title: Gro-seq-siJMJD6-E2-MCF7

description: Nascent RNA sequencing


title: FAP_G39

status: Public on Apr 16 2018

type: SRA

city: Houston

first: Eduardo

last: Vilar

state: TX

address: 1515 Holcombe Blvd., Unit 1360

department: Clinical Cancer Prevention

country: USA

institute: The University of Texas MD Anderson Cancer Center

description: colonic tubular adenoma from patient with Familial Adenomatous Polyposis (FAP)

library_source: transcriptomic

data_processing:
mRNA sequences were aligned with Bowtie 1.1.2 on hg19 and quantified with RSEM 1.2.21 using default settings. RSEM expected counts were used for analysis
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited RSEM output with expected counts, TPM, FPKM values

molecule: total RNA

organism: Homo sapiens

source_name: RNA

extract_protocol:
Total RNA was extracted using TRIzol (Invitrogen) and the RNeasy kit (Qiagen). RNA was quantified using Qubit (Life Technologies) and RNA quality was assessed with the Agilent 2100 Bioanalyzer to select samples with RNA integrity numbers (RIN) ≥8 for analysis.
Sample preparation, library construction, and sequencing was performed at the DNA Analysis Facility Core at MD Anderson

tag: tissue

value: tubular adenoma

tag: size


title: SUB2

city: Singapore

first: Lei

last: Sun

address: 8 College Road

country: Singapore

institute: Duke-NUS

data_processing:
Reads were aligned to Hg19, using Tophat (version tophat-2.0.11) and genes were quantified using Cufflinks (Version 2.1.1)
Genome_build: Hg19
Supplementary_files_format_and_content: FPKM

source_name: Adult subcutaneous fat

extract_protocol:
Qiagen miRNeasy Mini Kit
NEBNext® Ultra™ Directional RNA Library Prep Kit

value: subcutaneous fat


title: SUB1


title: OME2

source_name: Adult Omental fat

value: omental fat


title: BATD21

source_name: Cultured brown preadipocytes

tag: cell type

value: Cultured brown preadipocytes


title: BATD0

source_name: Cultured brown pre-adipocytes

value: Cultured brown pre-adipocytes


title: SUBD21

source_name: Cultured white preadipocytes

value: Cultured white preadipocytes


title: SUB3


title: SUBD0

source_name: Cultured white pre-adipocytes

value: Cultured white pre-adipocytes


title: OME1


title: OME3


title: fiBAT2

source_name: Fetal interscapular brown adispose tissues

value: Fetal interscapular brown adipose


title: fiBAT3


title: PolII-siCTL-E2-MCF7

city: Ganzhou

first: Wenjuan

last: Zhang

state: Jiangxi

address: No. 23, Qingnian Road

department: First Affiliated Hospital

country: China

institute: Gannan Medical University

library_source: genomic

data_processing:
Basecalling was performed using CASAVA version 1.4
Reads were aligned to the hg19 genome assembly using Bowtie version 2.2.1.
The bigwig files were generated by using Homer v4.7, in which the total tags are normalized to 1.00e+07.
Genome_build: hg19
Supplementary_files_format_and_content: bigwig

molecule: genomic DNA

growth_protocol: MCF7 cells were cultured in DMEM medium supplemented with 10% FBS.

extract_protocol:
Cells were then fixed with 1% formaldehyde (Sigma) for 10 mins at room temperature (RT) (for MED12 and Pol II ChIP), or fixed with disuccinimidyl glutarate (DSG) (2mM) (Proteochem) for 45 min at RT, washed twice with PBS and then double-fixed with 1% formaldehyde for another 10 min at RT (for JMJD6 ChIP). Fixation was stopped by adding glycine (0.125M) and incubated for 5 mins at RT, followed by washing with PBS twice. Chromatin DNA was sheared to 300~500 bp average in size through sonication. Resultant was immunoprecipitated with anti-MED12, anti-Pol II or anti-JMJD6 antibody overnight at 4°C, followed by incubation with protein G magnetic beads (Invitrogen) for an additional 2 hrs. After washing and elution, the protein-DNA complex was reversed by heating at 65°C overnight. Immunoprecipitated DNA was purified by using QIAquick spin columns (Qiagen) and subjected to high throughput sequencing.
The libraries were constructed following Illumina’s Chip-Seq Sample prep kit. Briefly, Chip DNA was end-blunted and added with an ‘A’ base so the adaptors from Illumina with a ‘T’ can ligate on the ends. Then 200–400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR.

