aleksandra

last: Trifunovic

address: Joseph-Stelzmann-Str. 26

institute: Cluster of Excellence in Cellular Stress Responses in Aging-Associated Diseases

data_processing:
Adapter and basic quality trimming (fastp v0.21.0) : `fastp --in1 in/<read1.fastq> --in2 in/<read2.fastq> --out1 out/<read1.fastq> --out2 out/<read2.fastq> --adapter_fasta CCG_adapters_combined.fasta --overrepresentation_analysis --json out/<name>_fastp.json --html out/<name>_fastp.html --report_title '<name> <sample N>'`
pseudo alignment (Kallisto v0.46.1, genome GRCm38.p6 Ensembl Release 97): `kallisto quant -i <index> -t <N cores> -b 100 in/<read1_trimmed.fq> in/<read1_trimmed.fq> -o out/<sample_name>_kallisto.out`
Differential gene expression analysis with EdgeR v3.32.1 using R v4.0.3, & tximport v1.18.0. VST for visualisation using DESeq2 v1.30.1
Genome_build: GRCm38.p6 Ensembl Release 97
Supplementary_files_format_and_content: counts_matrix_rawcounts.tsv, DGE files

source_name: cardiac tissue, clpp

growth_protocol: Mice were grown on normal chow diet (NCD )

extract_protocol:
Total RNA was isolated from mouse tissues using TRIzol reagent (Life Technologies). Initially, 50-100 mg of fresh or snap-frozen tissue was placed in 2 mL tubes (Peqlab) together with ceramic beads (Mobio, Dianova GmbH) and 1 mL TRIzol. The tissue was homogenized using a Precellys 24 fast-prep machine (Bertin) at 5500 rpm for 2x 20 sec.  Homogenates were then transferred to a new tube and it proceeded with the addition of chloroform and subsequent phase separation according to the manufacturer's instruction. The RNA pellet was resuspended in RNase/DNase free H2O (Gibco).
RNA isolation for RNAseq was performed using the 'Direct-zol RNA MiniPrep Kit' (Zymo Research) according to the manufacturer’s instruction. Purified RNA was submitted to the Cologne Centre for Genomics for further processing. Libraries were prepared using the 'TruSeq mRNA Stranded Sample Preparation Kit' (Illumina) and validated on a tape station (Agilent). Equimolar amounts of libraries were pooled, quantified (Peqlab KAPA Library Quantification Kit, Applied Biosystems 7900HT Sequence Detection System) and finally sequenced on a 'NovaSeq 6000 sequencer' (Illumina).

treatment_protocol: To generate full-body Clpp knockout mice, Clppfl/fl were mated with transgenic mice ubiquitously expressing Cre recombinase under the control of the β-actin promoter, resulting in Clpp+/-mice. Heterozygous Clpp+/- mice were further intercrossed to obtain homozygous. For the generation of double deficient Clpp and Fgf21 knockout animals, Clppfl/fl Fgf21fl/fl mice were mated with transgenic mice ubiquitously expressing Cre recombinase (β-actin Cre).

value: clpp

value: cardiac

tag: repeat


title: cardiac tissue, clpp, rep 2


title: cardiac tissue, dko, rep 3

source_name: cardiac tissue, dko

value: dko


title: Donor15_IFNG_high_Rep1

description: other (CRISPR sgRNA capture)

tag: donor

tag: group


title: UNFUSED joint BRB

city: New York

first: Matthew

middle: A

last: Lalli

state: NY

address: 1 Gustave L. Levy Pl

department: Psychiatry

institute: Icahn School of Medicine at Mount Sinai

data_processing:
Remove transposon bases from each read: cutadapt -g "^"$barcode$Long_PB_LTR -o $out_file".temp.fastq.gz" --minimum-length 1 --discard-untrimmed -e 0.2 --no-indel
Remove any remaining Illumina adpaters: cutadapt -a "CTGTCTCTTATACACATCTCCGAGCCCACGAGACTNNNNNNNNNNTCTCGTATGCCGTCTTCTGCTTG" -o $out_file".fastq --minimum-length 1 $out_file".temp.fastq.gz"
Identify SRT barcode: umi_tools extract --stdin $out_file".temp.fastq.gz" --bc-pattern=CCCCNGTTAA --whitelist=$safelist --stdout $out_file".fastq.gz"
Align reads to reference genome: novoalign -d /novoalign_indexes/$genome/$genome.nvx -f $out_file".fastq.gz"  -n 40 -o SAM -o SoftClip > $out_sam
Convert to sorted bam file: samtools view -bS -h -F 260 $out_sam | samtools sort - -o $out_bam_map
Tag bam file with sequencing indexes: python3 TagBam.py
Tag bam file with SRT barcodes: python3 TagBamWithSrtBC.py
Annotate SRT insertion sites: python3 AnnotateInsertionSites.py
Convert insertion sites to Calling Card Format: python3 BamToCallingCard.py -b XP XJ XQ -i $out_map_sort_prefix"_final.bam" -o $out_map_sort_prefix"_unsorted.ccf"
Sort CCF file: sort -k1V -k2n -k3n $out_map_sort_prefix"_unsorted.ccf" > $out_map_sort_prefix"_final.ccf"
Genome_build: hg38
Supplementary_files_format_and_content: qBED (calling cards format, .ccf) Chromosome start stop number of insertions strand barcode/index

source_name: HEK293T cells

growth_protocol: HEK293T cells were maintained in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were passaged every 3–4 d by enzymatic dissociation using trypsin.

extract_protocol:
Total RNA is harvested using Direct-zol RNA MiniPrep kit (Zymo Research). RNA concentration and integrity was assessed using Nanodrop.
Calling card libraries were prepared as described. We performed first-strand reverse transcription reactions in 20 μL total volume using 2 μg of RNA from each in vitro sample. RNA mixed with water and dNTPs was hybridized to oligo-dT primers (1 μL of 50 μM SMART_dTVN) by incubation at 65 ˚C for 5 minutes and immediately transferred onto ice. 0.5 μL of Maxima H Minus Reverse Transcriptase, RNasin RNAse inhibitor, and 5X RT buffer were added and samples are incubated at 50 °C for 60 min for reverse transcription. cDNA is then amplified with primers specific to the SRT before tagmentation.

treatment_protocol: Calling card experiments are performed as described with minor modifications. Twenty-four hours before transfection, 250,000 HEK293T cells are plated per well in a 12 well plate. The next day, cells are transfected using PEI (Polysciences) with 1 ug of total DNA comprising 500 ng of piggyBac (fused or unfused) and 500 ng of donor SRT (purified PCR product or miniprepped DNA). Medium is changed 24 hours after transfection. Three days after transfection, each well is trypsinized and replated into a T25 flask. For puromycin-resistance SRTs, puromycin is added 24 hours later (2 ug / mL). RNA is extracted three days after puromycin selection.

