title: DMSO, E. coli, ex vivo

city: Groningen

first: Danny

last: Incarnato

state: Netherlands

address: Nijenborgh 7

department: Molecular Genetics

institute: University of Groningen

description:
E. coli native deproteinized total RNA, DMSO treated
Ecoli_exvivo_DMSO_raw.wig

data_processing:
Library strategy: SHAPE-MaP
Analysis of SHAPE-MaP data has been conducted using RNA Framework v2.6.9 (https://github.com/dincarnato/RNAFramework). Reads pre-processing and mapping was performed using the rf-map module (parameters: -ctn -cmn 0 -cqo -cq5 20 -b2 -mp "--very-sensitive-local"). Reads were trimmed of terminal Ns and low-quality bases (Phred < 20). Reads with internal Ns were discarded. Mapping was performed using the “very-sensitive-local” preset of Bowtie2. The mutational signal was then derived using the rf-count module (parameters: -m -rd), enabling the right re-alignment of deletions. When processing SARS-CoV-2 data, a mask file containing the sequences of primer pairing regions was passed along (through the -mf parameter). Generated RC files from both the even and odd sets, containing per base mutation counts and coverage, were then combined in a single RC file using the rf-rctools module (mode: merge). Data normalization was performed using the rf-norm module (parameters: -sm 3 -nm 3 -n 1000 -mm 1), by setting the minimum base coverage to 1,000X and the maximum mutation frequency to 1, and by using the Siegfried et al., 2014 scoring scheme and box-plot normalization of SHAPE reactivities.
Supplementary_files_format_and_content: Additional data files (RC/XML files for analysis with RNA Framework [http://www.rnaframework.com]) are available at: http://www.incarnatolab.com/datasets/SARS_Manfredonia_2020.php

organism: Escherichia coli

source_name: DMSO, E. coli, ex vivo

extract_protocol:
Samples were then vortexed briefly and 1 mL of ice-cold QIAzol Lysis Reagent was added to each sample, followed by extensive vortexing. 200 µl of chloroform were added, followed by vigorous vortexing for 15 sec. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12,500 x g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 mL tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase at 37°C for 20 min.
Total E. coli RNA was first fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer [65 mM Tris-HCl pH 8.0; 95 mM KCl; 4 mM MgCl2], then purified with NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 2 μl NF H2O. Eluted RNA was supplemented with 0.5 μl 20 μM random hexamers and 0.25 μl dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 5 μl. Reactions were supplemented with 1 μl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 0.5 μl DTT 0.1 M, 0.25 μl MnCl2 120 mM, 5 U SUPERase•In™ RNase Inhibitor and 50 U SuperScript II RT. Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 42°C. SSII was heat-inactivated by incubating at 75°C for 15 min. 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, cat. E6111L). Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina, following manufacturer instructions.

treatment_protocol: A single colony of Escherichia coli K-12 DH10B was picked and inoculated in LB medium without antibiotics, then grown to exponential phase (OD600 ~ 0.5). 2 mL aliquots were collected by centrifugation at 1000 x g (4°C) for 5 min. Cell pellets were resuspended in 1 mL of Resuspension Buffer [15 mM Tris-HCl pH 8.0; 450 mM Sucrose; 8 mM EDTA], and lysozyme was added to a final concentration of 100 µg/mL. After incubation at 22°C for 5 min and on ice for 10 min, protoplasts were collected by centrifugation at 5000 x g (4°C) for 5 min. Pellets were resuspended in 120 µl Protoplast Lysis Buffer [50 mM HEPES pH 8.0; 200 mM NaCl; 5 mM MgCl2; 1.5% SDS], supplemented with 0.2 µg/µl Proteinase K. Samples were incubated for 5 min at 22°C and for 5 min on ice. Sample was then extracted 2 times with phenol:chloroform:isoamyl alcohol (25:24:1, pre-equilibrated 3 times with RNA folding buffer [50 mM HEPES pH 8.0; 200 mM NaCl; 5 mM MgCl2]), and once with chloroform. 20 U SUPERase•In™ RNase Inhibitor were then added and RNA was equilibrated at 37°C for 20 min prior to probing. 180 µl of deproteinized E. coli rRNAs in RNA folding buffer were mixed with 20 µl of each SHAPE compound (assuming complete conversion, stocks should be ~1 M, resulting in a final concentration of 100 mM in the probing reaction). RNA was then allowed to react at 37°C for 15 minutes, with moderate shaking, after which 200 µl of 1 M DTT were added, to quench the reaction.

value: Escherichia coli K12 DH10B

value: deproteinized total RNA

value: DMSO


title: NAI, E. coli, ex vivo

status: Public on Oct 30 2020

type: SRA

country: Netherlands

description:
E. coli native deproteinized total RNA, NAI treated
Ecoli_exvivo_NAI_raw.wig
Ecoli_exvivo_NAI_vs_DMSO_raw.wig

library_source: transcriptomic

molecule: total RNA

source_name: NAI, E. coli, ex vivo

tag: strain

tag: sample type

tag: treatment

value: NAI


title: 408_WholeSpleen

city: Stanford

first: Tawaun

middle: Autry

last: Lucas

state: CA

address: 1201 Welch Rd

country: USA

institute: Stanford University

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using trim galore
Trimmed reads were mappped to the mm10 genome using STAR --outSAMunmapped Within  --outFilterType BySJout --outSAMattributes NH HI AS NM MD --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1
Aligned reads were quantified using RSEM
Counts were modified using cellular deconvoultion and re-adjusted to reflect purity
Differential expression analysis conducted using DESeq
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: excel file of differential expression for genes that include our expression cutoffs include FPKM values for each Sample ...

organism: Mus musculus

source_name: Whole Spleen

extract_protocol:
Spleens were dissected, minced and resuspended in 4ml of dissociation buffer and incubated with constant agitation or 1 h. After the hour, Dispase II (2mg/ml) was added and the samples were incubated for an additional 30 min. Upon completion, 5 mM EDTA was added to the sample and incubated at room temperature for 5 min and subsequently centrifuged. Red blood cells were lysed and samples were diluted with 13 ml of cold PBS + FBS before another centrifugation at . The cell pellets were resuspended in of PBS +  FBS and passed through a cell strainer. After washing, the remaining cells on the filter (size-selected), were lysed using RLT buffer.
Libraries were prepared using standard Illumina protocols

tag: tissue

value: Spleen

tag: Sex

value: Male


title: 408_SizeSelected

source_name: Spleen Glia


title: RNA_C2_dKO_REP3

city: Berlin

first: Achim

last: Leutz

address: Robert-Rössle-Str. 10

department: Cancer Research

country: Germany

institute: MDC Berlin

data_processing:
RNA read alignment: STAR (version 2.5.3a)
RNA read quantification: HTSeq (version 0.10.0)
RNA differential gene expression: DESeq2 (version 1.26.0)
Genome_build: mm10
Supplementary_files_format_and_content: RNA: normalized read count, csv

extract_protocol:
For each cell line, three independent RNA samples were isolated at different time points.
The total RNA was isolated from cells using the RNeasy Kit (Qiagen). The Qiagen RLT lysis buffer was additionally supplied with β-mercapthethanol (10μl per 1ml buffer). The cell lysates were thoroughly homogenized by passing through syringe with 20-G needle. The concentration and purity of the extracted RNA was measured on Qubit 3 Fluorometer (Thermo Fisher Scientific). For each cell line, three independent RNA samples were isolated at different time points. The further analyze of RNA quality and integrity was assessed on Bioanalyzer 2100 (Agilent Technologies), using the Eukaryote Total RNA Nano assay. The subsequent libraries preparation and Next Generation Sequencing (NGS) on the NextSeq500 platform (Illumina) was performed in collaboration with Laboratory of Functional Genomics, Nutrigenomics and Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin (Dr. Tatiana A. Borodina)

treatment_protocol: MLL-ENL transformed cell lines were maintained in IMDM with 10% FCS, supplied with murine IL-3 (10 ng/ml). Cultures were passaged three times a week or when the cell number exceeded 1.5x10^6 per 1 ml.

tag: cell type

value: MLL-ENL transformed fetal liver cells

tag: genotype

value: Cebpa KO, Cebpb KO

tag: exogenous expression

value: GFP


title: RNA_C2_dKO_REP2


title: 407_WholeSpleen


title: 407_SizeSelected


title: Mouse cerebellar nuclei

first: Justus

middle: M

last: Kebschull

address: 327 Campus Drive

description: Mouse cerebellar nuclei

data_processing:
Basecalling through Illumina pipeline
STAR alignment to (I) mouse genome GRCm38, Ensembl 92 annotation, (ii) human genome GRCh38, annotation Ensemble 94, (iii) chicken genome GRCg6a Ensembl annotation 99
Count reads in exons and introns using Htseq
Produce count matrix
Analyse in Seurat
Genome_build: GRCm38, GRCh28, GRCg6a
Supplementary_files_format_and_content: seurat v3 object containing count matrix, dimensionality reduction, cluster assignment, metadata

source_name: Cerbellar nulcei

extract_protocol:
cerbellar nulcei were dissected from fresh frozen or accute sliced brains, flash frozen. Nuclei were released using standard protocols, stained for NeuN and FACS sorted into 384 well plates
smartseq2

value: Cerbellar nulcei


title: D425 WT rep2

city: St. Petersburg

first: Bongyong

last: Lee

state: Florida

address: 501 6th Ave S

institute: The Johns Hopkins University

description:
Sample 2
D425_HLX2_7_vs_D425.txt
D425_rep2

data_processing:
Adapter sequences were trimed from reads via Trimmomatic (v0.38), default parameters were used.
Reads were mapped to hg38 via STAR (2.7.0d), default parameters were used.
DESeq2 (v1.22.2) was used to compute the differential expression level between samples, default parameters were used.
samples 5, 6  were compared with sample 3, 4.
sample 7, 8 were compared with samples 5, 6.
sample 7, 8 were compared with sample 5, 6.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include reads

organism: Homo sapiens

source_name: Medulloblastoma cell line

growth_protocol: D425 cells were cultured in DMEM/F12 with 10% serum and 1% glutamate/penicillin/streptomycin. Intracranial xenografts were established by injecting D425 cells and D425 lnc-HLX-2-7 KD cells into the cerebellum of NOD-SCID mice (Jackson Laboratory). Cerebellar coordinates were -2 mm from lambda, +1 mm laterally, and 1.5 mm deep. After 30 days of injecting D425 cells and D425 lnc-HLX-2-7 KD cells into the cerebellum of NOD-SCID mice, tumors were harvested.

