title: T47D_siHNRNPC

status: Public on Aug 23 2018

city: Beijing

first: Xuerui

last: Yang

address: Haidian District

department: School of Life Sciences

institute: Tsinghua University

data_processing:
Basecalls performed using CASAVA version 1.8
Truseq library were first pre-processed using Cutadapt (version 1.11) to remove adaptors and trim low-quality bases from 5’ and/or 3’ ends. Reads shorter than 20bp were discarded.
Trimmed and filtered reads were aligned to GENCODE v23 reference transcriptome using RSEM (v.1.2.15) invoking Bowtie2 (v2.1.0) with default parameters. Expected read counts of each gene were also estimated with RSEM.
Genome_build: hg38
Supplementary_files_format_and_content: Tab-delimited text files include rounded count values for each Sample

growth_protocol:
T47D was grown in RIPM 1640 (Corning) with 10% FBS and 0.2U/ml insulin.
Culture medium for different cell lines are discribed in the SAMPLES section. All cells were maintained in a humidified atmosphere with 5% CO2 at 37°C.

extract_protocol:
Cells were collected 48 hours after siRNA transfection. The nuclear and cytoplasmic fractions were separated with Nuclear/Cytosol Fractionation Kit (Biovision, K266-25). Total RNA were then extracted from the cytoplasmic lysates, with Trizol reagent (Invitrogen). The residual genome DNA was removed by Turbo DNase (Ambion), and ribosomal RNA was removed with the ribozero kit for human (Epicentre).
About 100ng ribosomal RNA-depleted cytoplasmic RNA was used for cDNA library preparation with Truseq RNA sample Preparation Kit (Illumina).

treatment_protocol: Reverse transfections of the siRNAs (10nM) were performed using Lipofectamine RNAiMAX Reagent (Invitrogen) following the manufacturer’s instructions. siRNAs and RNAiMAX were separately diluted in optimem medium and mixed together for 20 minutes incubation. For 96-well plates, 100ul medium containing 10^5 cells were added to the transfection mixture. 6 hours after transfection, replace the cell supernatant with fresh cell medium.

tag: cell line

tag: genotype/variation

value: HNRNPC knock-down

tag: molecule subtype

value: cytoplasmic RNA


title: T47D_siNC

type: SRA

country: China

library_source: transcriptomic

molecule: total RNA

organism: Homo sapiens

value: control


title: RNA wild-type R1

status: Public on Aug 24 2018

city: Cologne

first: Konrad

middle: U.

last: Förstner

state: North Rhine-Westphalia

address: Gleueler Str. 60

department: Information Services

country: Germany

institute: ZB MED - Information Centre for Life Sciences

description: ERCC spike-in added

data_processing:
Cutadapt
Read mapping with BWA mem (BWA version 0.7.12)
Counting of read per feature with bedtools intersect (bedtools version 2.25.0)
Genome_build: Tb427v8 (the genome was assembled as part of this study)

organism: Trypanosoma brucei brucei

source_name: whole liquid cell culture

growth_protocol: All replicates were grown in HMI-11 (37 °C, 5% CO2) to a maximum of 1x10^6 cells/ml. Drug selection after transfection was applied if necessary.

extract_protocol:
Total RNA from 45 million cells was extracted in triplicates (NucleoSpin RNA, Macherey & Nagel) and spiked with a synthetic RNA control (ERCC, invitrogen). rRNA was depleted from the sample using custom-made anti-rRNA oligos that were hybridized to the targed RNA and subsequently digested with RNAseH. Residual DNA was digested away with a DNAse and the sample was purified using RNeasy Minelute columns (Qiagen).
Libraries were prepared as follows: cDNA was produced from 100 ng of rRNA depleted RNA using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® until second strand cDNA synthesis. The double-stranded cDNA was purified using Agencourt AMPure XP Beads and eluted in 0.1X TE Buffer. cDNA was end-repaired using T4 DNA polymerase (NEB),
Klenow large Fragment (NEB) and  T4 DNA Polynucleotide Kinase (NEB). Protruding A-bases were added to the 3` end using  Klenow fragment (3´ to 5´ exo minus) and dATP. TruSeq adapters were ligated to the cDNA fragments using Quick Ligase (NEB) and was purified using AMPure XP magnetic beads. Y-shaped adapters were converted to dsDNA with Illumina primers for 5 cycles, purified with magnetic beads  and library fragments of 200-500 bp were size selected from an agarose gel. The gel-purified library was further amplified for 10-17 cycles using Illumina primers and purified using magnetic beads. Libaries were quantified using Qubit and qPCR and were sequenced on an Illumina flow cell  in an Illumina NextSeq aparatus following the manufacturer's instructions