tag: transposase

value: unfused-hyper-piggyBac

tag: self-reporting transposon

value: mixed barcoded Puro SRT


title: cardiac tissue, clpp, rep 3

tag: genotype


title: cardiac tissue, dko, rep 4


title: cardiac tissue, dko, rep 2


title: GFP_S1_RNAseq

tag: cell type


title: nearSCR ΔSRR107-111/+ cast rep1

tag: hybrid generation

tag: restriction enzyme


title: nearSCR ΔSRR107-111/+ cast rep2


title: cardiac tissue, dko, rep 1


title: GFP_S3_RNAseq


title: nearSCR ΔSRR107,111/+ mus rep1


title: MYOD1 joint BRB


title: B3GNT2_S3_RNAseq

value: B3GNT2 ORF overexpression


title: cardiac tissue, clpp, rep 1


title: nearSCR ΔSRR109/+ cast rep1


title: cardiac tissue, fgf21, rep 4

source_name: cardiac tissue, fgf21


title: HEK293T par-clip from HDLBP_3

city: Berlin

first: Markus

last: Landthaler

address: Hannoversche Str. 28

department: Berlin Institute for Medical Systems Biology

institute: Max Delbrueck Center

data_processing:
Library strategy: PAR-CLIP
Basecalls were converted to fastq files using Bcl2Fastq (2.16.0.10). Reads were demultiplexed, stripped of the 3’adapter sequence by Flexbar (v2.5) and collapsed to remove PCR duplicates. This was followed by trimming of four nucleotides from both 5’ and 3’ end of the read using FASTX Toolkit v0.0.14. Next, the reads were aligned to the human genome (hg19 build) using BWA v0.7.15-r1140 by using the previously published computational PAR-CLIP pipeline (v0.97a) (Lebedeva et al. 2011; Jens 2016). https://github.com/marvin-jens/clip_analysis.
To obtain gene level binding information, we summed T-C transitions in reproducible clusters for each gene. We thus obtained the total number of crosslink positions within whole mRNAs, or within the CDS, 5’UTR or 3’UTR.
To assess the relative distribution of TC-transitions within the transcriptome and obtain accurate mapping to transcripts originating from repetitive genomic loci, reads were sequentially mapped to reference transcripts by Bowtie2 (v2.3.2)  in the following order by retaining the unmapped reads from the previous to the next mapping step. We started with human pre-rRNA (GenBank U13369.1), followed by rRNA (GenBank NR_023363.1, NR_003285.2, NR_003287.2, NR_003286.2), snRNA, snoRNA, other ncRNA (all from Ensembl, including RN7SL), tRNA (GtRNADb), mtDNA (GenBank AF347015.1) and finally the human genome (hg19, iGenomes). The last genome-mapping step was performed by the STAR aligner (v2.2.1) where only uniquely mapped reads (MAPQ=255) were retained. Except for the tRNA mapping (see below for details), we retained reads that mapped with a Bowtie2 MAPQ=20 or more and T-C transitions were extracted using row_mpile_coverage_plus_TC.pl script (Schueler et al. 2014).
For transcriptome level analysis, remaining reads after mtDNA mapping were aligned to the transcriptome sequence (GTF annotation file Gencode v19) by the STAR aligner (v2.2.1) by retaining only the reads that uniquely mapped to the hg19 genome (parameters --outFilterMultimapNmax 1 --outFilterMismatchNmax 5 --outFilterMatchNmin 15 --alignSJoverhangMin 5 --seedSearchStartLmax 20 --outSJfilterOverhangMin 30 8 8 8 --quantMode TranscriptomeSAM). T-C transitions with transcriptome coordinates were obtained from the BAM file using the row_mpile_coverage_plus_TC.pl script (Schueler et al. 2014) and written into a bed file.
3’ adaptor 4N-RA3, rApp-NNNNTGGAATTCTCGGGTGCCAAGG-InvdT; 5’ adaptor OR5-4N, rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrCrNrNrNrN
Genome_build: hg19
Supplementary_files_format_and_content: processed_data_parclip_pergene.txt: tab delimited matrix of T-C transitions, unique read counts and cluster counts in the coding sequence (CDS), 3' untranslated regions (3'UTR), 5' untranslated regions (5'UTR) and whole mRNA (transcript) (mean of 2 replicates) for all detected genes.
Supplementary_files_format_and_content: processed_data_parclip_per_transcript.txt: tab delimited matrix of T-C transitions in transcript coordinates, number of T-C transitions and total read coverage for every position in both replicates, sequence of the crosslink-centred 7-mer, normalized number of T-C transitions, lengths of mRNA, 5'UTR, CDS and 3'UTR (l_tr, l_utr5,l_cds,l_utr3), transcript coordinates of CDS start and stop, as well as corresponding gene id and transcript expression level (TPM_transcript).
Supplementary_files_format_and_content: processed_data_parclip_trna.txt: tab delimited matrix of T-C transitions in tRNA coordinates (tc_start, tc_stop), number of T-C transitions and total read coverage for every position in both replicates (TCnumber_hdlbp2 TCnumber_hdlbp3, allReadsPerPos_hdlbp2 allReadsPerPos_hdlbp3), sequence of the crosslink-centred 7-mer (seq_tc_middle_t), normalized number of T-C transitions, mean fraction of T-C transitions over total coverage, standard deviation of fraction of T-C transitions over total coverage, T-C transition signal in whole tRNA normalized to the total tRNA abundance.
Supplementary_files_format_and_content: processed_data_parclip_overlapping_clusters.txt: tab delimited matrix of HDLBP binding clusters for both replicates (1 and 2) in chromosome coordinates, including cluster start, stop, cluster id, number of T-C transitions in cluster (tc_rep), strand, number of unique reads in cluster, chromosome coordinate of cluster centered region, number of overlapping nucleotides between cluster replicates, corresponding gene and region (CDS, 3'UTR, 5'UTR, start codon, stop codon).

source_name: HEK293 Flp-In T-REx embryonic kidney cell line

growth_protocol: FLAG/HA-HDLBP-HEK293 Flp-In T-REx cells were cultured in standard Dulbeco´s modified Eagle´s medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% 2 mM L-glutamine (Thermo Fisher Scientific).

extract_protocol:
Cells were UV crosslinked (365 nm, 0.15 J/cm²), harvested, snap frozen and stored at -80°C. Cell pellets were incubated with 3 cell pellet volumes of lysis buffer (50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1 % IGEPAL CA-630, 0,1 % SDS, 0,5% sodium deoxycholate, complete EDTA-free protease inhibitor cocktail [Roche]) for 30 min on ice. Cell lysates were cleared (13.000 rpm, 15 min, 4°C), filtered and RNase T1 was added at final concentration of 1 unit/µl for 10 min at 22°C. 20 µl beads (Dynabeads Protein G, Thermo Fisher Scientific) per 1 ml lysis buffer were washed twice with lysis buffer and coated for 1 h with 0,25 µg/µl FLAG antibody (Sigma-Aldrich, F1804). Afterwards beads were washed with lysis buffer twice and added to the sample. After 2 h incubation at 4°C on a rotating wheel the beads were washed with IP wash buffer (50 mM HEPES-KOH [pH 7.5], 300 mM KCl, 0,05 % IGEPAL CA-630, 0,5 mM DTT, complete EDTA-free protease inhibitor cocktail) 3 times and a second RNase T1 treatment was carried out. For replicate 1 a final concentration of 1 unit/µl and for replicate 2 a final concentration of 15 unit/µl was used for 10 min at 22°C. Subsequently, beads were washed with high-salt was buffer (50 mM Tris-HCl [pH 7.4], 1 M NaCl, 1 % IGEPAL CA-630, 0,1 % SDS, 0,5 % sodium deoxycholate, 1 mM EDTA, complete EDTA-free protease inhibitor cocktail [Roche]) 3 times and 2 times with polynucleotide kinase buffer (50 mM Tris-HCl [pH 7.5], 50 mM NaCl,10mM MgCl2, 5 mM DTT). Samples were radiolabeled using T4 polynucleotide kinase (NEB) and resolved on a Novex 4-12% Bis Tris gel (Thermo Fischer Scientific). The protein-RNA complexes were transferred to a nitrocellulose membrane (Whatman), exposed to a phosphorimager screen for 30 min and excised at approximately 160 kDa. After proteinase K (Roche, 40 min at 50°C) digest the RNA was extracted by phenol-chloroform treatment and precipitation.
small RNA cloning and library generation (Hafner et al.,2012, PMID:22926237) using 3′ adaptor 4N-RA3, 5′ adaptor OR5-4N, RT primer RTP and PCR primers RP1 (forward primer) and RPI1-12 (reverse primer, containing barcodes, for sequences see Illumina Truseq small RNA oligonucleotides)

treatment_protocol: FLAG/HA-HDLBP-HEK293 Flp-In T-REx cells were incubated with 100 µM 4sU for 16 h.

tag: genotype/variation

value: FLAG/HA tagged HDLBP

tag: cell line

tag: replicate

value: replicate 2

value: HDLBP-bound RNA fragment


title: cardiac tissue, fgf21, rep 3


title: ASCL1 joint BRB


title: nearSCR ΔSRR109/+ mus rep2

source_name: Embryonic stem cells

value: DpnII - Csp6I


title: JUNB_S2_RNAseq

value: JUNB ORF overexpression


title: cardiac tissue, fgf21, rep 1


title: HEK293T par-clip from HDLBP_2

value: replicate 1

tag: molecule subtype


title: cardiac tissue, wt, rep 3

source_name: cardiac tissue, wt

value: wt


title: nearSCR ΔSRR109/+ cast rep2

value: Embryonic stem cells


title: HEK293T ribosome profiling from guide2_2

description: Ribosome profiling (Ribo-seq)

data_processing:
Library strategy: Ribo-seq
Basecalls were converted to fastq files using Bcl2Fastq (2.16.0.10). Reads were demultiplexed and adapter sequences removed by Flexbar (2.5). Reads were then collapsed to remove PCR duplicates, followed by removal of random nucleotides (four on both 5’ and 3’ end of the reads) using fastx_trimmer (FASTX Toolkit 0.0.14). Reads aligning to rRNA sequences and other sources of contamination using a custom index were removed by Bowtie2 (v2.3.2) and the remaining sequences were aligned to the human transcriptome (hg19) using STAR aligner (2.5.3a) using GTF annotation file Gencode v19 by retaining only the reads that uniquely mapped to the hg19 genome (parameters --outFilterMultimapNmax 1 --outFilterMismatchNmax 5 --outFilterMatchNmin 15 --alignSJoverhangMin 5 --seedSearchStartLmax 20 --outSJfilterOverhangMin 30 8 8 8 --quantMode TranscriptomeSAM). Transcriptome BAM files were converted to the bed format using BedTools bamToBed (v2.26.0). Bed files were then input into riboWaltz (v1.1.0) (PMID:30102689), which we used for downstream quality control and P-site coverage analysis.
To calculate translation efficiencies per gene we used RSEM (v1.2.2) which supplied us with read counts and TPM values per gene. Differences in translational efficacy, as well as in mRNA abundance and due to both effects (transcription and translation) were detected by DESeq2 (1.18.1) with an interaction term model as described previously (PMID: 31763789). Briefly, RPF read counts were normalized using the DESeq2 estimateSizeFactors function by taking into account all read counts. DESeq2 was run with default parameters. Log2-transformed fold changes for downstream comparisons were taken directly from the DESeq2 output.
3’ adaptor 4N-RA3, rApp-NNNNTGGAATTCTCGGGTGCCAAGG-InvdT; 5’ adaptor OR5-4N, rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrCrNrNrNrN
Genome_build: hg19
Supplementary_files_format_and_content: processed_data_riboseq_te.txt: tab delimited matrix of transcript per million (TPM) values per gene for ribosome profiling and rna-seq samples, translational efficiency (te) for all samples and log2-transformed fold changes and DESeq2 p-adjusted values between samples (ribo.rna=change in translational efficiency, rpf=change in ribosome protected fragments).
Supplementary_files_format_and_content: processed_data_riboseq_psite_coverage.txt: tab delimited matrix of ribosome raw and normalized (norm. prefix) P-site coverage per psite position within transcript, psite transcript coordinate (psite), start and stop codon transcript coordinate and distance of psite from start and stop codons in nucleotides.

growth_protocol: HEK293 and HEK293 HDLBP knockout cell lines (1 10 cm dish per replicate) were grown in DMEM + 10%FBS to ~90 % confluency.