extract_protocol:
Total RNA was prepared from cell line and orthotopic xenograft using a Direct-zol RNA Miniprep kit (Zymo Research). The quality of RNA was determined with the Agilent 2100 Bioanalyzer Nano Assay (Agilent Technologies).
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Human medulloblastoma cell line

value: WT


title: RNA_C2_dKO_REP1


title: RNA_C2_WT_REP2

value: wild-type


title: Nalm6-DMSO scRNA-seq

city: Kuopio

first: Merja

last: Heinäniemi

address: Yliopistonranta 1 E

department: School of Medicine

country: Finland

institute: University of Eastern Finland

data_processing:
Data was aligned with Cell Ranger 3.0.2 to human+mouse combined reference (hg19 & mm10) version 3.0.0 from 10x Genomics with default parameters.
Cells were filtered with Cell Ranger 3.0.2 following alignment.
The mouse cells were left out by filtering cells which cellranger characterized as mouse or doublet cells or had more than 500 counts assigned to mouse genes.
The mouse genes were filtered out from the count matrix.
Genome_build: hg19
Supplementary_files_format_and_content: barcode annotation: filtered barcode annotation; feature annotation: filtered feature list; count matrix: filtered UMI count matrix

molecule: polyA RNA

source_name: scRNAseq in control cell line

growth_protocol: Nalm-6 (ACC-128, DSMZ, Germany) cells, modified to carry inducible Tet-On construct or empty vector, were cultured at 1 million/ml in RPMI 1640 (Gibco, Thermo Fisher) supplemented with 2 mM  L-glutamine (Gibco, Thermo Fisher), penicillin (100 U/ml), and 10% Tet-system approved FBS (Clontech, Takara Bio).

extract_protocol:
scRNAseq: Nalm-6-RUNX1 was induced for 72 h using 500 ng/ml doxycycline (Sigma-Aldrich) before scRNA-seq cell collection. Excess dead cells were depleted from samples using bead-based Dead Cell Removal Kit (#130-090-101, MACS miltenyi Biotech)
scRNAseq: Cells were combined with reverse transcriptase Master Mix and partitioned into Gel Bead-In EMulsions (GEMs) using 10X GemCode Technology, where the poly-A transcripts are barcoded with an Illumina R1 sequence, a 16 bp 10X barcode and a 10 bp Unique Molecular Identifier (UMI). 11-12 cycles of PCR was used to amplify the cDNA.

treatment_protocol: To generate stable Nalm6-RUNX1 and Nalm6-ev (empty vector) cell lines, RUNX1 cDNA (NM_001754, originally from Origene Technologies Inc, MD, USA, cat #RG223809) was cloned into  pLVX-Tight-Puro vector (Clontech) using NotI and EcoRI restriction sites. Nalm6-cells were transduced with the regulatory vector TetOn and subsequently with pLVX response vectors (either without an insert or with RUNX1). The expression of RUNX1 in Nalm6-cells was induced with 500ng/mL doxycycline (Clontech) and confirmed by RT-qPCR using the following primers that recognize both endogenous and induced RUNX1: forward TTCCTTCGTACCCACAGTGC, reverse TGGCATCGTGGACGTCTCTA. The expression level of the RUNX1 induction was 3.3 times the level of a Nalm6-ev as measured by quantitative reverse-transcriptase PCR (LightCycler® 480 SYBR Green I Master, LightCycler® 480 apparatus, Roche Diagnostic) following ddCt calculations.

tag: cell line

value: pre-B-ALL cell line

tag: genotype/variation

value: empty vector

tag: time


title: D425 WT rep1

description:
Sample 1
D425_HLX2_7_vs_D425.txt
D425_rep1


title: Nalm6-lvRUNX1 scRNA-seq

source_name: scRNAseq in RUNX1 overexpressing cell line

value: induced RUNX1 expression


title: Lepr_pos_MPP_5

city: Indianapolis

first: James

last: Ropa

state: IN

address: 950 W Walnut St

institute: Indiana University

description: Ribosomal RNA depleted total RNA

data_processing:
Quality of sequences were confirmed using FastQC v0.11.8
Reads were trimmed using Cutadapt v2.1 with the following options: -b GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT -B GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT -b CACACACACACACACACACACACACACACACACACACACA -B CACACACACACACACACACACACACACACACACACACACA -b CTGTCTCTTATACACATCT -B CTGTCTCTTATACACATCT -a "A{10}" -a "T{10}" -A "A{10}" -A "T{10}" --nextseq-trim=20 --minimum-length 50
Indices for alignment were generated using the full sequence and annotation files from GENCODE M22 release of the GRCm38 mouse genome primary assembly with STAR v2.7.1a using the following options: --sjdbOverhang 99
Reads were aligned to the genome using STAR v2.7.1a with the following options: --outSAMtype BAM SortedByCoordinate --outSAMunmapped Within
Aligned reads were counted by gene (features = exons) using the htseq-count function from HTSeq v0.11.2, which only keeps uniquely aligned reads by default, using the following options: -s no -r pos -f bam
The R package "DESeq2" was used to determine gene level differences between Lepr+ and Lepr- cells. HSCs and MPPs were analyzed separately, so the comparisons were Lepr+ HSCs vs Lepr- HSCs and Lepr+ MPPs vs Lepr- MPPs. The design for the model built in DESeq2 built in batch effects for the isolated Lepr+ and Lepr- cells, so that samples coming from the same pool of cells are paired samples.
Genome_build: GRCm38
Supplementary_files_format_and_content: count files containing HTSeq raw counts assigned to genes

source_name: bone marrow

extract_protocol:
Bone marrow was harvested from two 7-9 week old mice per sample by crushing femurs,tibiae,hips, and spines; cells were sorted for hematopoietic stem cells or hematopoietic multipotent progenitor cells and were sorted based on Leptin Receptor cell surface expression. 1,000-8,000 total cells were obtained per sample. RNA was extracted using Qiagen Rneasy Micro Kit.
Takara SmartSeq v4 Ultra Low Input RNA Kit for cDNA synthesis and Nextera XT for library preparation and amplification.

value: Hematopoietic multipotent progenitor cells

tag: cell surface

value: Leptin Receptor positive


title: RNA_C1_WT_REP3


title: Lepr_neg_MPP_5

description: Ribosomal RNA depleted total RNA

value: Leptin Receptor negative


title: CF146_day40_mt

city: San Diego

first: Cynthia

last: Silveira

state: CALIFORNIA

address: 5500 Campanile Dr, North Life Sciences 302

department: Biology

institute: San Diego State University

data_processing:
Quality filtering using PRINSEQ ++
Cloning vector sequences were removed using SMALT with 80 % identity against the NCBI UniVec database.
Human genome sequences were removed using SMALT with 80 % identity against the human reference genome GRCh38
The transcripts were mapped to the NCBI RefSeq database of complete bacterial genomes using SMALT at 96 % identity.

source_name: Cystic fibrosis sputum

extract_protocol:
Total RNA extraction
Ovation¨ Complete Prokaryotic RNA-Seq Library

tag: exacerbation day


title: RNA_C1_WT_REP2


title: d28 1.2 Tcf7 -

city: Lausanne

first: Tania

last: Wyss

address: Rue du Bugnon 25A

country: Switzerland

institute: SIB Swiss Institute of Bioinformatics

description:
20
processed data file: d28_Tcf_neg_bampe_peaks.txt

library_source: genomic

data_processing:
The bcbio-nextgen pipeline (v. 19.03, https://github.com/bcbio/bcbio-nextgen) was used for initial computations of the analysis.
Reads were filtered for quality and aligned to the Mus musculus reference genome (mm10) using BWA (version 0.7.17-r1188).
The three alignment files of each sample were merged using samtools (version 1.9).
Peak calling was performed globally for each cell population using Macs2 (version 2.1.1) with the --broad argument.
Genome_build: Mus musculus mm10
Supplementary_files_format_and_content: *_bampe_peaks.txt: Tab-delimited text files containing the output of Macs2 --broad peaks. One file per cell population.

molecule: genomic DNA

source_name: Tcf7GFP- P14 cells at d28 of LCMV Arm infection

extract_protocol:
Cells were lysed in lysis buffer (NP-40)(0.1%) and crude nuclei were tagmented (TDE enzyme and buffer)(Illumina) and DNA was purified using minielute PCR Purification kit (Qiagen) according to Buenrostro et al., Curr Protoc Mol Biol (2015).
Library preparation was done using Illumina’s Unique Dual (UD) Indexes (R#20027213), and 10 PCR cycles according to Buenrostro et al., Curr Protoc Mol Biol (2015). Libraries were cleaned using Agencourt AMPure XP magnetic beads (A63880, Beckman) and sequenced on an Illumina HiSeq 4000, with paired end 150 nucleotides.

treatment_protocol: Splenocytes were enriched for CD8 T-cells using the EasySep Mouse CD8+ T Cell Isolation Kit (StemCell Technologies). Tcf7GFPhi and Tcf7GFP- P14 cells were flow sorted as CD45.2+ CD45.1- from the spleen at d8 or day28 post infection. In addition P14 cells were flow sorted from the spleen of naive mice as CD8+ CD462L+ cells (Naive).

value: CD8 T cells expressing a P14 TCR and a Tcf7GFP reporter

value: LCMV Arm infection

tag: time point

value: day 28 post-infection


title: Lepr_pos_HSC_5

description: Ribosomal RNA depleted total RNA

value: Hematopoietic stem cells


title: Lepr_neg_HSC_5

description: Ribosomal RNA depleted total RNA


title: RNA_C1_WT_REP1

value: MLL-ENL transformed fetal liver cells


title: CF146_day35_mt


title: CF146_day33_mt


title: Lepr_pos_MPP_4

description: Ribosomal RNA depleted total RNA


title: RNA_C1_LAPS_REP3

value: Cebpa KO, Cebpb KO

value: GFP, rat CEBP/b isoform LAP*


title: RNA_C1_LAPS_REP2


title: Lepr_pos_MPP_3

description: Ribosomal RNA depleted total RNA


title: d8 2.2 Tcf7 -

description:
18
processed data file: d8_Tcf_neg_bampe_peaks.txt

source_name: Tcf7GFP- P14 cells at d8 of LCMV Arm infection

value: day 8 post-infection


title: RNA_C1_LAPS_REP1


title: CF146_day30_mt


title: Lepr_neg_MPP_3

description: Ribosomal RNA depleted total RNA


title: Lepr_neg_MPP_4

description: Ribosomal RNA depleted total RNA


title: RNA_C1_dKO_REP3

value: GFP


title: d8 1.2 Tcf7 -

description:
13
processed data file: d8_Tcf_neg_bampe_peaks.txt


title: d8 1.2 Tcf7 hi

description:
12
processed data file: d8_Tcf_high_bampe_peaks.txt

source_name: Tcf7GFPhi P14 cells at d8 of LCMV Arm infection


title: RNA_C1_dKO_REP2


title: Lepr_pos_HSC_4

description: Ribosomal RNA depleted total RNA


title: CF146_day21_mt


title: CF146_day37_mt


title: d28 1.2 Tcf7 hi

description:
19
processed data file: d28_Tcf_high_bampe_peaks.txt

source_name: Tcf7GFPhi P14 cells at d28 of LCMV Arm infection


title: Lepr_pos_HSC_3

description: Ribosomal RNA depleted total RNA


title: RNA_C1_dKO_REP1


title: d28 2.1 Tcf7 -

description:
10
processed data file: d28_Tcf_neg_bampe_peaks.txt


title: CF146_day16_mt


title: d8 2.2 Tcf7 hi

description:
17
processed data file: d8_Tcf_high_bampe_peaks.txt


title: d28 2.1 Tcf7 hi

description:
09
processed data file: d28_Tcf_high_bampe_peaks.txt


title: 9-2607-C

city: New York

first: Bin

last: Zhang

state: NY

address: 1470 Madison Avenue

department: Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

description: processed data file: expression.log2cpm.tsv

data_processing:
Paired-end sequencing reads (100bp) were generated on a NovaSeq platform. Raw reads were aligned to hg19 using STAR aligner (v2.5.2a).
Following read alignment, featureCounts (1.6.3) was used to quantify the gene expression at the gene level based on Ensembl gene model GRCh37.70.
Genes with at least 1 count per million (CPM) in at least 1 sample were considered expressed and hence retained for further analysis, otherwise removed.
After filtering, the raw read counts were normalized by the voom function in limma.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: expression.log2cpm.tsv: Tab-separated text file.