treatment_protocol: MCF7 cells were maintained in stripping medium (phenol red free) for three days before treating with or without estrogen (E2, 10-7 M) for 1h.

tag: antibody

value: PolII

tag: sirna

value: siCTL

tag: treatment

tag: cell line

value: Michigan Cancer Foundation-7 cells(MCF-7 cells)

tag: antibody catalog/vendor

value: SC-899(Santa Cruz Biotechnology)


title: fiBAT1


title: Macrophages form 15Day AML Spleen

city: Tianjin

first: Guo Guang

last: Zheng

state: Tianjin

address: Blood Hospital,288 Nanjing Road,Heping District

department: Institute of Hematology & Blood Hospital

institute: Chinese Academy of Medical Science& Peking Union  Medical College

description: LAMs form 15Day AML Spleen

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mouse genes.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: n/a
Supplementary_files_format_and_content: RPKM

organism: Mus musculus

source_name: Macrophage

extract_protocol:
sorted Macrophages were harvested and resuspended in 350ul RLT and 3.5ul β-Me.Total RNA is exacted by QIAGEN Rneasy Mini Kit(CAT 74103). 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: GFP+ leukemia cells were isolated from leukemia mice and transplanted into 6-8 weeks old C57BL/6J mice (5×10^5 cells/mouse) without irradiation. All mice suffered AML with a survival time of 25 to 30 days. Macrophages were sorted from BM and spleen of leukemia mice at 15d.

tag: strain

value: Spleen

tag: age


title: fiBAT4


title: Macrophages form 15Day AML  BM

description: LAMs form 15Day AML BM

value: BM


title: 207Z_MSCB-187

status: Public on Apr 15 2018

city: Barcelona

first: giovanni

last: iacono

state: Spain

address: Baldiri Reixac, 4

department: single cell sequencing

country: Spain

institute: CNAG,Centro Nacional de Análisis Genómico

description:
fresh cells
Cell_1842
IPSC_Expression.xlsx

data_processing:
Illumina Real-Time Analysis (RTA) 1.18.66.3 software used for basecalling.
Sequenced reads were filtered for low-complexity or low-quality sequence.
Cells with less than 60% of reads mapping on the reference genome were discarded.
MARS-Seq libraries were mapped as single-end reads with the GEMTools 1.7.0 RNA pipeline on the genome reference for human cells.
Gene quantification was performed using UMI corrected transcript information to correct for amplification biases.
Genome_build: Human: Gencode release 24, assembly GRCh38.p5;
Supplementary_files_format_and_content: Excel (.xlsx) file containing gene expression values (rows) as UMI counts (MARS-Seq) for each cell (columns) in condition.
IPSC_Expression.xlsx: gene expression
Table_S1_Linarsson.xlsx: Markers of Zeisel/Linarsson dataset
Table_S2_10xfull.xlsx: Markers 10x genomics developmental pallium (change cluster number)
Table_S3_10x_Reln.xlsx: Markers of Reelin subtypes  (change cluster number)
iCell_10x_brain.h5: Expression data for the iCells, HDF5 format
gene_names_10x.txt: Gene names (rows) for the iCells in HDF5 format
The data of 10x genomics is public in HDF5 format at:
https://support.10xgenomics.com/single-cell-gene-expression/datasets/1.3.0/1M_neurons