extract_protocol:
Cells were lysed in mammalian polysome buffer, digested with RNase I, loaded on a MicroSpin S-400 HR colum (GE Healthcare) and RNA was extracted using Trizol LS as described in Calviello et al. 2016 (doi:10.1038/nmeth.3688)
libraries were constructed as described in Calviello et al. 2016 (doi:10.1038/nmeth.3688)

treatment_protocol: Cells were washed with ice cold PBS containing 100 µg/ml cycloheximide. The PBS was thoroughly removed and plates were put on liquid nitrogen for 10 sec and subsequently on ice.

value: HDLBP knockout guide 2

value: ribosome protected RNA


title: nearSCR +/ΔSCR cast rep2

value: +/delSCR


title: B3GNT2_S1_RNAseq

value: A375 melanoma cells

value: 7 days


title: HEK293T ribosome profiling from guide2_1

description: Ribosome profiling (Ribo-seq)


title: nearSCR +/ΔSCR cast rep1


title: MCL1_S3_RNAseq

value: MCL1 ORF overexpression


title: nearSCR +/ΔSCR mus rep2


title: B3GNT2_S2_RNAseq


title: HEK293T ribosome profiling from guide1_1

description: Ribosome profiling (Ribo-seq)

value: HDLBP knockout guide 1


title: cardiac tissue, fgf21, rep 2

value: cardiac


title: nearSCR +/ΔSCR mus rep1


title: cardiac tissue, wt, rep 2


title: MCL1_S2_RNAseq


title: nearSCR ΔSRR109/+ mus rep1


title: HEK293T ribosome profiling from 293_2

description: Ribosome profiling (Ribo-seq)

value: HDLBP WT


title: MCL1_S1_RNAseq


title: HEK293T ribosome profiling from guide1_2

description: Ribosome profiling (Ribo-seq)


title: nearSCR ΔSCR/+ mus rep2

value: delSCR/+


title: JUNB_S1_RNAseq


title: K-Ras G12D mutant [KMR19]

city: Houston

first: Jason

middle: T

last: Yustein

state: TX

address: 1102 Bates Ave

department: Pediatrics-Oncology

institute: Texas Children's Hospital

data_processing:
Samples were trimmed using TrimGalore
Trimmed samples were aligned to the mm10 genome using STAR
Features were quantified using featurecounts
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with feature counscounts for every gene and every sample

source_name: K-Ras G12D mutant

growth_protocol: cell lines were maintained in DMEM with 10%FBS and 1% penstrep

extract_protocol:
RNA was extracted with Qiagen RNAeasy Plus Mini kit
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: strain

value: cell line from tumor


title: cardiac tissue, wt, rep 1


title: CD274_S2_RNAseq

tag: perturbation

value: CD274 ORF overexpression


title: CD274_S3_RNAseq

tag: perturbation time


title: HEK293T ribosome profiling from 293_1

description: Ribosome profiling (Ribo-seq)


title: CD274_S1_RNAseq


title: nearSCR ΔSCR/+ cast rep1

value: DpnII - Csp6I


title: K-Ras G12D mutant [KMR72]

tag: tissue


title: ATCC25872-SNP45-converted (#9168) replicate 3

city: Lausanne

first: Melanie

last: Blokesch

address: Station 19 / EPFL-SV-UPBLO

department: School of Life Sciences

country: Switzerland

institute: Swiss Federal Institute of Technology Lausanne (EPFL)

data_processing:
Data processing was done by Microsynth (Balgach, Switzerland) using their pipeline "Standard differential prokaryotic gene expression analysis"
Single end reads (1x75bp), which passed Illumina’s chastity filter, were demultiplexed and trimmed off the Illumina adaptors using Illumina’s bcl2fastq software version v2.20.0.422 (no further refinement or selection).
Reads were mapped to the reference genomes (A1552 chromosome 1 [CP028894] and 2 [CP028895]; ATCC25872 chromosome 1 [CP047305] and 2 [CP047306]) via bowtie2 (version 2.3.5.1)
Uniquely mapped reads to annotated genes were counted using the software htseq-count (HTSeq version 0.11.2)
Normalization of the raw counts and differential gene expression analysis was carried out with help of the R software package DESeq2 (version 1.22.2)
Genome_build: Reference genomes were A1552 chromosome 1 [CP028894] and 2 [CP028895] or ATCC25872 chromosome 1 [CP047305] and 2 [CP047306]
Supplementary_files_format_and_content: .tsv files for normalized counts

organism: Vibrio cholerae

source_name: in vitro bacterial culture

growth_protocol: V. cholerae strains were back-diluted 1:100 from an overnight culture in LB medium and grown for 3 h at 30°C with agitation.

extract_protocol:
RNA was isolated using standard RNA isolation kits. The RNA was cleaned up after DNAse treatment using a RNA clean-up procedure (GenElute Mammalian Total RNA miniprep kit; Sigma). Next, Illumina RNA libraries incl. bacterial Ribo-depletion were prepared.
RNA libraries were prepared with a TruSeq Stranded mRNA sample prep kit (Illumina).

treatment_protocol: The harvested bacteria were washed in pre-chilled PBS buffer, lysed through resuspension in Tri Reagent, and stored at -80C until their processing.

value: ATCC25872-SNP45-converted (#9168)

value: ATCC25872 with SNP45 in intergenic region between VCA0106 and VCA0107 (vipA) converted from T to G by site-directed mutation

tag: growth medium

value: LB medium


title: ATCC25872-SNP45-converted (#9168) replicate 1


title: nearSCR ΔSCR/+ mus rep1

value: Embryonic stem cells


title: JUNB_S3_RNAseq

value: JUNB ORF overexpression


title: nearSCR ΔSCR/ΔSCR cast rep4

value: delSCR/delSCR


title: K-Ras G12D mutant [KMR46]

value: K-RasG12D p53F/+


title: K-Ras G12D mutant [KMR78]

value: K-RasWT p53F/+


title: ATCC25872-SNP45-converted (#9168) replicate 2


title: ChIP_JUNB_CFLAG_Input

description:
ChIP_JUNB_353I
Input control

data_processing:
bcl2fastq 2.20.0
Bowtie 1.2.3
MACS 1.4.2
Genome_build: hg38
Supplementary_files_format_and_content: bed files contain peaks called using MACS

extract_protocol:
ChIP was performed as described in the methods section of the manuscript
ChIP samples were prepared for NGS with NEBNext Ultra II DNA Library Kit for Illumina

value: A375 melanoma cells

value: C-term FLAG JUNB ORF expression

value: 7 days


title: ATCC25872 (#276) replicate 3

value: wild-type (ATCC25872)


title: nearSCR ΔSCR/ΔSCR mus rep3

data_processing:
Library strategy: 4C-seq
Image analysis and base calling were performed using RTA 2.7.7 and bcl2fastq
Individual 4C-seq experiments were demultiplexed according to bait primer sequence using sabre, with two mismatches permitted (-m 2)
Reads were aligned using Bowtie (v1.0.0) with the settings -a -m 1 --best --strata
Mapped reads were filtered and mapped to DpnII restriction fragments with the 4See utilities described in Ben Zouari et al. 4See: a flexible browser to explore 4C data. Front. Genet. 10: 1372.
Genome_build: mm10
Supplementary_files_format_and_content: Cis files, containing a header and two columns (restriction fragment midpoint coordinate, counts), the input format for 4See (Ben Zouari et al. 4See: a flexible browser to explore 4C data. Front. Genet. 10: 1372.)


title: K-Ras wild type  [MR17]

source_name: K-Ras wild type

value: K-RasWT p53R172H/+


title: cardiac tissue, wt, rep 4

value: wt

value: cardiac

tag: repeat


title: K-Ras wild type  [MR42]

value: cell line from tumor


title: ChIP_JUNB_CFLAG_FLAG

description:
ChIP_JUNB_353F
FLAG antibody pulldown


title: ChIP_JUNB_NFLAG_Input

description:
ChIP_JUNB_352I
Input control

value: N-term FLAG JUNB ORF expression


title: nearSCR ΔSCR/ΔSCR ns rep2

description: 4C-seq with nearSCR bait

value: DpnII - Csp6I


title: ATCC25872 (#276) replicate 1


title: nearSCR ΔSCR/ΔSCR ns rep1

description: 4C-seq with nearSCR bait


title: ChIP_JUNB_NFLAG_FLAG

description:
ChIP_JUNB_352F
FLAG antibody pulldown


title: nearSCR F1 cast rep4

value: Wild type


title: A1552-SNP45-converted (#9063) replicate 3

value: A1552 with SNP45 in intergenic region between VCA0106 and VCA0107 (vipA) converted from G to T by site-directed mutation