source_name: National Institute of Mental Health (NIMH) childhood-onset schizophrenia (COS) cohort

growth_protocol: NPCs were seeded as 4-6x10e5 cells/well in a 24-well plate coated with Matrigel (coverslips are put in a plate and coated with Matrigel for immunostaining). At day -1, cells were transduced with rtTA, pLV-TetO-hNGN2-eGFP-Neo and ATP6V1Ai gRNA or empty lentiguide-Hygro-mTagBFP2 (Addgene 99374) lentiviruses via spinfection. Medium was switched to non-viral medium three hours post-spinfection. At Day 0, 1 μg/ml dox was added to induce NGN2-expression. At Day 1, transduced hiPSC-NPCs were treated with corresponding antibiotics to the lentiviruses (300 ng/ml puromycin for dCas9-effectors-Puro, 1 mg/ml G-418 for hNGN2-eGFP-neo and 1 mg/ml HygroB for lentiguide-Hygro-mTagBFP2) in order to increase the purity of transduced NPCs. At day 3, NPC medium was switched to neuronal medium (Brainphys (Stemcell Technologies, #05790), 1x N2 (Life Technologies #17502-048), 1x B27-RA (Life Technologies #12587-010), 1 μg/ml Natural Mouse Laminin (Life Technologies), 20 ng/ml BDNF (Peprotech #450-02), 20 ng/ml GDNF (Peptrotech #450-10), 500 μg/ml Dibutyryl cyclic-AMP (Sigma #D0627), 200 nM L-ascorbic acid (Sigma #A0278)) including 1 μg/ml Dox, along with antibiotic withdrawal. 50% of the medium was replaced with fresh neuronal medium (lacking dox once every second day. At day 11, full medium change withdrew residual dox completely. At day 13, NGN2-neurons were treated with 200 nM Ara-C to reduce the proliferation of non-neuronal cells in the culture, followed by half medium change by day 17. At Day 17, Ara-C was completely withdrawn by full medium change, followed by half medium changes until the neurons were harvested around day 21-24.

extract_protocol:
Add 0.25 mL of TRIzol™ Reagent (Thermo Fisher Scientific, Cat#15596026) per 24-well well directly to the culture dish to lyse the cells. Transfer the lysate to 1.5 mL RNase-free eppendorf tube. Add 50 µL of chloroform in the hood, and then securely cap the tube. Invert 2-3 times and vortex 5-10 sec. Hold on ice for 10 min, inverting every 3 min. Centrifuge the sample for 15 minutes at 12,000× g at 4°C. Transfer clear supernatant to fresh RNase-free tubes with 150 µL of Isopropanol. Centrifuge the sample for 10 minutes at 12,000× g at 4°C. Discard the supernatant with a micropipettor. The pellet is washed in 0.5 mL of ice cold 70% ethanol from -20°C. Resuspend the pellet in 50 μL of RNase-free water. Store the RNA at -70°C.
RNA sequencing libraries were prepared using the Kapa Total RNA library prep kit.
NovaSeq, 100nt, Paired-End Read

treatment_protocol: Human β-amyloid (1-42) peptide was purchased from GenScript (#RP10017). 500 µM β-amyloid was prepared in the vehicle solution of Tris-HCl, pH 7.5 and stored at –20°C. 21-day isogenic pairs of ATP6V1A-manipulated NGN2-neurons were exposed to 5 µM β-amyloid for 24 hours, and then collected for RNA sequencing.

value: hiPSC-derived NGN2-neurons

value: Wild type


title: S4_bgluc_neu

city: Dresden

first: Triantafyllos

last: Chavakis

address: Fiedlerstraße 42

department: Klinische Pathobiochemie

institute: Medizinische Fakultät der TU Dresden

data_processing:
bcl2fastq2 2.19.1
processing of sequencing data with cellranger-atac (1.2.0) (cellranger-atac count –id= ${id} --reference= refdata-cellranger-atac-mm10-1.2.0  --fastqs=${fastqinput} –sample=${sample})
Genome_build: mm10
Supplementary_files_format_and_content: fastq files having R1 and R3 in the filename are standard fastq files which contain the genomic read data
Supplementary_files_format_and_content: fastq files having R2 in the filename are standard fastq files which contain the cell barcode information
Supplementary_files_format_and_content: Files ending on peak.zip contain the folder of the filtered peak-barcode output. It contains of three files. peaks.bed (For each peak, its chromosome, start and end positions are stored in); barcodes.tsv (Each barcode sequence includes a suffix with a dash separator followed by a number), matrix.mtx (matrix market exchange format, with the rows being cell barcodes). For more information, please read https://support.10xgenomics.com/single-cell-atac/software/pipelines/latest/output/matrices .
Supplementary_files_format_and_content: Files ending on tf.zip contain the folder of the filtered tf-barcode output. It contains of three files. motifs.tsv (For each transcription factor, its fully specified name in the reference and its common name are stored as first and second column); barcodes.tsv (Each barcode sequence includes a suffix with a dash separator followed by a number), matrix.mtx (matrix market exchange format, with the rows being cell barcodes). For more information, please read https://support.10xgenomics.com/single-cell-atac/software/pipelines/latest/output/matrices .

source_name: Neutrophils

extract_protocol:
Bone marrow and spleen were isolated and single cell suspensions were prepared. BM GMP and splenic neutrophils were FACS-sorted and nuclei were isolated using as described in Methods.
Nuclei isolation for single-cell ATAC sequencing was performed according to the protocol of 10xGenomics. Briefly, cells were sorted into V bottom plate, centrifuged at 300 rcf for 5 min at 4°C. Chilled Lysis buffer (2.5mM Tris-HCl (pH 7.4), 2.5mM NaCl, 0.9mM MgCl2, Tween-20 0.025%, Nonidet P40 Substrate (Sigma Cat#74385) 0.025%, Digitonin 0.0025%, BSA 0.25%, nuclease free water) was added to the pellet and cells were incubated on ice for 2 min.  Cells were washed once with Wash buffer (2.5mM Tris-HCl (pH 7.4), 2.5mM NaCl, 0.9mM MgCl2, Tween-20 0.025%), concentrated by centrifugation at 500 rcf for 5 min at 4°C;  after removing the supernatant, 7 µl 1x Nuclei Buffer (10x Genomics Cat#2000153) was added. One µl of the nuclei suspension was taken for evaluation (nuclei quality control and determining nuclei concentration). Nuclei suspension was then used according to the Chromium Single Cell ATAC Reagent Kits protocol (10xGenomics Cat# CG000168). Five µl of the nuclei suspension was mixed with the tagmentation mix and incubated for one hour at 37°C. After finishing the tagmentation, the still intact nuclei were mixed with a barcoding mix and loaded into a 10x chip H (Chromium Next GEM Chip H Single Cell Kit v1.1; PN-1000162, Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1; PN-1000176) together with barcoded beads and partitioning oil and encapsulated using the Chromium controller. The gel emulsion was transferred into a PCR tube to perform 12 cycles of amplification in a thermocycler. The gel emulsion containing barcoded DNA was broken, purified (Dynabeads™ MyOne™ SILANE; PN-2000048) and subjected to a final index PCR for 11 cycles (10xGenomics Cat# CG000168). After size selection (0.4x/1.2x volume of beads) (Beckman Coulter SPRIselect; B23318), the library was examined on a fragment analyzer (NGS High Sensitivity Fragment Analysis Kit; DNF-474) for its quality and quantity and sequenced on a Illumina NextSeq 500 (Illumina NextSeq 500/550 High Output Kit v2.5;150 Cycles; 20024907) in 72 PE mode (R1 72, R2 72, I1 8, I2 16) at an average depth of 5400 fragments/cell. More information on the library prep can be found at the site https://www.10xgenomics.com/resources/support-documentation/.

value: beta-glucan

tag: gender

value: male

tag: age

value: 8-10 weeks old


title: 8-2607-C


title: CF146_day2_mt


title: d28 1.1 Tcf7 -

description:
05
processed data file: d28_Tcf_neg_bampe_peaks.txt


title: Lepr_pos_MPP_2

description: Ribosomal RNA depleted total RNA

value: Hematopoietic multipotent progenitor cells


title: CF146_day28_mt


title: S2_bgluc_GMP

source_name: Bone marrow GMP


title: S3_PBS_neu

value: PBS control


title: Lepr_neg_HSC_3

description: Ribosomal RNA depleted total RNA

value: Leptin Receptor negative


title: S1_PBS_GMP


title: d8 1.1 Tcf7 -

description:
03
processed data file: d8_Tcf_neg_bampe_peaks.txt

value: CD8 T cells expressing a P14 TCR and a Tcf7GFP reporter


title: Naïve 2

description:
11
processed data file: naive_bampe_peaks.txt

source_name: Naïve P14 cells

value: CD8 T cells expressing a P14 TCR

value: Naive


title: 5-2607-C


title: d8 1.1 Tcf7 hi

description:
02
processed data file: d8_Tcf_high_bampe_peaks.txt

value: LCMV Arm infection


title: Bgl-B-T

data_processing:
bcl2fastq 2.19.1
alignment was done with GSNAP (2018-05-30) to the mm10 reference with support of Ensembl annotation version 87 (-d mm10 --gunzip -A sam -t 14 --use-sarray=1 --input-buffer-size=500000 --output-buffer-size=500000 -B 5 -N 0 -n 1 -s EnsemblGene-87.ss.mm10.iit)
alignment files were processed with featureCounts v1.6.2 and the support of annotation from Ensembl version 87 (featureCounts -a EnsemblGene-87.mm10.TR.gtf -s 2 -Q 1 -T 8)
Genome_build: mm10
Supplementary_files_format_and_content: lab1627.gene_count.csv.gz is a tab separated files which list the genes and their characteristics, followed by the counts of the samples

extract_protocol:
Bone marrow was isolated and single cell suspensions were prepared. GMP were FACS-sorted (Lin—c-Kit+Sca1—CD16/32+CD34+) and RNA was isolated using RNeasy Plus Microkit (Qiagen).
mRNA was isolated from 50 ng total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer’s instructions. Final elution was done in 15ul 2x first strand cDNA synthesis buffer (NEBnext, NEB). Samples were then directly subjected to the workflow for strand specific RNA-Seq library preparation (Ultra Directional RNA Library Prep II, NEB). For ligation custom adaptors were used 1: (Adaptor-Oligo 5'-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3', Adaptor-Oligo 2: 5'-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3'). After ligaton adapters were depleted by an XP bead purification (Beckman Coulter) adding the beads solution in a ratio of 1:1. Indexing was done during the following PCR enrichment (14 cycles, 65 °C) using custom amplification primers carring the i7 index sequence indicated with ‘NNNNNNNN’. (Primer1: AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T, primer2: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T. After two more XP beads purifications (1:1) libraries were quantified using Qubit dsDNA HS Assay Kit (Invitrogen).