molecule: polyA RNA

source_name: neuronal progenitors

growth_protocol: Skin fibroblasts from patients with WBS or 7DUP were reprogrammed to induced Pluripotent Stem Cells (iPSC) by retroviral delivery of the pluripotency factors OCT4, SOX2, KLF4 and MYC, at the Centre of Regenerative Medicine in Barcelona (CMR[B]). Individual iPSC were picked to generate single clone colonies that were expanded and fully characterized (Martí et al. 2013). Briefly, genomic stability was confirmed by karyotype; integration and silencing were verified by PCR and quantitative RT-PCR; pluripotency was demonstrated by Alkaline Phosphatase staining and expression of pluripotency markers by immunocytochemistry. Finally, the capacity to differentiate to mesoderm, ectoderm and endoderm germ layers both in vitro and in vivo was verified by embroid bodies and teratoma formation followed by immunostaining. All iPSC were deposited in the Stem Cell Bank repository of the Instituto de Salud Carlos III.  We generated Neural Stem Cells (NSCs) from iPSCs following the Gibco protocol based on PSC Neural Induction Medium (NIM). Briefly, differentiation of iPSC colonies was performed by seven days culture in NIM, followed by several passages of maturation in Complete Neural Expansion Medium (Neurobasal Medium, Advanced DMEM⁄F-12, and Neural Induction Supplement (Gibco)). Confirmation of expression of NSC markers was done by immunocytochemistry (Human Neural Stem Cell Immunocytochemistry Kit, Gibco). After 4-7 passages, NSCs were detached with accutase (gibco) and resuspended. Single-cells were sorted in a BD Influx cell sorter to MARS-seq plates for single cell-RNAseq (Flow Cytometry Core Facility, Univeritat Pompeu Fabra).

extract_protocol: To construct single-cell libraries from polyA-tailed RNA, we applied massively parallel single-cell RNA sequencing (MARS-Seq). Briefly, single cells were FACS-sorted into 384-well plates, containing lysis buffer (0.2% Triton (Sigma-Aldrich); RNase inhibitor (Invitrogen)) and reverse-transcription (RT) primers. The RT primers contained the single-cell barcodes and unique molecular identifiers (UMIs) for subsequent de-multiplexing and correction for amplification biases, respectively. Single-cell lysates were denatured and immediately placed on ice. The RT reaction mix, containing SuperScript III reverse transcriptase (Invitrogen) was added to each sample. In the RT reaction, spike-in artificial transcripts (ERCC, Ambion) were included at a dilution of 1:16x106 per cell. After RT, the cDNA was pooled using an automated pipeline (epMotion, Eppendorf). Unbound primers were eliminated by incubating the cDNA with exonuclease I (NEB). A second pooling was performed through cleanup with SPRI magnetic beads (Beckman Coulter). Subsequently, pooled cDNAs were converted into double-stranded DNA with the Second Strand Synthesis enzyme (NEB), followed by clean up and linear amplification by T7 in vitro transcription overnight. Afterwards, the DNA template was removed by Turbo DNase I (Ambion) and the RNA was purified with SPRI beads. Amplified RNA was chemically fragmented with Zn2+ (Ambion), then purified with SPRI beads. The fragmented RNA was ligated with ligation primers containing a pool barcode and partial Illumina Read1 sequencing adapter using T4 RNA ligase I (NEB). Ligated products were reversed transcribed using the Affinity Script RT enzyme (Agilent Technologies) and a primer complementary to the ligated adapter, partial Read1. The cDNA was purified with SPRI beads. Libraries were completed through a PCR step using the KAPA Hifi Hotstart ReadyMix (Kapa Biosystems); and a forward primer that contains Illumina P5-Read1 sequence and the reverse primer containing the P7-Read2 sequence. The final library was purified with SPRI beads to remove excess primers. Library concentration and molecular size were determined with High Sensitivity DNA Chip (Agilent Technologies). The libraries consisted of 192 single-cell pools. Multiplexed pools (2) were run in one Illumina HiSeq 2500 Rapid two lane flow cell following the manufacturer’s protocol. Primary data analysis was carried out with the standard Illumina pipeline. We produced 52 nt of transcript sequence reads.