value: LB medium


title: HEK293T rna-seq from guide2_2

description: polyA RNA from input lysate

data_processing:
Basecalls were converted to fastq files using Bcl2Fastq (2.16.0.10). Reads were demultiplexed and adapter sequences removed by Flexbar (2.5). Reads were then collapsed to remove PCR duplicates, followed by removal of random nucleotides (four on both 5’ and 3’ end of the reads) using fastx_trimmer (FASTX Toolkit 0.0.14). Reads aligning to rRNA sequences and other sources of contamination using a custom index were removed by Bowtie2 (v2.3.2) and the remaining sequences were aligned to the human transcriptome (hg19) using STAR aligner (2.5.3a) using GTF annotation file Gencode v19 by retaining only the reads that uniquely mapped to the hg19 genome (parameters --outFilterMultimapNmax 1 --outFilterMismatchNmax 5 --outFilterMatchNmin 15 --alignSJoverhangMin 5 --seedSearchStartLmax 20 --outSJfilterOverhangMin 30 8 8 8 --quantMode TranscriptomeSAM). Transcriptome BAM files were converted to the bed format using BedTools bamToBed (v2.26.0). Bed files were then input into riboWaltz (v1.1.0) (PMID:30102689), which we used for downstream quality control and P-site coverage analysis.
To calculate translation efficiencies per gene we used RSEM (v1.2.2) which supplied us with read counts and TPM values per gene. Differences in translational efficacy, as well as in mRNA abundance and due to both effects (transcription and translation) were detected by DESeq2 (1.18.1) with an interaction term model as described previously (PMID: 31763789). Briefly, RPF read counts were normalized using the DESeq2 estimateSizeFactors function by taking into account all read counts. DESeq2 was run with default parameters. Log2-transformed fold changes for downstream comparisons were taken directly from the DESeq2 output.
3’ adaptor 4N-RA3, rApp-NNNNTGGAATTCTCGGGTGCCAAGG-InvdT; 5’ adaptor OR5-4N, rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrCrNrNrNrN
Genome_build: hg19
Supplementary_files_format_and_content: processed_data_riboseq_te.txt: tab delimited matrix of transcript per million (TPM) values per gene for ribosome profiling and rna-seq samples, translational efficiency (te) for all samples and log2-transformed fold changes and DESeq2 p-adjusted values between samples (ribo.rna=change in translational efficiency, rpf=change in ribosome protected fragments).
Supplementary_files_format_and_content: processed_data_riboseq_psite_coverage.txt: tab delimited matrix of ribosome raw and normalized (norm. prefix) P-site coverage per psite position within transcript, psite transcript coordinate (psite), start and stop codon transcript coordinate and distance of psite from start and stop codons in nucleotides.

extract_protocol:
Cells were lysed in mammalian polysome buffer and RNA was extracted using Trizol LS
Illumina TruSeq Stranded mRNA Kit

value: HDLBP knockout guide 2

value: replicate 2

value: poly(A)+ RNA


title: HEK293T rna-seq from guide2_1

description: polyA RNA from input lysate

value: replicate 1


title: nearSCR F1 mus rep3

extract_protocol:
Cells were fixed with 2% paraformaldehyde in 10% FBS in PBS for 10 min at 23°C. The fixation was quenched with cold glycine at a final concentration of 125 mM, then cells were washed with PBS and permeabilized on ice for 1 h with 10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.1% NP-40, and protease inhibitors. Nuclei were resuspended in DpnII restriction buffer at 10 million nuclei/mL concentration, and 5 million nuclei aliquots were further permeabilized by treatment for 1 h with 0.4% SDS at 37°C, then incubating for a further 1 h with 3.33% Triton X-100 at 37°C. Nuclei were digested overnight with 1500 U DpnII at 37°C, then washed twice by centrifuging and resuspending in T4 DNA ligase buffer. In situ ligation was performed in 400 μL T4 DNA ligase buffer with 20,000 U T4 DNA ligase overnight at 23°C. DNA was purified by reverse cross-linking with an overnight incubation at 65°C with proteinase K, followed by RNase A digestion, phenol/chloroform extraction, and isopropanol precipitation. The DNA was digested with 5 U/μg Csp6I at 37°C overnight, then re-purified by phenol/chloroform extraction and isopropanol precipitation. The DNA was then circularized by ligation with 200 U/μg T4 DNA ligase under dilute conditions (3 ng/μL DNA), and purified by phenol/chloroform extraction and isopropanol precipitation. For allele-specific 4C from the SCR-proximal bait, samples of the DNA were digested with BveI or Alw26I, cutting specifically at the region between the DpnII site and the 4C reading primer annealing site on the 129 or castaneus allele, respectively. For 129-specific 4C from the SCR bait, the material was digested with AvaIII, cutting specifically at the region between the Csp6I site and the 4C non-reading primer annealing site on the castaneus allele. There were no SNPs allowing castaneus-specific 4C from this bait. No digestion was required for allele-specific 4C from the human insertion sequence, since this was only present on the 129 allele. 100 ng aliquots of treated DNA were then used as template for PCR with bait-specific primers containing Illumina adapter termini. PCR reactions were pooled, primers removed by washing with 1.8× AMPure XP beads, then quantified on a Bioanalyzer (Agilent) before sequencing with a HiSeq 4000 (Illumina).
4C PCR products are quantified in a Bioanalyzer, and pooled in equimolar quantities for direct cluster generation on an Illumina HiSeq4000

value: Embryonic stem cells


title: A1552 (#1) replicate 3

value: wild-type (A1552)


title: A1552-SNP45-converted (#9063) replicate 1


title: nearSCR F1 ns rep1

description: 4C-seq with nearSCR bait


title: HEK293T rna-seq from guide1_2

description: polyA RNA from input lysate

value: HDLBP knockout guide 1


title: FLAMs_SiglecF_Hi_Rep2

city: East Lansing

first: Andrew

last: Olive

state: MI

address: 567 Wilson Rd, BPS 5100

department: Microbiology and Molecular Genetics

institute: Michigan State University

data_processing:
Illumina software was used for Basecalls
FastQC (v0.11.5) was used to assess the quality of raw data.
Cutadapt (v2.9) was used to removeadapter sequences
Genome_build: sgRNA counts were determined using MAGeCK "count" function with trimmed FASTq files as input
significant changes in gene representation were quantified using MAGeCK "test" using the robust rank algorithm
Supplementary_files_format_and_content: Normalized Count table was generated using MAGeCK

source_name: Macrophage Fetal Liver Cells

growth_protocol: Macrophages were grown in DMEM with 10% FBS 30ng/ml GM-CSF 15 ng/ml TGFB for the duration of the experiment

extract_protocol:
Cells were lysed and total genomic DNA was isolated using aluminum acetate purification
Libraries were constructed using the targeted PCR-mediated amplification of sgRNAs from purified gDNA. This amplification for each sample incorporated adaptors and sample indexes.

treatment_protocol: Transduced library was cultuted for two weeks then gDNA was isolated or cells were sorted for SiglecF expression

value: Fetal Liver

tag: derivation

value: cultured primary fetal liver cells

value: C57BL/6J Cas9+

tag: surface expression

value: SiglecF High


title: FLAMs_SiglecF_Low_Rep2

value: SiglecF Low


title: FLAMs_Input_Rep2

value: Input


title: FLAMs_SiglecF_Low_Rep1


title: ATCC25872 (#276) replicate 2

value: wild-type (ATCC25872)


title: HEK293T rna-seq from guide1_1

description: polyA RNA from input lysate


title: HEK293T rna-seq from 293_2

description: polyA RNA from input lysate

value: HDLBP WT


title: A1552 (#1) replicate 1


title: Qa2- uterus_2

city: Tübingen

first: Jakob

last: Admard

address: Calwerstr. 7

department: Institut für Medizinische Genetik und Angewandte Genomik

institute: Universitätsklinikum Tübingen

data_processing:
Basecalling of raw sequencing output was performed with Illumina bcl2fast2 version 2.19.1.
For subsequent adapter sequence trimming in FASTQ files, ngs-bits SeqPurge version 2020_06 was used.
Cleaned read sequences were aligned with STAR version 2.7.3a
Read counting for exonic regions was performed with subread featureCounts version 2.0.0.
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: Raw gene-level count data in featureCounts default output format, see description at http://bioinf.wehi.edu.au/subread-package/SubreadUsersGuide.pdf

source_name: uterus

extract_protocol:
Uteri were removed, snap-frozen in liquid nitrogen, and RNA was harvested using Qiagen Rneasy Mini Kit.
mRNA libraries were prepared using the NEB Next Ultra II Directional RNA Library Prep Kit for Illlumina.

tag: age

value: gestational age E18.5

value: uterus


title: FLAMs_Input_Rep1


title: Qa2- uterus_1


title: FLAMs_SiglecF_Hi_Rep1


title: A1552 (#1) replicate 2

tag: genotype

value: LB medium


title: HEK293T rna-seq from 293_1

description: polyA RNA from input lysate


title: CFS414 cell line, passage 50, 10x seq

last: Biegler

address: 1230 York Ave

institute: The Rockefeller University

description:
Zebra finch (Taeniopygia guttata castanotis)
Passage 50, CFS414 cell line, 10x sequencing data

data_processing:
The Cell Ranger 10x Genomics Pipeline was used to demultiplex the raw base calls into FASTQ files for subsequent alignment to the genome, filter for quality reads, and generate count matrices.
Associated supplementary files were further processed using the Seurat RStudio package (v3.2.0). A detailed breakdown of code and processing steps used may be found at https://github.com/Neurogenetics-Jarvis/CFS414_scRNAseq
Integration Seurat object of the Passage 24 and 50 10x datasets. Rstudio Notebook for data processing steps and data visualization available on https://github.com/Neurogenetics-Jarvis/CFS414_scRNAseq
Genome_build: bTaegut1.pri.v2, with W chromosome from bTaegut2.pat.W. The UTR portions of the gene annotations were extended using previously collected RNAseq data to enhance read alignment.
Supplementary_files_format_and_content: gzipped tsv and mtx outputs from the Cell Ranger pipeline for use in downstream processing.

organism: Taeniopygia guttata

growth_protocol: CFS414 cells at passage 24 (from cryopreserved stock of Passage 18) and passage 50 (ongoing) were plated onto 10cm cell culture dishes at a subculture of 1:15 from the previous passage and plated in complete media (DMEM high-glucose, no glutamine (Cat. #11960044) with 10% (v/v) fetal bovine serum (Cat. #21640079), 1x GlutaMAX (Cat. #35050061), 1x Antibiotic-Antimycotic (Cat. #15240062), 1x NEAA (Cat. #1140050), and 1x Sodium pyruvate (Cat. #11360070), all from GibcoTM).