value: WT


title: 28-2607-i1-Ab

value: ATP6V1A knock down

value: amyloid-beta


title: Lepr_neg_MPP_1

description: Ribosomal RNA depleted total RNA


title: Naïve 1

description:
01
processed data file: naive_bampe_peaks.txt


title: 24-2607-i1-Ab


title: Lepr_neg_HSC_4

description: Ribosomal RNA depleted total RNA

value: Hematopoietic stem cells


title: Bgl-A-T


title: 23-2607-i1-Ab


title: Lepr_pos_HSC_1

description: Ribosomal RNA depleted total RNA

value: Leptin Receptor positive


title: PBS-D-T


title: 30-2607-i1-Ab

value: hiPSC-derived NGN2-neurons


title: PBS-C-T


title: d28 1.1 Tcf7 hi

description:
04
processed data file: d28_Tcf_high_bampe_peaks.txt

value: day 28 post-infection


title: 22-2607-i1-Ab


title: 20065FL-01-02-16

city: Balice

first: Kacper

last: Zukowski

address: Krakowska 1 Street

country: Poland

institute: National Research Institute of Animal Production

data_processing:
The raw sequences were quality controlled using FastQC.
Flexbar software was used to remove Illumina adapters and poly-A sequences.
The raw reads were aligned to the Sus scrofa genome (Sscrofa11.1) as a reference, while the whole procedure was followed by alignment parameters from ENCODE3's STAR-RSEM pipeline
The quality control metrics across aligned reads per sample were obtained using RNA-SeQC software
The differential expression estimation was proceed with utilization of DeSeq2 Bioconductor R-project package.
Genome_build: Sscrofa11.1
Supplementary_files_format_and_content: genes_fat.csv.gz
Supplementary_files_format_and_content: genes_liver.csv.gz

organism: Sus scrofa

source_name: fat

growth_protocol: All pigs were female and they were maintained at Pig Test Station under the same feeding and housing conditions and were slaughtered when they reached 100±2 kg.

extract_protocol:
RNA was isolated by RNA was isolated by the PureLink™ RNA Mini Kit (Ambion, Cambridge, UK) according to the manufacturer’s recommendations. The RIN values were over 7 what was estimated by TapeStation2200 and RNA tapes (Agilent, Palo Alto, USA).
The cDNA libraries were prepared by TruSeq RNA Sample Preparation Kit v2 (Illumina) according to the supplied protocol.

tag: breed

value: Zlotnicka White

tag: day

value: female

tag: shear force

value: high fat content

tag: replicant


title: 20-2607-C-Ab

value: Wild type

tag: treatment


title: 2-2607-C


title: Lepr_neg_MPP_2

description: Ribosomal RNA depleted total RNA

value: Hematopoietic multipotent progenitor cells

tag: strain


title: PBS-B-T

value: PBS control

tag: Sex

value: male

value: 8-10 weeks old


title: Lepr_pos_HSC_2

description: Ribosomal RNA depleted total RNA

tag: cell type


title: 20065FL-01-02-13

value: low fat content


title: PBS-A-T

tag: genotype


title: ALL12-d15

data_processing:
Data was aligned with Cell Ranger 3.0.2 to human reference (hg19) version 3.0.0 from 10x Genomics with default parameters.
Cells were filtered with Cell Ranger 3.0.2 following alignment.
Genome_build: hg19
Supplementary_files_format_and_content: barcode annotation: 10x Cell Ranger filtered barcode list; feature annotation: 10x Cell Ranger feature list; count matrix: 10x Cell Ranger filtered UMI count matrix

source_name: Primary pre-B-ALL BM

extract_protocol:
Mononuclear cells (MNC) were extracted from fresh BM at diagnosis using Ficoll-Paque Plus (GE Healthcare, #17-1440-02). Bone marrow MNCs were also extracted from two patients (ALL10 and ALL12) during the induction therapy at day 15 after initiation of therapy. MNCs were viably frozen in 15% DMSO/40% FBS in RPMI in liquid nitrogen. Cells from primary BM samples were processed for scRNA-seq in the Finnish Functional Genomics Center, Turku, Finland, in 4 batches: 1) ALL3, 2) ALL1 3) ALL10 and ALL10-d15, and 4) ALL8, ALL9, ALL12, and ALL12-d15. Before applying the cells into the Chromium cartridge, their viability was checked using Trypan blue. PI-negative (live) cells were selected from sample ALL3 using FACS. Samples ALL1, ALL10, and ALL10-d15 were processed directly after thawing the MNC fraction without further processing. Excess dead cells were depleted from samples ALL8 and ALL9 using bead-based Dead Cell Removal Kit (#130-090-101, MACS miltenyi Biotech), increasing the percentage of viable cells from 43 % to 72 % and from 63 % to 78 %, respectively. For samples ALL12 and ALL12-d15, enrichment of leukemic cells was carried out by depleting non-B-cells using streptavidin-beads (BD Streptavidin Particles Plus, BD Biosciences, Franklin Lakes, NJ, USA) and biotinylated antibodies against human CD16 (clone 3G8), CD14 (HCD-14), CD11c (3.9), CD56 (HCD56), CD3 (UCHT1), and CD66 (G10F5) (Biolegend), all with final concentrations of 2 ug/ml, following the manufacturer's instructions and as previously described (Good et al. 2018 Nat Meth). Depletion efficiency was estimated by flow cytometry using CD3 (BV421, BD Biosciences, #56287, RRID:AB_27378607) and CD19 (Thermo Fisher Scientific, # 25-0199-41, RRID:AB_1582279) antibodies, with a viability dye (eBioscience, Fixable Viability Dye eFluor™ 506, #65-0866-14). Depletion decreased the proportion of T-cells (CD3+) from 30 to 2%, increased the proportion of B-cells (CD19+) from 23 to 50%, and increased the percentage of viable cells from 50 to 80% in a test BM sample.
Cells were combined with reverse transcriptase Master Mix and partitioned into Gel Bead-In EMulsions (GEMs) using 10X GemCode Technology, where the poly-A transcripts are barcoded with an Illumina R1 sequence, a 16 bp 10X barcode and a 10 bp Unique Molecular Identifier (UMI). 11-12 cycles of PCR was used to amplify the cDNA.

treatment_protocol: All cases received standard induction therapy according to the NOPHO ALL-2008 protocol, with prednisolone 60 mg/m2/day p.o. days 1-29, vincristine 2.0 mg/m2 i.v. days 1, 8, 15, 22, and 29, doxorubicin 40 mg/m2 i.v. days 1 and 22, and methotrexate i.t. days 1, 8, 15, and 29

tag: tissue

value: bone marrow


title: 20065FL-01-02-12


title: 18-2607-C-Ab

tag: genotype/variation


title: ALL10-d15


title: 19-2607-C-Ab


title: Lepr_pos_MPP_1

description: Ribosomal RNA depleted total RNA


title: 20065FL-01-02-11


title: 20065FL-01-02-15


title: 17-2607-C-Ab


title: Bgl-C-BM

data_processing:
bcl2fastq 2.17.1.14 and 2.19.1
alignment was done with GSNAP (2018-05-30) to the mm10 reference with support of Ensembl annotation version 87 (-d mm10 --gunzip -A sam -t 14 --use-sarray=1 --input-buffer-size=500000 --output-buffer-size=500000 -B 5 -N 0 -n 1 -s EnsemblGene-87.ss.mm10.iit)
gene counts: alignment files were processed with featureCounts v1.6.2 and the support of annotation from Ensembl version 87 (featureCounts -a EnsemblGene-87.mm10.TR.gtf -s 2 -Q 1 -T 8)
Genome_build: mm10
Supplementary_files_format_and_content: lab1404.gene_count.csv.gz is a tab separated files which list the genes and their characteristics, followed by the counts of the samples

value: beta-glucan


title: Lepr_neg_HSC_2

description: Ribosomal RNA depleted total RNA

value: Leptin Receptor negative


title: 20065FL-01-02-14


title: Lepr_neg_HSC_1

description: Ribosomal RNA depleted total RNA


title: 20065FL-01-02-10


title: 16-2607-C-Ab

value: amyloid-beta


title: Spinal Cord Cervical control 1

city: Foster City

first: Ricardo

last: Ramirez

address: 333 Lakeside Drive

institute: Gilead Sciences, Inc.

description: 8382-643 SC1 P0003 1M

data_processing:
Fastq reads were clipped from illumina barcodes and poor quality bases
Clipped fastq files were aligned to a reference genome using STAR_2.5.3a with these options:--outSAMtype BAM SortedByCoordinate --outSAMattributes NH --outSAMunmapped Within --alignSJoverhangMin 8 --sjdbScore 3 –twopassMode Basic
Genes were quantified using featureCounts
Genome_build: Macaca fascicularis 5.0.94
Supplementary_files_format_and_content: processed data is a matrix of read counts produced by featureCounts

organism: Macaca fascicularis

source_name: Spinal Cord (Cervical)

extract_protocol:
Samples were stored in RNAlater. RNA was extracted using the Qiagen miRNeasy mini kit
Samples were created using the Illumina TruSeq stranded mRNA kit. Sequencing was performed using an Illumina HiSeq 2500, 50bp paired-end reads

value: Spinal Cord (Cervical)

value: Control (0 mg/kg/day)

value: Male


title: 20065FL-01-02-09


title: Bgl-B-BM


title: Bgl-A-BM


title: 20065FL-01-02-08


title: ALL10


title: ALL9


title: 15-2607-i1

value: hiPSC-derived NGN2-neurons

value: ATP6V1A knock down


title: ALL8


title: Bgl-D-BM


title: 14-2607-i1


title: ALL12


title: PBS-D-BM


title: 20065FL-01-02-06

value: Zlotnicka White

value: female


title: ALL1


title: 13-2607-i1


title: PBS-E-BM


title: 20065FL-01-02-07

value: high fat content


title: Nalm6_ETV6RUNX1

source_name: Nalm6 pre-B-ALL cell line with inducible TEL-AML1 construct

growth_protocol: Nalm-6 (ACC-128, DSMZ, Germany) cells, modified to carry inducible Tet-On constructs, were cultured at 1 million/ml in RPMI 1640 (Gibco, Thermo Fisher) supplemented with 2 mM  L-glutamine (Gibco, Thermo Fisher), penicillin (100 U/ml), and 10% Tet-system approved FBS (Clontech, Takara Bio).

extract_protocol:
Excess dead cells were depleted from samples using bead-based Dead Cell Removal Kit (#130-090-101, MACS miltenyi Biotech)
Cells were combined with reverse transcriptase Master Mix and partitioned into Gel Bead-In EMulsions (GEMs) using 10X GemCode Technology, where the poly-A transcripts are barcoded with an Illumina R1 sequence, a 16 bp 10X barcode and a 10 bp Unique Molecular Identifier (UMI). 11-12 cycles of PCR was used to amplify the cDNA.