tag: strain

value: Lister 427 MITat 1.2

tag: genotype

value: wild-type

tag: life cycle stage

value: Bloodstream form


title: bak1-4/serk4-1_R1

city: College Station

first: Libo

last: Shan

state: Texas

address: Norman Borlaug Center 136, TAMU2123

department: Department of Plant Pathology and Microbiology

country: USA

institute: Texas A&M University

data_processing:
Sequence cluster identification, quality prefiltering, base calling and uncertainty assessment were done in real time using Illumina's HCS 2.2.38 and RTA 1.18.61 software with default parameter settings
Sequencer .bcl basecall files were formatted into .fastq files using bcl2fastq 1.8.4 script configureBclToFastq.pl
Samples are demultiplexed during the execution of the configureBclToFastq.pl script. Three metrics are examined upon sample demultiplexing: (1) demultiplexing balance, (2) rate of prefiltering, and (3) the presence of technical adapter sequence at the ends of reads using cutadapt 1.0.
Reads were aligned to the Arabidopsis genome using Tophat v2.0.12 with default parameter settings
Feature reads counting was performed using the tool htseq-count of the Python package HTSeq 0.5.4. Differentially expressed genes were identified using the DESeq 1.12.0 package.
Genome_build: Arabidopsis TAIR10 genome annotation
Supplementary_files_format_and_content: txt format, gene relative expression level baseMean.

organism: Arabidopsis thaliana

source_name: seedlings

growth_protocol: Seedlings were germinated on ½ Murashige and Skoog (MS) plate containing 1% sucrose, 0.8% Agar.Plants were grown in a growth room at 23°C, 45% humidity and 75μE m-2s-1 light with a 12 hr light/12 hr dark photoperiod.

extract_protocol:
Total RNA from 10-day-old seedlings was extracted by TRIzol reagent (Invitrogen) and quantified with NanoDrop (Thermo Scientific).
cDNA libraries were made using Illumina TruSeq RNA prep kit as per the manufacturer's protocol and 1 μg total RNA was used for one sample.

treatment_protocol: Ten-day-old seedlings were transferred to a 6-well tissue culture plate with 2 ml water for overnight recover, and then harvest samples.

tag: ecotype

tag: ages

value: 10-day old

tag: tissue

value: whole plant


title: WT_R2

value: WT


title: MCF7_siLAMN_rep2

growth_protocol:
MCF7 was cultured in Dulbecco's modified Eagle's medium (DMEM) low glucose (Corning) supplemented with 10% FBS (HyClone).
Culture medium for different cell lines are discribed in the SAMPLES section. All cells were maintained in a humidified atmosphere with 5% CO2 at 37°C.

value: LAMN knock-down


title: MDM_6h_mock_r2

city: La Jolla

first: Max

last: Chang

state: CA

address: 9500 Gilman Drive

institute: University of California, San Diego

library_source: genomic

data_processing:
library strategy: Hi-C
Raw sequencing reads were analyzed with Juicer
Genome_build: hg19
Supplementary_files_format_and_content: hic files viewable by Juicebox

molecule: genomic DNA

growth_protocol: Human CD14+ monocytes, isolated from healthy donor blood, were plated at a density of 3 x 10^6 cells in a 10 cm dish and cultured for 10 days in RPMI 1640 + 10% fetal bovine serum, Penicillin-Streptomycin, and 1000U/ml GM-CSF.