extract_protocol:
Confluent CFS414 cells were dissociated from the surface of the dish using 0.25% Trypsin-EDTA, washed with PBS three times and counted on a Countess II FL (Life Technologies). A single cell suspension was diluted to around 1,000 cells/µL in 1xPBS. Approximately 5,000 cells were run on a Chromium Chip (10x Genomics).
cDNA synthesis and library preparation was performed using the Chromium Single Cell 3’ Reagent Kits (version 3, 10x Genomics) according to manufacturer’s instructions. Sequencing was performed by Novogene Co., Ltd. on an Illumina HiSeq 4000.

tag: cell type

value: embryonic fibroblast culture

tag: treatment

value: Lentiviral transduction of SV40 Large and small T antigen

tag: passage


title: CFS414 cell line, passage 24, 10x seq

description:
Zebra finch (Taeniopygia guttata castanotis)
Passage 24, CFS414 cell line, 10x sequencing data


title: nearSCR F1 ns rep2

last: Sexton

address: 1 rue Laurent Fries

institute: IGBMC (Institute of Genetics and Molecular and Cellular Biology)

description: 4C-seq with nearSCR bait

tag: hybrid generation

value: Wild type

tag: restriction enzyme

value: DpnII - Csp6I


title: PCOS 3

city: Beijing

first: Yanli

last: Pang

state: Beijing

address: No.49 Huayuan-Bei Road , Haidian District

country: China

institute: Reproductive Medicial Center , Peking University Third Hospital

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing poly-N and low quality reads from raw data.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Genome_build: Homo_sapiens_Ensemble_98
Supplementary_files_format_and_content: gene_fpkm.txt: Matrix table with FPKM values for each Sample.
Supplementary_files_format_and_content: gene_count.txt: Matrix table with raw gene counts for every gene and every sample.

source_name: granulosa cells

extract_protocol:
Human granulosa cells were separated with density gradient centrifugation and cultured in DMEM-F12 supplemented with 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (5,000 U/mL) for 12h, and subsequently collected for RNA extraction.
A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample.

value: ovary

value: granulosa cell

tag: diagnosis

value: Polycystic ovary syndrome (PCOS)


title: A1552-SNP45-converted (#9063) replicate 2

value: A1552 with SNP45 in intergenic region between VCA0106 and VCA0107 (vipA) converted from G to T by site-directed mutation


title: mtlRdel-M9YEG- n1

city: Vermillion

first: SRIVISHNUPRIYA

last: ANBALAGAN

state: SD

address: 414 East Clark Street

department: Basic Biomedical Sciences

institute: University of South Dakota

description: RNA-seq

data_processing:
original fastq format reads were trimmed by fqtrim tool (https://ccb.jhu.edu/software/fqtrim) to remove adapters, terminal unknown bases (Ns), and low quality 3’ regions (Phred score < 30)
trimmed fastq files were processed by FastQC
fastq files were mapped to Enterococcus faecalis OG1RF by CLC Genomics Workbench 12 for RNAseq analyses
read counts were modeled using Generalized Linear Model (GLM), assuming a negative binomial distribution, and reported as transcripts per million (TPM)
Wald test was used for statistical analysis of two-group comparisons
Genome_build: Enterococcus faecalis OG1RF
Supplementary_files_format_and_content: Excel file showing fold changes and statistical significance between different growth conditions

organism: Enterococcus faecalis

growth_protocol: For growth curves and RNA preparation E. faecalis cultures were grown in M9YE base supplemented with either 20 mM glucose or mannitol. E. faecalis liquid cultures were grown at 37 °C with shaking at 25 rpm.

extract_protocol:
Cell pellets were resuspended in Bacteria RNA Protect (Qiagen) and incubated at room temperature for 5 min. Cells were then pelleted and stored at −80°C. Once all samples were collected, pellets were thawed, resuspended in 200 μl of Tris/EDTA-mutanolysin-lysozyme solution (10 mM Tris [pH 8.0], 1.0 mM EDTA [pH 8.0], mutanolysin [500 U ml−1], lysozyme [30 mg ml−1]), and incubated at 37°C for 10 min. To the resuspensions, 700 μl of buffer RLT (Qiagen RNeasy minikit) with 10 μl of β-mercaptoethanol (β-ME) was added, with vortexing to mix. RNA was then purified using the Qiagen RNeasy minikit according to the manufacturer's instructions. Total RNA from biological replicates was treated with TURBO DNase (Ambion/Thermo Scientific, Waltham, MA) using the rigorous protocol according to the manufacturer's instructions. DNase was removed from treated samples using the RNA Clean & Concentrator-25 (Zymo Research).
TruSeq® Stranded Total RNA Library Prep

treatment_protocol: As indicated in the Characteristics column

tag: growth media

tag: genotype/variation

value: mtlR deletion mutant


title: mtlRdel-M9YEG- n2

description: RNA-seq


title: PCOS 2


title: PCOS 1


title: WT uterus_2

tag: strain


title: OG1RF-M9YEM- n2

description: RNA-seq


title: WT uterus_1


title: Control 2

value: Healthy


title: OG1RF-M9YEG- n2

description: RNA-seq


title: OG1RF-M9YEM- n1

description: RNA-seq


title: Control 3


title: Control 1


title: LSK cells-KO1 [K6_4]

city: Chicago

first: Susan

last: Ross

address: 835 South Wolcott Ave

department: Microbiology & Immunology

institute: University of Illinois at Chicago

data_processing:
Instrument model is Illumina Novaseq 6000 Platform using 2 X 150 paired-end sequence reads.
For gene quantification, raw reads were aligned to reference genome mm10 in a splice-aware manner using the STAR aligner. ENSEMBL gene annotations were quantified using FeatureCounts. Differential expression statistics was computed using edgeR.
rMATS was run with the default settings for the detection of differential alternative splicing events.
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with normalized abundance measurements for every gene and every sample
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: LSK cells-KO

extract_protocol:
LSK cells were sorted into lysis buffer. RNA was isolated by Qiagen RNeasy Micro Kit.
SMART-Seq® v4 Ultra® Low Input RNA Kit was used for library construction.

tag: strain background

value: knockout

value: Fetal liver

value: LSK cells


title: OG1RF-M9YEG- n1

description: RNA-seq


title: LSK cells-KO2 [K6_5]

tag: tissue


title: LSK cells-KO3 [K_R2]


title: LSK cells-het2 [H_L3]

source_name: LSK cells-het

value: heterzygous


title: LSK cells-het3 [H_L5]


title: mRNA-stim-well4

last: Steinhart

address: 1650 Owens St

institute: J. David Gladstone Institutes

description:
matrix.mtx.gz
features.tsv.gz
barcodes.tsv.gz

data_processing:
Sequencing reads were demultiplexed with bcl2fastq. Alignments and count aggregation of gene expression and sgRNA reads were completed with Cell Ranger version 6.1.1. Gene expression and sgRNA reads were aligned using cellranger count, with default settings. Gene expression reads were aligned to the “refdata-gex-GRCh38-2020-A” human transcriptome reference downloaded from 10x Genomics. sgRNA reads were aligned to the Perturb-seq library using the pattern (BC)GTTTAAGAGCTATG. Counts were aggregated with cellranger aggr with default arguments. To assign sgRNAs to cells, cellranger count output files “protospacer_calls_per_cell.csv” were manually aggregated in R using the bind_rows() function from the dplyr package to generate the “cellranger-guidecalls-aggregated-unfiltered.txt” file.
Genome_build: GRCh38-2020-A
Supplementary_files_format_and_content: cellranger aggr filtered feature barcode matrix output files (matrix.mtx.gz, features.tsv.gz, barcodes.tsv.gz), which can be loaded into Seurat or other similar software for scRNA-seq analysis
Supplementary_files_format_and_content: cellranger count “protospacer_calls_per_cell.csv" files manually aggregated across wells (cellranger-guidecalls-aggregated-unfiltered.txt)

source_name: Primary bulk T cells

growth_protocol: Primary human bulk T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641). Primary T cell activation was done by anti-humanCD3/CD28 CTS dynabeads (Fisher Scientific cat 40203D) at a 1:1 cell to bead ratio at 1e6 cells/ml.

extract_protocol:
After sorting, cells were processed with the 10x Genomics Chromium Next GEM Single Cell 3′ Reagent Kit v3.1 with Feature Barcoding technology for CRISPR Screening, following manufacturer’s protocol (CG000205 Rev D). Before loading the Chromium chip, sorted cells from two blood donors were normalized to 1000 cells/µl and mixed at a 1:1 ratio, for each condition. Twenty µl of cell suspension was loaded into four replicate wells per condition, for a total 80,000 cells loaded per condition.
Libraries were constructed using the 10x Genomics Chromium Next GEM Single Cell 3′ Reagent Kit v3.1 with Feature Barcoding technology for CRISPR Screening, following manufacturer’s protocol (CG000205 Rev D). Final sgRNA sequencing libraries were further purified for the correct size fragment by 4% agarose E-Gel EX Gels (ThermoFisher Scientific) and gel extracted using the NucleoSpin Gel and PCR Clean-up Mini kit (Machery-Nagel).

treatment_protocol: One day after activation, T cells were infected with dCas9-VP64 (CRISPRa) lentivirus. Two days after activation, T cells were split into two populations and infected with a 154 sgRNA Perturb-seq library an MOI of 0.3. The following day 2ug/ml puromycin was added to the culture medium and cells were passaged every two days maintaining a minimum concentration of 0.3e6cells/ml. Eight days after initial activation, cells were harvested, spun down and resuspended at 2e6 cells/ml X-VIVO 15 without supplements. Next day, cells were split into two conditions: 1) re-stimulated with anti-CD3/CD28/CD2 immunocult or 2) left. 24 hours later cells were sorted for mCherry+ (marking dCas9-VP64) before droplet generation.