treatment_protocol: The expression of the E/R fusion or luciferase (control) was induced for 24 h using 500 ng/ml doxycycline

tag: time

tag: construct


title: PBS-C-BM


title: 20065FL-01-02-05


title: 11-2607-i1


title: 20065FL-01-02-04


title: Nalm6_LUC

source_name: Nalm6 pre-B-ALL cell line with inducible LUC construct

value: LUC control


title: YCC11 Capture-C

city: Singapore

first: Wen Fong

last: Ooi

address: 60 Biopolis

country: Singapore

institute: Genome Institute of Singapore

description: Capture-C in YCC11

data_processing:
Library strategy: Capture-C
Pre-processing of raw reads was performed to remove adaptor sequences (trim_galore, http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and overlapping reads were merged using FLASH. In order to achieve short read mapping to the hg19 reference genome, the resulting pre-processed reads were then in-silico digested with DpnII and aligned using Bowtie (using p1, m2, best and strata settings). Aligned reads were processed using Capture-C analyser to (i) remove PCR duplicates, and (ii) classify sub fragments as ‘capture’ if they were contained within the capture fragment; ‘proximity exclusion’ if they were within 1 Kb on either side of the capture fragment; or ‘reporter’ if they were outside of the ‘capture’ and ‘proximity exclusion’ regions. We additionally used the r3Cseq package on the capture and reporter fragments to identify significant interactions of the viewpoint against a scaled background (q-value <0.05, FDR).
Genome_build: hg19
Supplementary_files_format_and_content: Space delimited with columns starting with chromosome, start, end, width, nReads, RPMs, z, p.value and q.value

source_name: gastric cancer cell line

extract_protocol: Capture-C probes at CCNE1 promoter regions was designed using CapSequm. Approximately 1 × 107 cells were crosslinked by 2% formaldehyde, followed by lysis, homogenization, DpnII digestion, ligation and de-crosslinking. DNA was sonicated using a Covaris to 150–200 bp to produce DNA suitable for oligo capture. Three micrograms of sheared DNA was used for sequencing library preparation (New England Biolabs). The promoter sequence of CCNE1 was double captured by sequential hybridization to customized biotinylated oligos (IDT, Supplementary Table 14) and enrichment with Dynabeads (LifeTech). Captured DNA was sequenced on the Illumina HiSeq 3000 instrument with the 2x150 bp configuration.

tag: cell line


title: 10-2607-i1


title: 1-2607-C

value: Wild type


title: 20065FL-01-02-02


title: AGS-EBF1-WT [RNA-seq]

description: Overexpressing WT EBF1 in AGS.

data_processing:
RNA-seq reads were processed using the star-rsem pipeline published at: https://github.com/gis-rpd/pipelines
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *_hg19.genes.results.desc.txt: Tab-delimited text files; Please refer to the self-explanatory header of each file.

source_name: Gastric Cancer Line

extract_protocol:
RNA of GC cell line was obtained using the RNeasy Mini Kit (Qiagen).
RNAseq libraries were prepared using Illumina Tru-Seq RNA Sample Preparation v2 protocol, according to the manufacturer’s instructions.

treatment_protocol: EBF1 cDNAs were cloned into MIGR1 vector (#27490, Addgene) between the HpaI and Bgl 2 sites. Retrovirus packaging was performed using Platinum-A (Plat-A) retroviral packaging cell lines by transfection using Lipofectamin 2000 (Thermo Fisher Scientific). GC cells were infected with retrovirus packaged with EBF1 genes for 48 hours and sorted for GFP.

value: AGS

value: gastric cancer cell line

value: WT EBF1 overexpression


title: 20065FL-01-02-03

value: low fat content


title: Bgl-C-T

value: beta-glucan

value: male

value: WT

value: 8-10 weeks old


title: 20065FL-01-02-01


title: AGS-EBF1-Q196P [RNA-seq]

description: Overexpressing mutant EBF1 in AGS.

value: mutant EBF1 overexpression


title: bgl4-m

data_processing:
bcl2fastq 2.17.1.14
alignment was done with GSNAP (2017-08-15) to the mm10 reference with support of Ensembl annotation version 87 (-d mm10 --gunzip -A sam -t 8 --use-sarray=1 --input-buffer-size=500000 --output-buffer-size=500000 -B 5 -N 0 -n 1 -s EnsemblGene-87.ss.mm10.iit)
gene counts: alignment files were processed with featureCounts v1.5.3 and the support of annotation from Ensembl version 87 (featureCounts -a EnsemblGene-87.mm10.TR.gtf -s 0 -Q 1 -T 8)
Genome_build: mm10
Supplementary_files_format_and_content: lab1321_1.gene_count.csv is a tab separated files which list the genes and their characteristics, followed by the counts of the samples

source_name: monocytes

extract_protocol:
Tumor tissue was isolated and single cell suspensions were prepared. Monocytes were FACS-sorted (CD45+CD11c-CD11b+Ly6g-Ly6c+) and RNA was isolated using RNeasy Plus Microkit (Qiagen).
Complete cDNA was synthesized from 5 ng total RNA using the SmartScribe reverse transcriptase (Takara Bio) with a universally tailed poly-dT primer and a template switching oligo followed by amplification for 12 cycles with with the Advantage 2 DNA Polymerase (Takara Bio). After ultrasonic shearing (Covaris LE220), amplified cDNA samples were subjected to standard Illumina fragment library preparation using the NEBnext Ultra DNA library preparation chemistry (New England Biolabs). In brief, cDNA fragments were end-repaired, A-tailed and ligated to indexed Illumina Truseq adapters. Resulting libraries were PCR-amplified for 15 cycles using universal primers, purified using XP beads (Beckman Coulter) and then quantified with the Fragment Analyzer.


title: AGS-EV [RNA-seq]

description: Empty vector in AGS.

value: empty vector


title: bgl1-m


title: 20065FL-01-01-15

source_name: liver


title: AGS_Input

description: Input (background) for AGS EBF1 ChIP-seq

data_processing:
Sequencing reads were mapped to the human reference genome (hg19) using BWA-MEM. Reads with MAPQ<10 and potential PCR duplication were filtered using SAMTools. Peaks were predicted using MACS2 with default parameters.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *_peaks.txt: Please refer to the website: https://github.com/taoliu/MACS for details.

extract_protocol:
30 million cells were cross-linked with 1% formaldehyde for 10 min at room temperature, and reactions were stopped by adding glycine to a final concentration of 0.2 M. Chromatin was extracted and sonicated to 500 bp. EBF1 (sc-137065, Santa Cruz Biotechnology; H00001879-M01, Abnova) and EZH2 (#5246, Cell Signaling) antibodies were used for chromatin immunoprecipitation (ChIP). Input DNA from cells prior to immunoprecipitation were used to normalize ChIP-seq peak calling.
ChIPed DNA (10 ng) was used for ChIP with DNA sequencing (ChIP-seq) library construction following manufacturer protocols (New England Biolabs).

value: EBF1 overexpression

tag: chip antibody


title: bgl2-m


title: AGS_EBF1_overexpression

description: EBF1 ChIP-seq in AGS


title: 20065FL-01-01-14


title: PBS-B-BM

value: PBS control


title: P5m


title: PBS-A-BM


title: P4m


title: SNU484_Capture-C

description: Capture-C in SNU484

data_processing:
Library strategy: Capture-C
Pre-processing of raw reads was performed to remove adaptor sequences (trim_galore, http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and overlapping reads were merged using FLASH. In order to achieve short read mapping to the hg19 reference genome, the resulting pre-processed reads were then in-silico digested with DpnII and aligned using Bowtie (using p1, m2, best and strata settings). Aligned reads were processed using Capture-C analyser to (i) remove PCR duplicates, and (ii) classify sub fragments as ‘capture’ if they were contained within the capture fragment; ‘proximity exclusion’ if they were within 1 Kb on either side of the capture fragment; or ‘reporter’ if they were outside of the ‘capture’ and ‘proximity exclusion’ regions. We additionally used the r3Cseq package on the capture and reporter fragments to identify significant interactions of the viewpoint against a scaled background (q-value <0.05, FDR).
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *.txt: Space-delimited text files with columns starting with chromosome, start, end, width, nReads, RPMs, z, p.value and q.value.

extract_protocol: Capture-C probes at CCNE1 promoter regions were designed using CapSequm. Approximately 1 × 10e7 cells were crosslinked by 2% formaldehyde, followed by lysis, homogenization, DpnII digestion, ligation and de-crosslinking. DNA was sonicated using a Covaris to 150–200 bp to produce DNA suitable for oligo capture. Three micrograms of sheared DNA was used for sequencing library preparation (New England Biolabs). The promoter sequence of CCNE1 was double captured by sequential hybridization to customized biotinylated oligos (IDT, Supplementary Table 14) and enrichment with Dynabeads (LifeTech). Captured DNA was sequenced on the Illumina HiSeq 3000 instrument with the 2x150 bp configuration.


title: bgl3-m


title: shPiwil1#2_rep2

type: RNA

city: Cleveland

first: Haidong

last: Huang

state: Ohio

address: 9500 Euclid Avenue

department: Cancer Biology

institute: Cleveland Clinic Foundation

description: J

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: The data were normalized using quantile normalization with beadarray in R

label: biotin

source_name: GSC line 3832

label_protocol: Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays.

growth_protocol: GSCs were cultured in neural basal medium supplemented with B-27, EGF, FGF, Sodium Pyruvate and glutamate at 37oC with 5% CO2.

extract_protocol: RNAs were extracted using Rneasy mini kit.

treatment_protocol: GSCs were transduced with shNT or shPiwil1 lentivirus.

value: GSC line 3832


title: 20065FL-01-01-13

value: Zlotnicka White

value: female


title: P3m


title: shPiwil1#2_rep4

description: L


title: shPiwil1#2_rep1

description: I


title: P2m


title: 20065FL-01-01-12


title: shPiwil1#1_rep4

description: H


title: SNU16_Capture-C

description: Capture-C in SNU16


title: 20065FL-01-01-16


title: shPiwil1#1_rep3

description: G


title: auxin free BY-2 rep3

city: Praha 6

first: Karel

last: Muller

address: Rozvojová 263

department: Lab. of hormonal regulations in plants

country: Czech Republic

institute: Institute of Experimenal Botany of the Czech Academy of Sciences

data_processing:
rough reads processed using Trimmomatic (parameters: leading:3, trailing:3, slidingwindow:4:20, minlen:40)
transcript quantification and normalization done by Salmon with parameters –seqBias, --gcBias, --numBootstraps 30
Genome_build: Nicotiana tabacum v1.0 cDNA (Edwards, K.D., Fernandez-Pozo, N., Drake-Stowe, K., et al. (2017) A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genomics, 18, 448)
ftp://ftp.solgenomics.net/genomes/Nicotiana_tabacum/edwards_et_al_2017
Genome browser available at: https://solgenomics.net
Supplementary_files_format_and_content: sf files generated by Salmon software, include TPM values

organism: Nicotiana tabacum

source_name: suspension cell culture

growth_protocol: Tobacco cell line BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) was maintained in liquid Murashige and Skoog (MS) medium (3% sucrose (w/v), 4.3 g/l MS salts, 100 mg/l myo-inositol, 1mg/l thiamin, 0.2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), and 200 mg/l KH2PO4; pH 5.8), in the dark, at 27°C, under continuous shaking (150 rpm; orbital diameter 30 mm) and sub-cultured every 7 days.