extract_protocol:
In situ Hi-C was performed as described (Rao et al., 2014) with modifications. Cells were pelleted by centrifugation and resuspended and cross-linked in 1% formaldehyde/PBS for 10 minutes at room temperature (RT). After quenching the reaction by adding 1/20th volume of 2.625 M glycine, cells were pelleted by centrifugation and washed twice with ice-cold 0.5% BSA/PBS. Aliquots of 2 M cells each were snap-frozen and stored at -80°C. After thawing cells on ice, nuclei were isolated by resuspending the cell pellet in 200 µl wash buffer (50 mM Tris/HCl pH 7.5, 10 mM NaCl, 1 mM EDTA, 0.5% SDS, 1x protease inhibitor cocktail (Sigma P8340)). Nuclei were spun down at 2500g for 5 minutes at room temperature. Most of the supernatant was discarded, leaving the nuclei in 10 µl liquid. Samples were resuspended in Dpn II buffer, 195 µl water and rotated at 37°C for 15 minutes.
Chromatin was digested overnight (ON) with 2 µl (100 U) Dpn II (NEB) at 37°C, rotating overhead with 8 RPM. The next day, Mbo I digests (not Dpn II digests) were inactivated by incubating for 20 minutes at 62°C, and nuclei were collected centrifugation (5 minutes, 500 g, RT). Two-hundred µl of the supernatant were discarded, leaving the nuclei in 50 µl liquid, and overhangs were filled in with Biotin-14-dATP by adding 50 µl Klenow Master Mix (31.95 µl water, 5 µl NEBuffer 2, 0.35 µl 10 mM dATP (or dCTP), 0.35 µl 10 mM dTTP, 0.35 µl 10 mM dGTP, 7.5 µl 0.4 mM Biotin-14-dCTP (or Biotin-14-dATP)(Thermo), 2 µl 10% Triton X-100, 2.5 µl (12.5 U) Klenow fragment (Enzymatics) and rotating overhead at RT, 8 RPM for 40 minutes. Reactions were stopped by adding 2.5 µl 0.5 M EDTA and placed on ice. Proximity ligation was performed by transferring the entire reaction into 1.5 ml Eppendorf tubes containing 400 µl ligase mix (320 µl water, 40 µl 10x T4 DNA ligase buffer (Enzymatics), 36 µl 10% Triton X-100, 0.5 µl 10 % BSA, 1 µl (600 U) T4 DNA ligase (HC, Enzymatics) and rotating ON at 16°C, 8 RPM. Reactions were stopped with 20 µl 0.5 M EDTA, treated with 1 µl 10 mg/ml DNase-free RNAse A for 15 minutes at 42°C, 33 µl 5 M NaCl and 55 µl 10 % SDS were added and crosslinks reversed for 4 h to ON at 65°C. Proteins were digested with 10 µl 20 mg/ml DNase-free proteinase K (Thermo) for 2 h at 55°C while shaking at 800 RPM, then 90 minutes at 65°C. After extraction with 600 µl ph 8-buffered phenol/chloroform/isoamyl alcohol (Ambion) followed by 300 µl chloroform, DNA was precipitated with 1.5 µl (22.5 µg) Glycoblue (Ambion) and 1400 µl 100% ethanol ON at -20°C, pelleted