tag: stimulation

value: yes


title: LSK cells-het1 [H_L1]


title: mRNA-stim-well2


title: mRNA-stim-well3


title: SAI3 NPC_mid_hind, biological replicate 2

city: Quebec City

first: Samer

last: Hussein

state: Quebec

address: 9 rue Mcmahon

department: Departement of medecine

country: Canada

institute: CHU de Québec - Université Laval

hyb_protocol: Purified and labeled RNA was prepared for analysis on Affymetrix Human Transcriptome Array 2.0 (Affymetrix) according to the manufacturer’s instructions, at Microarray Facility of The Centre for Applied Genomics at Sickkids, Toronto, Ontario.

scan_protocol: Prepared RNA was analyzed on Affymetrix Human Transcriptome Array 2.0 (Affymetrix) according to the manufacturer’s instructions, at Microarray Facility of The Centre for Applied Genomics at Sickkids, Toronto, Ontario.

data_processing: RMA method normalization, background substraction, and summarization was performed using Oligo (v1.34.2) R package from bioconductor. Normalized gene expression intensities were log2-scaled for all subsequent analyses. Unless otherwise stated, all data presented are representative of at least two independent experiments. A linear model approach and the empirical Bayes statistics were implemented for differential gene expression analysis, as described in limma (v3.26.9) R package user guide. P-values were adjusted using the Benjamini–Hochberg method and significance cut-off was set at 0.01.

label: biotin

source_name: Mid-hindbrain specified Neural progenitor cells derived from embryo

label_protocol: Purified RNA was labeled for analysis on Affymetrix Human Transcriptome Array 2.0 (Affymetrix) according to the manufacturer’s instructions, at Microarray Facility of The Centre for Applied Genomics at Sickkids, Toronto, Ontario.

growth_protocol: H1 and CA1 human pluripotent stem cells (hPSCs) were routinely cultured on mitomycin C treated mouse embryonic fibroblast feeder cells in KSR media with 10ng/ml FGF2. KSR media contains 80% DMEM-F12 (GIBCO), 20% Knockout serum replacement (GIBCO), 0.1mM 2-mercaptoethanol, 2mM Glutamax, 0.1mM Non-essential amino acids. Prior to neural differentiation hPSCs were transferred to geltrex (GIBCO) coated surface in the absence of feeders and were cultured in mTESR2 (Stem cell technologies) media. On the day of neural induction, hPSCs were replated on geltrex coated surface in N2B27 media supplemented with 10µM SB431542 and 100nM LDN193189 and 10µM Y27632. Neural cultures were detached from Geltrex first on day 8-10 with Accutase or PBS-EDTA, and replated on laminin (Sigma) coated surface, in neural maintenance media (NES) supplemented with 10µM Y27632 at 3x105 cells per cm2 and 6F : 10ng/ml FGF2, 3μM CHIR99021, 1μM SB431542 (Peprotech), 100nM LDN193189, 100nM K02288 (Selleckchem), 100nM AKTiVIII, 75nM MK2206 (Selleckchem), and 1 μM XAV939 (Selleckchem). NES media contains 50% DMEM-F12, 50% Neurobasal, 0.1mM 2mercaptoethanol, 2mM Glutamax, 1x N2 supplement, 0.05x B27 minus vitamin A supplement (GIBCO). 4F media is made by supplementing NES media with 10ng/ml FGF2, 3μM CHIR99021, 1μM SB431542 or 50ng/ml FST (Peprotech), 100nM LDN193189 or 50 ng/ml NOGGIN. 6F media contains components of the 4F media with 100nM K02288 (Selleckchem), 100nM AKTiVIII and 75nM MK2206 (Selleckchem), and 1-2 μM XAV939 (Selleckchem). XAV939 concentrations need to be adjusted to each cell line, 1μM for H1, CA1, H9, CTRL hPSCd-erived cNESCs, 2 μM for Shef6 derived cNESCs. For single cell clone isolations, H1 (passage 12) cNES cells were cultured in 4F or 6F medium as described in section ‘Neuroepithelial stem cell maintenance and differentiation’. Three to four days after plating the cells were rinsed with PBS once and detached with 5-minute Accutase digestion at 37°C. The cells were collected with 10fold volume DMEM-F12 with 0.1% BSA and centrifuged at 300xg for 3.5 minutes. The pelleted cells were resuspended in 1 ml fresh 6F media and triturated by pipetting with 1000μl tip for 20 times with moderate force without generating bubbles. Cells were centrifuged with additional 5fold volume DMEM-F12 with 0.1% BSA at 300xg for 3.5 minutes. For single cell cloning cNES cells were plated in 6F media at 100 cells per cm2 culture surface area in a 10 cm dish coated with poly-D-lysine and laminin. FGF was added daily at 20ng/ml concentration without media change for the first 4 days. Media was completely changed after 6 days. Colonies were manually picked at day 10 to 96 wells. CA1 and H1 hESCs were also differentiated to dorsal forebrain neuroepithelium for 8-10d without culture in 6F condition. Mid-hindbrain specified NESCs (mhbNESCs) were derived in FGF and EGF from hPSCs (AF22, 23) or from human embryos (SAI1,3).

extract_protocol: Total RNA was isolated from cultured cells with Qiagen RNeasy kit according to manufacturer’s instructions and was assessed for quality and quantity on a Bio-analyzer

treatment_protocol: No treatment

value: Mid-hindbrain specified NESCs


title: SAI1 NPC_mid_hind, biological replicate 1


title: mRNA-nostim-well4


title: AF23 NPC_mid_hind, biological replicate 2

source_name: Mid-hindbrain specified Neural progenitor cells derived from iPSC


title: LSK cells-WT2 [W_L2]

source_name: LSK cells-WT

value: wild type


title: mRNA-stim-well1


title: mRNA-nostim-well3


title: LSK cells-WT1 [W_1]


title: mRNA-nostim-well2


title: Mutant Rep3

data_processing:
The RNA-Seq analysis was performed with RaNA-Seq (Prieto and Barrios, 2019).
For transposable elements (TE) analysis, raw reads were trimmed using TrimGalore (version 0.6.6; http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and mapped to the reference genome (GRCm38, PRI) with annotation (PRI, https://www.gencodegenes.org/mouse/) using STAR (v2.7.6a) (Dobin et al., 2013).
TE families were quantified using featureCounts (v2.0.1) (Liao et al., 2014) with following options (-M -T 1 -s 0 -p) and TE annotation (http://labshare.cshl.edu/shares/mhammelllab/www-data/TEtranscripts/TE_GTF/).
The differentially expressed TEs were extracted by DESeq2.
Genome_build: mm10
Supplementary_files_format_and_content: DE.tsv, Counts_TE.txt, DESeq2_TE.txt

source_name: Skeletal muscle stem cells

growth_protocol: Isolated satellite cells were resuspended in the DMEM/F12 (gibco, Cat# 10565-018) containing 20% FBS, 2% Ultroser G (Pall, Cat# 15950-017), and Antibiotic-Antimycotic. Cells were cultured for 3 days in 8-well chamber slide (Thermo Scientific, Cat# 177445) with 1 mg/ml of Matrigel (Corning, Cat# 354230) at 5000 cells/cm2.

extract_protocol:
Total RNA was extracted from satellite cells with RNeasy Plus Micro Kit (Qiagen, Cat# 74034).
RNA-seq libraries were prepared using Ultra II Directional RNA Library Prep Kit for Illumina (NEB, Cat# E7760L) with NEBNext rRNA Depletion Kit (NEB, Cat# E6310X) according to the manufacturer’s instructions. The libraries were sequenced using NovaSeq 6000 Sequencing System (Illumina) with 150 bp paired end reads.

treatment_protocol: Tamoxifen (150 µl of 20 mg/ml) (Sigma, Cat# T5648) dissolved in corn oil (Sigma, Cat# C8267) were injected intraperitoneally for a total of 5 consecutive days to induce Cre-mediated recombination.

value: Hindlimb muscles

value: 9-week old

value: Tamoxifen


title: Mutant Rep2


title: mRNA-nostim-well1


title: guide-stim-well4

description: cellranger-guidecalls-aggregated-unfiltered.txt

molecule: other


title: LSK cells-WT3 [W_L5]


title: CA1_d8 ESC_diff, biological replicate 2

source_name: CA1 human embryonic stem cell differentiation at day 8

value: Differentiating ESCs


title: AF22 NPC_mid_hind, biological replicate 1


title: H1-D10 ESC_diff, biological replicate 1

source_name: H1 human embryonic stem cell differentiation at day 10


title: Control Rep3

value: Oil


title: H9_d10 ESC_diff, biological replicate 2

source_name: H9 human embryonic stem cell differentiation at day 10


title: ∆rpoN_0min_afterInd_26C_rep3

city: Umea

first: Maria

last: Fallman

address: Petrus Laestadius väg

department: Molecular Biology

country: Sweden

institute: Umea university

data_processing:
Reads were trimmed from 5' and 3' ends until all the adapter and low-quality bases were removed and the sequences passed QC checking by FastQC
Reads were aligned to a reference genome of Y. pseudotuberculosis (accession number NC_010465) using bowtie2 with the unique-mapping option.
Post-mapped read quality checking was done using RseQC, and the numbers of reads for each gene were counted using feaatureCount
Aligned reads were converted to BAM and wig by SAMtool and custom script
Differential gene expression was determined using DEseq2, using the shrinkage estimation of dispersion option (lfcSrink =True) to generate more accurate estimates of differential expression in fold changes
Genome_build: NC_010465
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: Matrix table with normalized counts and differential expression statistics for every gene and every sample

organism: Yersinia pseudotuberculosis

source_name: bacteria grown in Lauria broth

growth_protocol: All strains were routinely grown at 26ºC in LB medium containing kanamycin (50 µg/ml) to the desired OD600.