extract_protocol:
Total RNA was isolated using RNeasy Plant Mini kit (Qiagen) from 50-100 mg of BY-2 cells and treated with DNA-Free kit (Thermo Fischer Scientific)
RNA libraries were prepared for sequencing using standard Illumina protocols by GATC Biotech service company.

treatment_protocol: Auxin free culturred BY-2 cells were innoculated into MS medium without 2,4-dichlorophenoxyacetic acid.

tag: cultivar

value: Bright Yellow 2

tag: growth cycle stage

value: exponential growth phase

tag: cultivation conditions

value: auxin (2,4-D) starved conditions


title: bgl4-g

extract_protocol:
Tumor tissue was isolated and single cell suspensions were prepared. Neutrophils were FACS-sorted (CD45+CD11c-CD11b+Ly6c-Ly6g+) and RNA was isolated using RNeasy Plus Microkit (Qiagen).
Complete cDNA was synthesized from 5 ng total RNA using the SmartScribe reverse transcriptase (Takara Bio) with a universally tailed poly-dT primer and a template switching oligo followed by amplification for 12 cycles with with the Advantage 2 DNA Polymerase (Takara Bio). After ultrasonic shearing (Covaris LE220), amplified cDNA samples were subjected to standard Illumina fragment library preparation using the NEBnext Ultra DNA library preparation chemistry (New England Biolabs). In brief, cDNA fragments were end-repaired, A-tailed and ligated to indexed Illumina Truseq adapters. Resulting libraries were PCR-amplified for 15 cycles using universal primers, purified using XP beads (Beckman Coulter) and then quantified with the Fragment Analyzer.

tag: age


title: auxin free BY-2 rep2


title: P1m


title: 20065FL-01-01-10

tag: breed

tag: day

tag: shear force

value: high fat content


title: shPiwil1#2_rep3

description: K

tag: treatment


title: 20065FL-01-01-11

tag: Sex

tag: replicant


title: 20065FL-01-01-09


title: shPiwil1#1_rep2

description: F


title: bgl3-g

tag: strain


title: 20065FL-01-01-08


title: shNT_rep3

description: C

value: shNT


title: ctrl BY-2 rep7

value: auxin (2,4-D) supplemented conditions


title: AGS_Capture-C

description: Capture-C in AGS

value: AGS

tag: cell type

value: gastric cancer cell line


title: auxin free BY-2 rep1


title: bgl2-g

value: beta-glucan

value: male


title: 20065FL-01-01-07


title: P5g

value: 8-10 weeks old


title: 20065FL-01-01-06


title: ctrl BY-2 rep6


title: bgl1-g


title: NAI, even set (SARS-CoV-2), replicate #2

description:
In vitro re-folded SARS-CoV-2 RNA, NAI treated
SARS_NAI_raw_rep2.wig
SARS_NAI_vs_DMSO_raw_rep2.wig
SARS_NAI_vs_DMSO_raw_merge.wig

data_processing:
Library strategy: SHAPE-MaP
Analysis of SHAPE-MaP data has been conducted using RNA Framework v2.6.9 (https://github.com/dincarnato/RNAFramework). Reads pre-processing and mapping was performed using the rf-map module (parameters: -ctn -cmn 0 -cqo -cq5 20 -b2 -mp "--very-sensitive-local"). Reads were trimmed of terminal Ns and low-quality bases (Phred < 20). Reads with internal Ns were discarded. Mapping was performed using the “very-sensitive-local” preset of Bowtie2. The mutational signal was then derived using the rf-count module (parameters: -m -rd), enabling the right re-alignment of deletions. When processing SARS-CoV-2 data, a mask file containing the sequences of primer pairing regions was passed along (through the -mf parameter). Generated RC files from both the even and odd sets, containing per base mutation counts and coverage, were then combined in a single RC file using the rf-rctools module (mode: merge). Data normalization was performed using the rf-norm module (parameters: -sm 3 -nm 3 -n 1000 -mm 1), by setting the minimum base coverage to 1,000X and the maximum mutation frequency to 1, and by using the Siegfried et al., 2014 scoring scheme and box-plot normalization of SHAPE reactivities.
Supplementary_files_format_and_content: Additional data files (RC/XML files for analysis with RNA Framework [http://www.rnaframework.com]) are available at: http://www.incarnatolab.com/datasets/SARS_Manfredonia_2020.php

organism: Severe acute respiratory syndrome coronavirus 2

extract_protocol:
Probed RNA was purified on an RNA Clean & Concentrator-5 column (Zymo Research, cat. R1013).
For multiplex SHAPE-MaP, 70 oligonucleotide pairs, tiling the entire length of the SARS-CoV-2 genome (29,903 nt), were automatically designed using Primer3 and the following parameters: amplicon size between 480 and 520 bp, maximum poly(N) length between 2 and 3, minimum/optimal/maximum oligonucleotide size of 20/25/30, minimum/optimal/maximum Tm of 56/60/62 degrees, minimum/optimal/maximum GC content of 30/50/65 %. Also, pairs were designed in such a way that the target amplicon would include the target region of the reverse primer from the previous set and of the forward primer of the following set. Primers were then searched against the GENCODE v33 human transcriptome, keeping only those with less than 60% predicted base-pairing, or more than 60% predicted base-pairing and more than 2 mismatched bases at the 3’ end. For reverse transcription, all reverse primers were equimolarly pooled to yield a 100 μM RT primer mix. An additional anchored oligo-dT primer (TTTTTTTTTTTTTTTTTTTTVN) was added to the mix. For each sample, two 20 μl reverse transcription reactions were carried out. 3 μg of total RNA were mixed with 0.5 μl RT primer mix and 1 μl dNTPs (10 mM each), incubated at 70°C for 5 min and then transferred immediately to ice for 1 min. Reactions were then supplemented with 4 μl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 2 μl DTT 0.1 M, 1 μl MnCl2 120 mM, 20 U SUPERase•In™ RNase Inhibitor and 200 U SuperScript II RT (ThermoFisher Scientific, cat. 18064014). Reactions were incubated at 42°C for 2 h and then RT was heat-inactivated at 75°C for 15 min. RNA was then degraded by addition of 5 U RNase H (New England Biolabs, cat. M0297S) and cDNA from each 2 reactions was purified on a single RNA Clean & Concentrator-5 column and eluted in 36 μl NF H2O. For targeted amplification, primer pairs were split into 2 non-overlapping sets, odd and even. For each of these sets, pairs were pooled in smaller sets of 3-4 pairs, as follows: 1-19-37-55, 2-20-38-56, 3-21-39-57, 4-22-40-58, 5-23-41-59, 6-24-42-60, 7-25-43-61, 8-26-44-62, 9-27-45-63, 10-28-46-64, 11-29-47-65, 12-30-48-66, 13-31-49-67, 14-32-50-68, 15-33-51-69, 16-34-52-70, 17-35-53, 18-36-54. The complete primer list can be found in Table S1. PCR reactions were carried out in 50 μl, using 2 μl of eluted cDNA, 0.3 mM final each dNTP, 0.1 μM final each primer in the set and 2.5 U TaKaRa Taq™ DNA Polymerase (TaKaRa, cat. R001A). Cycling was performed using a touch-down approach. Briefly, for the first 10 cycles the annealing temperature was lowered by 0.5°C per cycle, starting at 58°C. Then, 20 additional cycles were performed at the lowest temperature. PCR products were purified using 1 volume of NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), checked on a 2% agarose gel. Each primer set gave a single specific band of the expected size. PCR products were equimolarly pooled to yield the final odd and even sets. 500 ng of each set were fragmented in a final volume of 20 μl, using 2 μl of NEBNext dsDNA Fragmentase (New England Biolabs, cat. M0348), supplemented with 1 μl 200 mM MgCl2, by incubating at 37°C for 25 min. This yielded fragments in the range of ~50-200 bp. Reactions were purified using 1 volume of NucleoMag NGS Clean-up and Size Select beads. 5 ng of fragmented DNA were then used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), as per manufacturer instructions.

treatment_protocol: Approximately 5x10^6 infected cells were resuspended in 1 mL of QIAzol Lysis Reagent (Qiagen, cat. 79306) and 200 μl of chloroform were added. Sample was vigorously vortexed for 15 sec and then incubated for 2 min at room temperature, after which it was centrifuged for 15 min at 12,500 x g (4°C). The upper aqueous phase was collected in a clean 2 mL tube, supplemented with 1 mL (~2 volumes) of 100% ethanol and then loaded on an RNA Clean & Concentrator-25 column (Zymo Research, cat. R1017). For in vitro folding, ~2 μg of total RNA in a volume of 39 μl were denatured at 95°C for 2 min, then transferred immediately to ice and incubated for 1 min. 10 μl of ice-cold 5X RNA Folding Buffer [500 mM HEPES pH 7.9; 500 mM NaCl] supplemented with 20 U of SUPERase•In™ RNase Inhibitor (ThermoFisher Scientific, cat. AM2696) were added. RNA was then incubated for 10 min at 37 °C to allow secondary structure formation. Subsequently, 1 μl of 500 mM MgCl2 (pre-warmed at 37°C) was added and RNA was further incubated for 20 min at 37°C to allow tertiary structure formation. For probing of RNA, NAI was added to a final concentration of 50 mM and samples were incubated at 37°C for 5 min. A negative control reaction was also prepared, by adding an equal amount of DMSO. Reactions were then quenched by addition of 1 volume DTT 1 M.

tag: sample type

value: In vitro re-folded SARS-CoV-2 RNA

value: NAI


title: ctrl BY-2 rep5


title: NAI, odd set (SARS-CoV-2), replicate #2

source_name: NAI, odd set (SARS-CoV-2), replicate #2


title: 20065FL-01-01-05

value: low fat content


title: P4g

value: PBS control


title: ctrl BY-2 rep3


title: NAI, even set (SARS-CoV-2), replicate #1

description:
In vitro re-folded SARS-CoV-2 RNA, NAI treated
SARS_NAI_raw_rep1.wig
SARS_NAI_vs_DMSO_raw_rep1.wig
SARS_NAI_vs_DMSO_raw_merge.wig


title: shNT_rep1

description: A

value: GSC line 3832


title: shNT_rep2

description: B


title: shPiwil1#1_rep1

description: E


title: shNT_rep4

description: D


title: P3g


title: 20065FL-01-01-04

value: Zlotnicka White

value: female


title: NAI, odd set (SARS-CoV-2), replicate #1

source_name: NAI, odd set (SARS-CoV-2), replicate #1


title: 20065FL-01-01-03


title: ctrl BY-2 rep2

value: Bright Yellow 2

value: exponential growth phase


title: P2g


title: 20065FL-01-01-02

value: high fat content


title: P1g


title: ctrl BY-2 rep1


title: 20065FL-01-01-01


title: DMSO, even set (SARS-CoV-2)

description:
In vitro re-folded SARS-CoV-2 RNA, DMSO treated
SARS_DMSO_raw.wig

source_name: DMSO, even set (SARS-CoV-2)