for 20’ at 16000g, 4°C, washed twice with 80% ethanol, and the pellet air-dried and dissolved in 131 µl TT (0.05% Tween 20/Tris pH 8). DNA (800 ng) was sheared to 300 bp in 130 µl TT on a Covaris M220 or S220, using the manufacturer’s protocol. DNA was then directly bound to streptavidin-coated Dynabeads. Biotinylated DNA captured on Dynabeads MyOne Streptavidin T1 (Thermo) by combining the sample (50 µl or 130 µl) with 20 µl Dynabeads that had previously been washed once with 1x B&W buffer (2X B&W: 10 mM Tris-HCl pH=7.5, 1 mM EDTA, 2 M NaCl), and had been resuspended in 50 µl or 130 µl 2x B&W containing 0.2% Tween 20. The binding reaction was rotated at RT for 30 minutes, and DNA-bound beads were washed once with 1 ml 1x B&W/0.1% Triton-X 100, once with TET (0.2% Tween 20, 10 mM Tris pH 8, 1 mM EDTA). Beads were resuspended in 100 µl Blunting Mix (74.5 µl water, 0.5 µl 10% Tween 20, 10 µl 10x T4 DNA ligase buffer (Enzymatics), 4 µl 10 mM dNTP, 2 µl (6 U) T4 DNA polymerase (Enzymatics), 0.4 µl (2 U) Klenow fragment (Enzymatics), 2 µl (20 U) T4 polynucleotide kinase (Enzymatics)) and incubated for 30 minutes at 20°C. Reactions were stopped by adding 2.5 µl 0.5 M EDTA, beads collected on a magnet and washed twice with 150 µl 1x B&W/0.1% Triton-X 100, once with 180 µl TET. Beads were resuspended in 50 µl A-tail Mix (44.25 µl water, 0.5 µl 10% Tween 20, 5 µl NEBuffer 2, 0.2 µl 100 mM dATP, 0.3 µl (15 U) Exo--Klenow (Enzymatics) and incubated for 30 minutes at 37°C. Reactions were stopped by adding 1.5 µl 0.5 M EDTA, beads collected on a magnet and the SN was discarded. Beads were resuspended on ice in 48 µl ice-cold Ligation mix (25 µl 2x T4 DNA ligation buffer (Enzymatics), 0.5 µl 10% Tween 20, 22.5 µl water), 2 µl 0.6 µM Bioo Nextflex DNA sequencing adapters were added, and the reaction incubated for 20 minutes at RT. Reactions were stopped by adding 5 µl 0.5 M EDTA, and beads were washed twice with 1x B&W/0.1% Triton-X 100, once with TET and resuspended in 30 µl 0.05% Tween 20/LoTE (TE dilute 1:4 with water). Libraries were amplified by PCR for 10 or 12 cycles (98°C, 30s; 10/12x[98°C, 10s; 60°C, 25s; 72°C, 30s]; 72°C, 5 min; 4°C, ∞), using 10 µl of the bead suspension in a 50 µl reaction with NEBNext High-Fidelity 2X PCR Master Mix (NEB) and 0.5 µM each Solexa 1GA/1GB primers (Solexa 1GA: AATGATACGGCGACCACCGA, Solexa 1GB: CAAGCAGAAGACGGCATACGA). Libraries were isolated adding  38 µl 20% PEG8000/2.5 M NaCl + 2 µl SpeedBeads (8.6 % PEG final), washed twice with 180 µl 80% EtOH, air-dried, and elute with 20 µl TT.