extract_protocol:
For isolation and purification of RNA, the Trizol method (Ambion Life Technologies, Carlsbad, CA) and the Direct-zol RNA kit (Zymo Research, USA) were used. To remove unwanted DNA, purified RNA was treated with DNAse for 30 min at room temperature according to the manufacturer’s instructions. RNA quantity was measured by Qubit
RNA libraries were prepared for sequencing using RNA-tag seq protocol

treatment_protocol: To reach the virulence induction condition, overnight bacterial cultures were diluted to OD600 0.05 in LB and grown at 26ºC. After 1 h, calcium was depleted by adding EGTA to 5 mM and MgCl2 to 20 mM , and cultures were shifted to 37ºC

value: YPIII

value: mutant

tag: time (minutes)


title: H1_d10 ESC_diff, biological replicate 2


title: CA1_d8 ESC_diff, biological replicate 1


title: H9 ESC, biological replicate 2

source_name: H9 human embryonic stem cell line

value: ESCs


title: H7_d10 ESC_diff, biological replicate 1

source_name: H7 human embryonic stem cell differentiation at day 10


title: H7 ESC, biological replicate 1

source_name: H7 human embryonic stem cell line


title: Control Rep2

last: Sakai

address: Shitsukawa

institute: Ehime University


title: Control Rep1

data_processing:
The RNA-Seq analysis was performed with RaNA-Seq (Prieto and Barrios, 2019). For transposable elements (TE) analysis, raw reads were trimmed using TrimGalore (version 0.6.6; http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and mapped to the reference genome (GRCm38, PRI) with annotation (PRI, https://www.gencodegenes.org/mouse/) using STAR (v2.7.6a) (Dobin et al., 2013).
TE families were quantified using featureCounts (v2.0.1) (Liao et al., 2014) with following options (-M -T 1 -s 0 -p) and TE annotation (http://labshare.cshl.edu/shares/mhammelllab/www-data/TEtranscripts/TE_GTF/).
The differentially expressed TEs were extracted by DESeq2.
Genome_build: mm10
Supplementary_files_format_and_content: DE.tsv, Counts_TE.txt, DESeq2_TE.txt


title: Mutant Rep1


title: guide-stim-well3

city: San Francisco

first: Zachary


title: guide-stim-well1


title: guide-nostim-well4


title: H1 ESC, biological replicate 2

source_name: H1 human embryonic stem cell line


title: H1JA_E4 cNES, biological replicate 2

source_name: Cortically specified neuroepithelial stem cell clone derived from H1 hESCs

value: cNESCs


title: CA1 ESC, biological replicate 1

source_name: CA1 human embryonic stem cell line


title: guide-stim-well2


title: ∆rpoN_0min_afterInd_26C_rep2


title: ∆rpoN_0min_afterInd_26C_rep1


title: H1JA_D4 cNES, biological replicate 2


title: OIL, methylated DNA

data_processing:
Raw reads were down-sampled to 50 million reads for each condition to avoid biases due to different sequencing depths.
The reads were trimmed using TrimGalore and were mapped using HISAT2 (Kim et al., 2015).
Peaks were obtained using MACS2 (version 2.2.6) (Zhang et al., 2008) using TMX treated mice-derived cells as ‘control’ with following options (--slocal 0 --llocal 0 -q 0.0001).
ChIPpeakAnno (Zhu et al., 2010) was used to annotate and visualize the peaks.
Genome_build: mm10
Supplementary_files_format_and_content: peaks file

extract_protocol:
Genomic DNA was isolated and purified with NucleoSpin Tissure Kit (MACHEREY-NAGEL, Cat# 740952).
Purified DNA was sheared with Covaris S220 with microTUBE AFA (Covaris, Cat# 520045). Methylated and non-methylated DNA fragments were separated using EpiXplore Methylated DNA Enrichment Kit (Clontech, Cat# PT5034-2). Libraries were generated with DNA SMART ChIP-Seq Kit (Takara, Cat# 634865). The libraries were selected into 250-350-bp fragments by E-Gel 2% SizeSelect electrophoresis (Invitrogen, Cat# G661012).
MBD2-Seq


title: guide-nostim-well1


title: guide-nostim-well3


title: OIL, non-methylated DNA


title: uLPS + MRS R14143 [104747-001-008]

city: Rijswijk

first: Raissa

last: Timmerman

address: Lange Kleiweg 161

department: Alternatives

country: Netherlands

institute: Biomedical Primate Research Centre

data_processing:
Clustering and sequencing: Illumina NextSeq 6000 was used
Reads were trimmed for adapter sequences using Trimmomatic v0.30
Presumed adapter sequences were removed from the read when the bases matched a sequence in the adapter sequence set (TruSeq adapters) with 2 or less mismatches and an alignment score of at least 12
The Macaca mulatta genomic reference (Macaca_mulatta.Mmul_10.dna.toplevel.fa) was used for alignment of the reads for each sample.
The reads were mapped to the reference sequence using a short read aligner based on Burrows-Wheeler Transform (Tophat v2.0.14) with default settings.
SAMtools v1.3 package was used to sort and index the BAM files. Based on the mapped locations in the alignment file the frequency of how often a read was mapped on a transcript was determined with HTSeq v0.6.1p1.
Genome_build: Macaca_mulatta.Mmul_10.dna.toplevel.fa
Supplementary_files_format_and_content: .count files containing raw gene counts for each sample
Supplementary_files_format_and_content: .tsv files containing FPKM values for each sample

organism: Macaca mulatta

source_name: Primary cultured microglia

growth_protocol: Microglia were cultured in 1:1 DMEM/HAMF10 medium containing 10% v/v heat inactivated FBS, 2 mM glutamax, 50 units/mL penicillin, 50 µg/mL streptomycin and 10 ng/mL M-CSF. Cells were kept in culture for 8 days without passaging. Half of the medium was replaced by fresh medium containing M-CSF every 3-4 days

extract_protocol:
Total cellular RNA was isolated using the RNeasy minikit (Qiagen, Cat#74104) according to manufacturer’s protocol
the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Cat#E7420L) was used to prepare and process the samples

treatment_protocol: At day 7l,microglia were exposed to 5 µM P2RY6 antagonist (MRS2578) 1hr prior to exposure to uLPS (10 µg/mL), or exposed to uLPS alone. 16 hr after uLPS exposure, mRNA was extracted

value: Subcortical white matter

value: Microglia

tag: donor nr

value: 5 µM MRS2578 + 10 µg/mL uLPS


title: H1JA_E4 cNES, biological replicate 1


title: uLPS R14143 [104747-001-007]

value: 10 µg/mL uLPS


title: C9orf78 rep2

first: IBRAHIM

middle: AVSAR

last: ILIK

state: Berlin

address: Ihnestraße 63-73

institute: Max-Planck-Institut für molekulare Genetik

data_processing:
Basecalling carried out by NovaSeq 6000: 3.4.4 and bcl2fastq v2.20.0.422
Paired-end reads were merged, trimmed and filtered by size using bbmerge and bbduk, merged reads longer and 34 base-pairs were retained for analysis
umi_tools was used to separate UMI from reads with the following command: umi_tools extract --bc-pattern=NNNXXXXXXNNNNN -I {Input.fastq.gz} --3prime
flexbar was used to split biological replicates from each other with the command: flexbar -r {reads.fastq.gz} -b  new_flash_barcodes.fa --barcode-trim-end --barcode-error-rate 0.2 --zip-output GZ
Internal replicates were merged (taR1, taR10, taR15, taR16 for rep1 of C9orf78 and GFP; taR2, taR4, taR7, taR9 for rep2 of C9orf78 and GFP, these files are provided)
Reads were mapped to the human genome (hg38) with bowtie2 and bbmap
umi_tools was used remove PCR duplicates: umi_tools dedup -I {in.bam} -S {out.bam} --spliced-is-unique --soft-clip-threshold 3
Genome_build: hg38
Supplementary_files_format_and_content: bigwig files were generated using bamCoverage from deepTools2

source_name: Flp-In™ T-REx™ 293 Cell Line

growth_protocol: Flp-In T-REx HEK293 expression cell lines are maintained in DMEM with Glutamax, high glucose and Na-pyruvate (Thermo Fisher #31966), that is supplemented with 10% FBS and penicillin-streptomycin, and kept under blasticidin (15µg/ml) and hygromycin (150µg/ml) selection.