extract_protocol: For multiplex SHAPE-MaP, 70 oligonucleotide pairs, tiling the entire length of the SARS-CoV-2 genome (29,903 nt), were automatically designed using Primer3 and the following parameters: amplicon size between 480 and 520 bp, maximum poly(N) length between 2 and 3, minimum/optimal/maximum oligonucleotide size of 20/25/30, minimum/optimal/maximum Tm of 56/60/62 degrees, minimum/optimal/maximum GC content of 30/50/65 %. Also, pairs were designed in such a way that the target amplicon would include the target region of the reverse primer from the previous set and of the forward primer of the following set. Primers were then searched against the GENCODE v33 human transcriptome, keeping only those with less than 60% predicted base-pairing, or more than 60% predicted base-pairing and more than 2 mismatched bases at the 3’ end. For reverse transcription, all reverse primers were equimolarly pooled to yield a 100 μM RT primer mix. An additional anchored oligo-dT primer (TTTTTTTTTTTTTTTTTTTTVN) was added to the mix. For each sample, two 20 μl reverse transcription reactions were carried out. 3 μg of total RNA were mixed with 0.5 μl RT primer mix and 1 μl dNTPs (10 mM each), incubated at 70°C for 5 min and then transferred immediately to ice for 1 min. Reactions were then supplemented with 4 μl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 2 μl DTT 0.1 M, 1 μl MnCl2 120 mM, 20 U SUPERase•In™ RNase Inhibitor and 200 U SuperScript II RT (ThermoFisher Scientific, cat. 18064014). Reactions were incubated at 42°C for 2 h and then RT was heat-inactivated at 75°C for 15 min. RNA was then degraded by addition of 5 U RNase H (New England Biolabs, cat. M0297S) and cDNA from each 2 reactions was purified on a single RNA Clean & Concentrator-5 column and eluted in 36 μl NF H2O. For targeted amplification, primer pairs were split into 2 non-overlapping sets, odd and even. For each of these sets, pairs were pooled in smaller sets of 3-4 pairs, as follows: 1-19-37-55, 2-20-38-56, 3-21-39-57, 4-22-40-58, 5-23-41-59, 6-24-42-60, 7-25-43-61, 8-26-44-62, 9-27-45-63, 10-28-46-64, 11-29-47-65, 12-30-48-66, 13-31-49-67, 14-32-50-68, 15-33-51-69, 16-34-52-70, 17-35-53, 18-36-54. The complete primer list can be found in Table S1. PCR reactions were carried out in 50 μl, using 2 μl of eluted cDNA, 0.3 mM final each dNTP, 0.1 μM final each primer in the set and 2.5 U TaKaRa Taq™ DNA Polymerase (TaKaRa, cat. R001A). Cycling was performed using a touch-down approach. Briefly, for the first 10 cycles the annealing temperature was lowered by 0.5°C per cycle, starting at 58°C. Then, 20 additional cycles were performed at the lowest temperature. PCR products were purified using 1 volume of NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), checked on a 2% agarose gel. Each primer set gave a single specific band of the expected size. PCR products were equimolarly pooled to yield the final odd and even sets. 500 ng of each set were fragmented in a final volume of 20 μl, using 2 μl of NEBNext dsDNA Fragmentase (New England Biolabs, cat. M0348), supplemented with 1 μl 200 mM MgCl2, by incubating at 37°C for 25 min. This yielded fragments in the range of ~50-200 bp. Reactions were purified using 1 volume of NucleoMag NGS Clean-up and Size Select beads. 5 ng of fragmented DNA were then used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), as per manufacturer instructions.

value: DMSO


title: DMSO, odd set (SARS-CoV-2)

source_name: DMSO, odd set (SARS-CoV-2)

extract_protocol:
Cells pellets were lysed by direct addition of 1 mL ice-cold QIAzol, followed by a brief incubation at 56°C for 5 min to completely dissolve cell aggregates. 200 µl of chloroform were added, followed by vigorous vortexing for 15 sec. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12,500 x g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 mL tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase at 37°C for 20 min.
For multiplex SHAPE-MaP, 70 oligonucleotide pairs, tiling the entire length of the SARS-CoV-2 genome (29,903 nt), were automatically designed using Primer3 and the following parameters: amplicon size between 480 and 520 bp, maximum poly(N) length between 2 and 3, minimum/optimal/maximum oligonucleotide size of 20/25/30, minimum/optimal/maximum Tm of 56/60/62 degrees, minimum/optimal/maximum GC content of 30/50/65 %. Also, pairs were designed in such a way that the target amplicon would include the target region of the reverse primer from the previous set and of the forward primer of the following set. Primers were then searched against the GENCODE v33 human transcriptome, keeping only those with less than 60% predicted base-pairing, or more than 60% predicted base-pairing and more than 2 mismatched bases at the 3’ end. For reverse transcription, all reverse primers were equimolarly pooled to yield a 100 μM RT primer mix. An additional anchored oligo-dT primer (TTTTTTTTTTTTTTTTTTTTVN) was added to the mix. For each sample, two 20 μl reverse transcription reactions were carried out. 3 μg of total RNA were mixed with 0.5 μl RT primer mix and 1 μl dNTPs (10 mM each), incubated at 70°C for 5 min and then transferred immediately to ice for 1 min. Reactions were then supplemented with 4 μl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 2 μl DTT 0.1 M, 1 μl MnCl2 120 mM, 20 U SUPERase•In™ RNase Inhibitor and 200 U SuperScript II RT (ThermoFisher Scientific, cat. 18064014). Reactions were incubated at 42°C for 2 h and then RT was heat-inactivated at 75°C for 15 min. RNA was then degraded by addition of 5 U RNase H (New England Biolabs, cat. M0297S) and cDNA from each 2 reactions was purified on a single RNA Clean & Concentrator-5 column and eluted in 36 μl NF H2O. For targeted amplification, primer pairs were split into 2 non-overlapping sets, odd and even. For each of these sets, pairs were pooled in smaller sets of 3-4 pairs, as follows: 1-19-37-55, 2-20-38-56, 3-21-39-57, 4-22-40-58, 5-23-41-59, 6-24-42-60, 7-25-43-61, 8-26-44-62, 9-27-45-63, 10-28-46-64, 11-29-47-65, 12-30-48-66, 13-31-49-67, 14-32-50-68, 15-33-51-69, 16-34-52-70, 17-35-53, 18-36-54. The complete primer list can be found in Table S1. PCR reactions were carried out in 50 μl, using 2 μl of eluted cDNA, 0.3 mM final each dNTP, 0.1 μM final each primer in the set and 2.5 U TaKaRa Taq™ DNA Polymerase (TaKaRa, cat. R001A). Cycling was performed using a touch-down approach. Briefly, for the first 10 cycles the annealing temperature was lowered by 0.5°C per cycle, starting at 58°C. Then, 20 additional cycles were performed at the lowest temperature. PCR products were purified using 1 volume of NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), checked on a 2% agarose gel. Each primer set gave a single specific band of the expected size. PCR products were equimolarly pooled to yield the final odd and even sets. 500 ng of each set were fragmented in a final volume of 20 μl, using 2 μl of NEBNext dsDNA Fragmentase (New England Biolabs, cat. M0348), supplemented with 1 μl 200 mM MgCl2, by incubating at 37°C for 25 min. This yielded fragments in the range of ~50-200 bp. Reactions were purified using 1 volume of NucleoMag NGS Clean-up and Size Select beads. 5 ng of fragmented DNA were then used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), as per manufacturer instructions.


title: paternal nucleus isolated from 2PN zygote 8

status: Public on Oct 29 2020

type: genomic

city: North Brunswick Township

first: Jia

last: Xu

state: NJ

address: 675 US Highway One

institute: Genomic Prediction, Inc.

description:
2PN pMII-8
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)

data_processing:
Affymetrix UK Biobank Axiom Array Manufacturer's Protocol
processed data files include DNA copy number and genotyping

source_name: single nucleus isolated from 2PN zygote at 20h post Cas9 RNP injection and fertilization

growth_protocol: fertilization of human metaphaseII oocytes was performed using standard intracytoplasmic sperm injection (ICSI), followed by culture in GlobalTotal (LifeGlobal)

extract_protocol: single cells were placed into single PCR tubes containing 2microliter of PBS or other hypotonic collection buffer. Genomic DNA amplification was performed using amplification kit as indicated for each sample.

treatment_protocol: Cas9 guide RNA ribonucleoprotein complex was assembled in vitro and injected either with sperm at MII stage, or at the 2-cell stage on day2 after ICSI

tag: tissue

value: 2PN zygote

tag: gender


title: ctrl BY-2 rep4

tag: cell line

value: auxin (2,4-D) supplemented conditions


title: paternal nucleus isolated from 2PN zygote 7

description:
2PN pMII-7
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)

hyb_protocol: Affymetrix UK Biobank Axiom Array Manufacturer's Protocol


title: paternal nucleus isolated from 2PN zygote 5

description:
2PN pMII-5
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)

scan_protocol: Affymetrix UK Biobank Axiom Array Manufacturer's Protocol


title: paternal nucleus isolated from 2PN zygote 6

description:
2PN pMII-6
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)

label_protocol: Affymetrix UK Biobank Axiom Array Manufacturer's Protocol


title: paternal nucleus isolated from 2PN zygote 2

description:
2PN pMII-2
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: paternal nucleus isolated from 2PN zygote 4

description:
2PN pMII-4
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: paternal nucleus isolated from 2PN zygote 3

description:
2PN pMII-3
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: maternal nucleus isolated from 2PN zygote 8

description:
2PN mMII-8
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: paternal nucleus isolated from 2PN zygote 1

description:
2PN pMII-1
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: maternal nucleus isolated from 2PN zygote 4

description:
2PN mMII-4
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: maternal nucleus isolated from 2PN zygote 5

description:
2PN mMII-5
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: maternal nucleus isolated from 2PN zygote 6

description:
2PN mMII-6
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: maternal nucleus isolated from 2PN zygote 3

description:
2PN mMII-3
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: maternal nucleus isolated from 2PN zygote 2

description:
2PN mMII-2
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2PN stage on day1_rep1

description: kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)

source_name: blastomere from gamete combination egg donor A and sperm donor

value: nd


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2PN stage on day1 rep2


title: maternal nucleus isolated from 2PN zygote 1

description:
2PN mMII-1
kit used for genomic DNA amplification: REPLI-g single cell kit (Qiagen Cat #150345)


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2PN stage on day1 rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2PN stage on day1_rep2


title: cumulus cells gDNA sample 2

description: amplified cumulus cells egg donor A sample 2

source_name: somatic cells from egg donor with wild type allele at rs758109813


title: cumulus cells gDNA sample 1

description: amplified cumulus cells egg donor A sample 1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryo 1_biopsy 8

description: kit used for genomic DNA amplification: Illustra GenomePhi V2 DNA amplification kit

source_name: blastomere from gamete combination egg donor A and sperm donor , possible interhomolog repair


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2-cell stage on day2 blastomere 2 embryo 2 rep 2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2-cell stage on day2 blastomere 2 embryo 2 rep 1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2-cell stage on day2 embryo 1_biopsy3_rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2-cell stage on day2 embryo 1_biopsy 3_rep2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 8_biopsy2_rep4

description: amplified by multiple displacement amplification

source_name: Trophectoderm biopsy from  gamete combination egg donor B and sperm donor


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 8_biopsy2_rep3


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 8_biopsy2_rep1


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 8_biopsy2_rep2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 8_biopsy1_rep3