value: neuronal progenitors

tag: patient type


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title: CS14 BAL_IPF

city: Roma

first: Ivan

last: Arisi

address: viale Regina Elena 295

department: Bioinformatics Facility

country: Italy

institute: European Brain Research Institute

data_processing:
Raw sequence data in FASTQ format were assessed for Per Base Sequence Quality (Phred average score =33, ribosomal RNA content <2%) using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
NovoAlign (Novocraft, http://novocraft.com) was used to align reads to a set of transcript sequences and also to the reference genome (hg19).
Raw read counts were mapped on UCSC mRNA transcript human database based on the GRCh37/hg19 human genome version. NovoAlign parameters were set to allow multiple alignments to the transcriptome set to allow for isoforms, but only unique alignments to the genome.
The raw counts per transcript were then aggregated onto unique UCSC gene symbols of protein-coding mRNAs, to obtain a final table with non-normalized expression levels (counts) for 19723 unique gene symbol IDs. These  data are contained in the file processed_data_matrix_counts.txt
Raw non-normalized data were fed into the R-Bioconductor tool edgeR to assemble differentially expressed gene lists.
Genome_build: hg19
Supplementary_files_format_and_content: raw read counts aggregated on UCSC mRNA transcripts database based on the GRCh37/hg19 human genome

source_name: lung bronchoalveolar lavage(BAL)

extract_protocol:
Fiberoptic bronchoscopy (FOB) and bronchoalveolar lavage(BAL) were carried out at first evaluation of IPF patients and control subjects. All FOBs and BALs were carried out following established procedures recommended by international guidelines, by instillation of 100 ml of room temperature normal saline solution per lobe in one to three lung lobes (right middle lobe, lingula or left lower lobe). Portions of BAL samples remaining after aliquots for diagnostic exams were taken, then used to generate the RNA sample bank, after informed consent was obtained. Lavage samples were maintained on ice and processed for RNA extraction within one hour of the endoscopic procedure. Total RNA from BAL cells (5x106) was obtained using the RNA/DNA/Protein Purification Kit (Norgen Biotech Corp., Thorold, ON, Canada). For the purpose of RNA analysis, study samples were selected based upon alveolar macrophage differential counts ( >80%), cell viability ( >95%),  epithelial cells counts (<5%).  Samples of BAL differential cell counts were done on Diff Quick (Bio-Optica, Milan, Italy) stained cytopreps (Cytospin Shandon 4, Thermo Scientific, Waltham, MA, USA). Samples were further selected after RNA quality assessment based upon RNA integrity (R.I.N.> 6.0) analyzed by Agilent 2100 Bioanalyzer with the Total RNA pico kit (Agilent Technologies, SantaClara CA).
For library preparation and high-throughput sequencing (Cofactor Genomics, Saint Louis, MO), cDNA was synthesized by random priming using the NuGEN Ovation FFPE RNA-seq system (NuGEN Technologies, San Carlos, CA), as recommended for clinical samples potentially affected by low RNA integrity [35],  and fragmented to an average size of 200 bp using the Covaris S2 (Covaris, Woburn, MA). Fragmented cDNA was end repaired, A-tailed and ligated with barcoded Illumina adapters. Ligated DNA was treated with NuGEN mouse Insert Dependent Adaptor Cleavage (InDA-C) reagent to deplete cDNA corresponding to ribosomal RNA, and PCR amplified. Final library yield was measured using the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific, Waltham, MA)  and library size assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

value: BAL macrophages

tag: Sex

value: male

tag: disease

value: Idiopathic pulmonary fibrosis

tag: smoker

value: Smoking


title: 207Z_MSCB-122

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title: CS16 BAL_IPF

value: Yes [ex]


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