extract_protocol:
Cells were solubilized with 1X PBS with additional 0.3M NaCl 1% Triton™ X-100 and 0.2% TWEEN® 20
Flp-In™ T-REx™ 293 cells expressing either 3xFLAG-BIO tagged C9ORF78 or 3xFLAG-BIO tagged GFP were induced with 0.1µg/mL doxycycline for ~16hrs and UV-crosslinked with 0.2mJ/cm2 UV-C light. Target proteins were purified using MyONE C1 streptavidin beads, after a partial RNase digestion using RNAse1 and end-repair with T4 PNK, uniquely barcoded s-oligos were ligated to each sample (2 biological replicates). After several stringent washes (up to 1% SDS), biological replicates are merged, and bead-bound RNA was released with proteinase K and purified with Oligo Clean and Concentrator columns. RNA was then reverse-transcribed and treated with RNaseH to phosphorylate 5’-ends of cDNA. The cDNA was then circularized with CircLigaseII, amplified with Q5 polymerase and sequenced on an Illumina platform (100bp, PE sequencing).
FLASH (Ilik et al., 2020)

treatment_protocol: Cells were UV-crosslinked with 200mJ/cm2 UV-C on 15 cm plates

tag: cell line

value: Flp-In T-REx 293 Human Embryonic Kidney Cell Line

value: 200mJ/cm2 UV-C

tag: expressing

value: 3xFLAG-BIO tagged C9ORF78


title: TMX, methylated DNA

first: Hiroshi

tag: age


title: H1JA_C4 cNES, biological replicate 2

tag: cell type


title: TMX, non-methylated DNA

tag: treatment


title: H1JA_D4 cNES, biological replicate 1


title: guide-nostim-well2

source_name: Primary bulk T cells


title: uLPS + MRS R02046 [104747-001-006]


title: GFP rep1

value: 3xFLAG-BIO tagged GFP


title: C9orf78 rep1


title: uLPS R02046 [104747-001-005]


title: ChrSeq_siLuc_R2

city: Leicester

first: Michael

last: Tellier

address: Lancaster Road

institute: University of Leicester

data_processing:
Adapters were trimmed with Cutadapt v1.13
Mapping to the hg38 genome was performed with STAR v2.6.1d.
Extraction of properly paired and properly mapped reads and Bam files sorting and indexing were done with SAMtools v1.3.1
Mapped reads were de-duplicated with Picard (ChIP-seq and DRIP-seq)
Bigwig files were generated with the Deeptools2 v2.5.0.1 bamCoverage tool
Genome_build: hg38
Supplementary_files_format_and_content: bigwig files: genome-coverage files for all samples

growth_protocol: HeLa cells were maintained in DMEM with 10% fetal bovine serum

extract_protocol:
RNA were purified from the chromatin fraction. DNA were purified from chromatin IP products.
For Chromatin RNA-seq, libraries were prepared using Illumina's TruSeq Stranded Total RNA library Preparation Kit according to Illumina's instructions. For ChIP-seq and DRIP-seq, libraries were prepared using Illumina's TruSeq ChIP library Preparation Kit according to Illumina's instructions.

treatment_protocol: siRNAs were transfected for 72 hours according to Lipofectamine 2000 reagent protocol.

value: HeLa

tag: antibody

tag: sirna

value: Scramble siRNA


title: uLPS + MRS R12016 [104747-001-004]


title: uLPS R12016 [104747-001-003]


title: WT_0min_afterInd_26C_rep2

tag: genotype

value: wild type


title: ChrSeq_siH2A_R2

value: RNaseH2A siRNA


title: ChrSeq_siLuc_R1


title: EV-T

city: Suita

first: Daisuke

last: Okuzaki

state: Osaka

address: Yamadaoka 3-1

department: Immunology Frontier Research Center

institute: Osaka univ.

description: total mOB-derived large extracellular vesicless without filtration

data_processing:
The sequencing was generated >10,000,000 raw reads from the sample.
CLC genomics workbench v9.5.3 (Qiagen) was used for miRNA analysis.
Adapters were  trimmed under default parameter setting to retain only reads with lengths 15-25 bp. 832,524 of  1,927,614 trimmed reads were then mapped and annotated against the miRBase (version 21).
Read counts of the annotated miRNA were exported from CLC genomics workbench.
Genome_build: mm10
Supplementary_files_format_and_content: Read count

source_name: total mOB-derived large EVs without filtration

extract_protocol:
RNAs in EVs from primary osteoblasts were isolated using a miRNeasy Mini Kit (Qiagen).
Small RNA libraries were constructed according to the manufacturer’s instructions using the NEBNext Small RNA Library Prep Set for Illumina (NEB).
Libraries were sequenced on the HiSeq 2500 platform (Illumina) in 75-base pair single-end reads.

value: mOB-derived large EVs


title: H1JA cNES, biological replicate 1

source_name: Cortically specified neuroepithelial stem cell population derived from H1 hESCs


title: H1JA_C4 cNES, biological replicate 1


title: H1JA_B4 cNES, biological replicate 2


title: H1JA_D2 cNES, biological replicate 1


title: H1JA_D2 cNES, biological replicate 2


title: H1JA cNES, biological replicate 2


title: 48hour R2

status: Public on Feb 03 2022

city: Belfast

first: Aditi

last: Singh

state: Please Select State

address: 97 Lisburn Road, Belfast

department: The Wellcome-Wolfson Institute for Experimental Medicine, SMDBS

institute: Queen's University Belfast

description:
ES ND1 48h +Dox H3K27ac R2
ChIP-seq in ES

data_processing:
ChIP-sequencing files in fastq format were subjected to quality control using FASTQC. Bowtie2 was used with local parameter for soft clipping of reads and align the reads to the mouse mm10 genome using UCSC.
Aligned sam files were sorted, indexed, and converted to the bam format using SAMTOOLS v0.1.19. Peak calling was performed using MACS2 and taking Input as a control.
Peaks were merged from all samples as well as replicates to retain all the regions of interest for differential occupancy.
qCount from QuasR was used to count reads from all samples in the merged peak regions.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: K27Reads_mergedPeaks.csv: Comma-separated text file includes read counts from all samples.

source_name: A2Lox cell line

growth_protocol: Murine ESCs were cultured at 37°C, 7% CO2 and 88% relative air humidity in 8 ml ES medium (DMEM supplemented with 10% fetal calf serum, 1X MEM NEAA, 2mM L-glutamine and LIF on feeders (inactivated MEFs)). For further experiments, feeders were removed by splitting mESCs every 2 days on tissue-culture dishes coated with 0.2% gelatin with daily media change. Experiments were performed at feeder-free five state.

extract_protocol:
Cells were cross-linked in medium containing 1% formaldehyde for 10 min at room temperature, neutralized with 0.125M glycine, scraped off and rinsed twice with cold 10 ml 1X PBS. Cells were pelleted by centrifugation for 7 min at 4°C at 600g. Pellets were resuspended in 10 mL of Buffer L1 (50mM Hepes KOH, pH 7.5, 140mM NaCl, 1mM EDTA pH 8.0, 10% glycerol, 5% NP-40, 0.25% Triton-X 100) and incubated at 4°C for 10 min. This was followed by centrifugation for 5 min at 4°C at 1300g. The pellet was again resuspended in 10 ml of Buffer L2 (200mM NaCl, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 10mM Tris pH 8.0) and incubated at room temperature for 10 min, followed by centrifugation for 5 min at 4°C at 1300g. The pellet was then resuspended in 900 µl Buffer L3 (1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 10mM Tris pH 8.0, 100mM NaCl, 0.1% Na-deoxycholate, 0.17mM N-Lauroyl sarcosine) containing protease inhibitors and incubated at 4°C for overnight following sonication using Bioruptor plus (Diagenode). After clearing cellular debris by spinning at 14,000 g for 10 min at 4°C DNA 60 µg of chromatin were incubated at 4 °C overnight with respective antibodies after 1h of preclearing. The mixture was then incubated with 40 µl protein A-Sepharose beads preblocked with tRNA and BSA for 3 h at 4°C. Beads were washed twice with 1 ml buffer L3 and once with 1 ml DOC buffer (10 mM Tris (pH 8.0), 0.25 M LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA), and bound chromatin was eluted in 1% SDS/0.1 M NaHCO3. This followed treatment with RNase A (0.2mg/ml) for 30 min at 37°C and then with Proteinase K (50 µg/ml) for 2.5 h at 55°C. The crosslinking was reversed at 65°C overnight with gentle shaking. DNA was purified by phenol-chloroform extraction followed by ethanol precipitation and recovered in 40 µl TE buffer.
ChIP-seq libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: Ectopic NeuroD1 expression was induced with Doxycycline (500 ng/ml final. conc.) for 48 hours.

value: ES cells

value: plusDox (NeuroD1 induction)

tag: time point

value: 48 hour

tag: chip antibody


title: 48hour R1

description:
ES ND1 48h +Dox H3K27ac R1
ChIP-seq in ES


title: WT_0min_afterInd_26C_rep3

value: YPIII


title: CA1J cNES, biological replicate 1

source_name: Cortically specified neuroepithelial stem cell population derived from CA1 hESCs

value: cNESCs


title: uLPS + MRS R07110 [104747-001-002]

value: Subcortical white matter

value: Microglia


title: H1JA_B4 cNES, biological replicate 1


title: ChrSeq_siH2A_R1

department: Department of Molecular and Cell Biology


title: uLPS R07110 [104747-001-001]

value: 10 µg/mL uLPS


title: CA1J cNES, biological replicate 2


title: 0hour R2

description:
ES ND1 0h -Dox H3K27ac R2
ChIP-seq in ES

value: minusDox (Uninduced)

value: 0 hour


title: Input

description: Input


title: 0hour R1

description:
ES ND1 0h -Dox H3K27ac R1
ChIP-seq in ES


title: Control, 293T

city: La Jolla

first: Max

last: Chang

address: 9500 Gilman Drive

institute: University of California, San Diego

data_processing:
Library strategy: CRISPR screen
extraction of sgRNA sequences with cutadapt
sgRNA counting and comparison with MAGeCK and the Brunello CRISPR library
Genome_build: hg38
Supplementary_files_format_and_content: gene-level summary

extract_protocol:
Genomic DNA (gDNA) of cell pellets was isolated using a Quick-DNA Miniprep Plus kit (Zymo Research).
PCR was performed on gDNA to construct Illumina sequencing libraries, each containing 5 μg gDNA, according to the Broad Institute protocol for PCR of sgRNAs for Illumina sequencing.

tag: rounds of infection


title: 12hour R2

description:
ES ND1 12h +Dox H3K27ac R2
ChIP-seq in ES

value: 12 hour


title: WT_0min_afterInd_26C_rep1

tag: strain


title: Survivor


title: Control