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 8_biopsy1_rep4


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 8_biopsy1_rep2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy3_rep3


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy3_rep2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy3_rep4


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy3_rep1


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy2_rep4


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy2_rep3


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy2_rep2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy2_rep1


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy1_rep4


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 5_biopsy2_rep4


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 5_biopsy2_rep2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy1_rep1


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy1_rep2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 5_biopsy2_rep3


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 6_biopsy1_rep3


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 5_biopsy2_rep1


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 5_biopsy1_rep1


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 5_biopsy1_rep4


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryo 1_biopsy 6


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryo 1_biopsy 3


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 5_biopsy1_rep2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryo 1_biopsy 2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 5_biopsy1_rep3


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo 8_biopsy1_rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoC_biopsy 2

source_name: blastomere from gamete combination of egg donor D and sperm donor


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoC_biopsy 3


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoC_biopsy 6_rep2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoC_biopsy 7_rep2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoC_biopsy 7_rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoC_biopsy 6_rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoC_biopsy 4_rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoB_biopsy 4_rep2

source_name: blastomere from gamete combination egg donor D and sperm donor


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoB_biopsy 3_rep2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoB_biopsy 4_rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoB_biopsy 3_rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoB_biopsy 2_rep2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoA_biopsy 4_ rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoA_biopsy 4_ rep2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryo A_biopsy 2_repeat 2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryo A_biopsy 2_repeat 1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryo A_biopsy 3_ rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryo A_biopsy 3_ rep2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoC_biopsy 8


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo10_rep 2

description: MII injection TE biopsy embryo 10 rep 2

source_name: Trophectoderm biopsy from gamete combination egg donor B and sperm donor

value: Trophectoderm


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES8_rep 3

description: eysES8 rep 3

source_name: pluripotent stem cell line eysES8, gamete combination egg donor C and sperm donor

value: pluripotent stem cell line

value: male


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo10_rep 3

description: MII injection TE biopsy embryo 10 rep 3


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo10_rep 1

description: MII injection TE biopsy embryo 10 rep 1

source_name: Trophectoderm from gamete combination egg donor B and sperm donor


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo10_rep 4

description: MII injection TE biopsy embryo 10 rep 4


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES8_rep 4

description: eysES8 rep 4


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoC_biopsy 4_rep2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoB_biopsy 1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoB_biopsy 2_rep1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryo A_biopsy 1


title: single embryonic stem cell from eysES6_REPLIG amplification_cell A

description: single control eysES6 cell cell A

source_name: pluripotent stem cell line eysES6, gamete combination egg donor C and sperm donor


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES6_rep 2

description: eysES6 rep 2


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES6_rep 3

description: eysES6 rep 3


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES8_rep 2

description: eysES8 rep 2


title: single embryonic stem cell from eysES6_REPLIG amplification_cell B

description: single control eysES6 cell cell B


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES6_rep 4

description: eysES6 rep 4


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES5_rep 2

description: eysES5 rep 2

source_name: pluripotent stem cell line eysES5, gamete combination egg donor C and sperm donor


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES5_rep 4

description: eysES5 rep 4


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES8_rep 1

description: eysES8 rep 1


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES5_rep 1

description: eysES5 rep 1


title: single embryonic stem cell from eysES4_REPLIG amplification_cell B

description: single gene NHEJ edited eysES4 cell B

source_name: pluripotent stem cell line eysES4, gamete combination egg donor C and sperm donor


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES5_rep 3

description: eysES5 rep 3


title: single embryonic stem cell from eysES4_REPLIG amplification_cell A

description: single gene NHEJ edited eysES4 cell A


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES4_rep 4

description: eysES4 rep 4


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES4_rep 3

description: eysES4 rep 3


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES4_rep 1

description: eysES4 rep 1


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES4_rep 2

description: eysES4 rep 2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo4_rep 4

description: MII injection TE biopsy_embryo 4 rep 4

source_name: Trophectoderm biopsy from same blastocyst as eysES4, gamete combination egg donor C and sperm donor


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo4_rep 3

description: MII injection TE biopsy_embryo 4 rep 3


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo4_rep 2

description: MII injection TE biopsy_embryo 4 rep 2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoC_biopsy 5

description: MII injection single 'wt' blastomere embryo C-5

value: blastomere


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo4_rep 1

description: MII injection TE biopsy_embryo 4 rep 1


title: genomic DNA egg donor B rep 1 [69]

description: egg donor B rep 1

value: somatic cells from egg


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryo 1_biopsy 4

description: MII injection single 'wt' blastomere embryo 1_biopsy 4


title: human embryonic stem cell line derived after Cas9 RPN injection at fertilization_eysES6_rep 1

description: eysES6 rep 1


title: 2PN zygote_20h_Cas9 RNP injection at fertilization

description: MII injection 'wt' 2PN zygote 1

source_name: 2PN zygote from gamete combination egg donor A with sperm donor


title: genomic DNA egg donor B rep 1 [85]

description: egg donor B rep 1


title: genomic DNA egg donor B rep 2 [93]

description: egg donor B rep 2


title: genomic DNA egg donor B rep 2 [77]

description: egg donor B rep 2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo F_biopsy2

description: MII injection TE BIOPSY embryo F biopsy 2

source_name: Trophectoderm from gametes combination of egg donor D and sperm donor


title: genomic DNA egg donor C rep 2

description: egg donor C rep 2


title: genomic DNA egg donor C rep 1

description: egg donor C rep 1


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo E

description: MII injection TE BIOPSY embryo E


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo D biopsy 2

description: MII injection TE BIOPSY embryo D biopsy 2


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo F_biopsy1


title: Trophectoderm biopsy day6_Cas9 RNP injection at fertilization_embryo D biopsy 1

description: MII injection TE BIOPSY embryo D biopsy 1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at fertilization_embryoA_biopsy 5

description: MII injection single blastomere embryo A cell 5


title: genomic DNA egg donor D

description:
genomic DNA egg donor D
na


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2-cell stage on day2 blastomere 3 embryo 2 rep 1

description: 2-cell injection, embryo 2_biopsy 3_rep 1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2-cell stage on day2 blastomere 2 embryo 1 rep 2

description: 2-cell injection, embryo 1 biopsy 2_rep 2


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2-cell stage on day2 blastomere 3 embryo 2 rep 2

description: 2-cell injection, embryo 2_biopsy 3_rep 2


title: genomic DNA egg donor A

description: egg donor A


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2-cell stage on day2 blastomere 2 embryo 1 rep 1

description: 2-cell injection, embryo 1 biopsy 2_rep 1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2-cell stage on day2 blastomere 1 embryo 1 rep 1

description: 2-cell injection, embryo 1 biopsy 1_ rep 1


title: Cleavage stage blastomere_day4_Cas9 RNP injection at 2-cell stage on day2 blastomere 1 embryo 1 rep 2

description: 2-cell injection, embryo 1 biopsy 1_ rep 2


title: genomic DNA sperm donor rep 2

description: sperm donor rep 2

source_name: semen from subject with homozygous frame shift mutation at rs758109813

value: sperm


title: genomic DNA sperm donor rep 3

description: sperm donor rep 3


title: genomic DNA sperm donor rep 1

description: sperm donor rep 1


title: AP2IX-5 IP 2

city: Lille

first: Mathieu

last: Gissot

address: 1, rue du Pr. calmette

country: France

institute: CNRS

description:
RH ∆Ku80 AP2IX-5-HA
AP2IX-5.merged.cleaned.bowtie2_ME49.mark_duplicates.macs2_callpeak_extsize220_controlAP2IX5_peaks.narrowPeak

data_processing:
Clusters were generated on a flow cell within a cBot using the Cluster Generation Kit (Illumina), and libraries were sequenced as 50-bp reads on a HiSeq 2000 using a Sequence By Synthesis (SBS) technique (Illumina).
Image analysis and base calling were performed using the HiSeq Control Software and Real-Time Analysis component. Demultiplexing was performed using Illumina’s conversion software (bcl2fastq 2.17). The quality of the data was assessed using FastQC from the Babraham Institute and the Illumina software Sequence Analysis Viewer (SAV). Potential contaminants were investigated with the FastQ Screen software from the Babraham Institute.
genome build: T. gondii_ ME49 genome from ToxoDB-39
Supplementary_files_format_and_content: narrowPeak

organism: Toxoplasma gondii

source_name: HFF cell culture infected with T. gondii

growth_protocol: Intracellular parasite were grown 30h on HFF cells.

extract_protocol:
Genomic DNA was sonicated and process for immunoprecipitation. DNA was purified on Qiagen Minielute columns.
Library preparation was performed using the Nugen Ovation® Ultralow System V2 kit according to the manufacturer’s instructions. Libraries were validated using a Fragment Analyzer and quantified by qPCR (ROCHE LightCycler 480).

value: RH deltaKu80 AP2IX-5-HA

value: HFF cells


title: AP2IX-5 input 1


title: AP2IX-5 input 2


title: iKD AP2IX-5 +Auxin 1

data_processing:
Clusters were generated on a flow cell within a cBot using the Cluster Generation Kit (Illumina), and libraries were sequenced as 50-bp reads on a HiSeq 2000 using a Sequence By Synthesis (SBS) technique (Illumina).
Image analysis and base calling were performed using the HiSeq Control Software and Real-Time Analysis component. Demultiplexing was performed using Illumina’s conversion software (bcl2fastq 2.17). The quality of the data was assessed using FastQC from the Babraham Institute and the Illumina software Sequence Analysis Viewer (SAV). Potential contaminants were investigated with the FastQ Screen software from the Babraham Institute.
RNASeq reads were aligned to the Toxoplasma gondii genome (ToxoDB-25_TgondiiGT1_Genome.fasta from the ToxoDB Toxoplasma Genomics Resource, downloaded on 08/31/15) with a set of gene model annotations (ToxoDB-25_TgondiiGT1.gff from the ToxoDB Toxoplasma Genomics Resource, downloaded on 08/31/15) using the splice junction mapper TopHat 2.0.13. Final read alignments having more than three mismatches were discarded. Gene counting was performed using HTSeq-count 0.6.1p1 (union mode). Because the data come from a strand-specific assay, the read must be mapped to the opposite strand of the gene. Before statistical analysis, genes with less than 15 reads (combining all the analyzed samples) were filtered out. Differentially expressed genes were identified using the Bioconductor package edgeR 3.6.7. The data were normalized using the Relative Log Expression (RLE) normalization factors. Genes with adjusted p-values less than 5% (according to the FDR method from Benjamini-Hochberg) were declared differentially expressed.

growth_protocol: Intracellular parasite were grown 6 hours with or without Auxin.

extract_protocol:
Total RNA was extrated using TRIzol reagent according to manufacturer’s protocol.
Library preparation was performed using the TruSeq Stranded mRNA Sample Preparation kit (Illumina) according to the manufacturer’s instructions. Libraries were validated using a Fragment Analyzer and quantified by qPCR (ROCHE LightCycler 480).

value: RH delta-Ku80 Tir1 iKD AP2IX-5


title: iKD AP2IX-5 -Auxin 3


title: iKD AP2IX-5 +Auxin 2


title: iKD AP2IX-5 +Auxin 3