treatment_protocol: For infection, growth medium was replaced with virus diluted to the appropriate concentration in serum-free RPMI 1640 medium at a total volume of 2 mL. Dishes were incubated in a cell culture incubator with gentle rocking every 15 minutes. After 60 minutes, the inoculum was removed and cells were washed twice washed gently with 5 mL serum-free RPMI 1640. 10 mL of RPMI 1640 + 10% fetal bovine serum and Penicillin-Streptomycin was added and cells were incubated for the specified length of time.

tag: cell type

value: monocyte-derived macrophages (MDM)

tag: infection

value: uninfected (mock control)

tag: hours post-infection

tag: biological replicate


title: WT_R1


title: MDM_6h_mock_r1


title: G.LB.I.17

status: Public on Aug 26 2018

city: Shanghai

first: Denghui

last: Liu

state: Shanghai

address: ShengLi Building, 320 Yueyang Road

institute: CAS-MPG Partner Institute for Computational Biology (PICB)

data_processing:
Reads quality was examined by “fastQC”
Filtered reads were aligned to rhesus monkey genome database (AV et al. 2014) by tophat v2.1.0
Only uniquely mapped reads were used to calculate the counts of each gene by HTSeq (Anders et al. 2015).
Reads counts were normalized to library size by DESeq2 , and normalized reads were then used for calculate the FPKM (Fragments Per Kilobase of transcript per Million fragments mapped).
Genome_build: MacaM
Supplementary_files_format_and_content: Contains the expression level of each gene in all samples, measured by FPKM

molecule: polyA RNA

organism: Macaca mulatta

source_name: later blastocyst, inner cell

growth_protocol: Cycling female monkeys were ovarian stimulated by injection of rhFSH (recombinant human follitropin alfa, GONAL-F, Merck Serono) 18 IU twice-daily for 8 days from the onset of menstrual cycle. On day 9, 1000 IU of rhCG (recombinant human chorionic gonadotropin alfa, OVIDREL, Merck Serono) was injected. After 32–35 hr of rhCG administration, mature oocytes were collected by laparoscopic follicular aspiration 63. Oocytes were removed of cumulus cells and those with extrusion of the first polar body (metaphase II stage) were collected for downstream experiments. Successful fertilization was assessed by the presence of two pronuclei. Embryos were then cultured at 37℃, 5% CO2 in 50 μl drops of equilibrated HECM-9 containing 10% FBS (HyClone Laboratories Inc.). Culture medium was changed every other day. Morphologically normal embryos with few fragments were used in the study. 16-cell embryos with good morphology were collected at about E3. Early morulae with compacted blastomeres were collected at about E4. Late morulae with more blastomeres were collected at about E4.5. Early blastocysts having a small cavity were collected at about E5. Middle blastocysts with a cavity occupying around half size of embryos were collected at about E5.5. Late blastocysts with fully expanded cavity were collected at about E6. Hatched blastocysts from the zona pellucida were collected at about E7.

extract_protocol:
To collect single blastomeres, embryos with good quality were transferred into 0.5% Pronase in TL-HEPES to remove zona pellucida, followed by three washes in PVP-BSA. To distinguish the outer and inner cells, embryos were incubated with PKH2 dye (Sigma) which labels the live outer cells with red fluorescence. After labeling, embryos were rinsed with Ca2+/Mg2+ free PVP-PBS and treated with 0.125% trypsin-EDTA for at least 5 min at 37℃ until the cell surface looks rough. Disaggregating was done by a mouth pipette aided by several finely pulled glass capillaries of different internal diameter. After most cells were separated apart, single cells were individually transferred into small droplets of PVP-PBS and the inner/outer cell identity was identified by the fluorescent microscopy. Cells showing no fluorescence were inner cells, whereas those with red fluorescence were outer cells.
Briefly, single cells were washed three times in PVP-PBS and those with a clear round shape were added into cell lysis buffer, followed by cDNA synthesis, primer removal, poly(A) tailing, second-strand cDNA synthesis, and PCR amplification. PCR products were purified by Ampure XP beads and used for libraries construction.
Libraries were deep sequenced on Illumina HiSeq 2500 platform, using 100-bp single-end strategy.

tag: Stage

value: LB

tag: position

value: Inner


title: RNA ΔH3.V R1

description: ERCC spike-in added

value: Lister 427 MITat 1.2 single marker


title: MDM_6h_H5N1-UV_r2

value: UV-inactivated H5N1 influenza


title: MDM_6h_H5N1-UV_r1


title: MDM_6h_H5N1_r2

value: active H5N1 influenza


title: bak1-4/serk4-1_R2


title: MDM_6h_H5N1_r1


title: MCF7_siHNRNPC_rep2

value: HNRNPC knock-down


title: MCF7_siNC_rep2

value: cytoplasmic RNA


title: MDM_12h_H5N1_r2


title: MDM_12h_mock_r2


title: MDM_18h_H5N1-UV_r2


title: MDM_18h_mock_r2


title: MDM_12h_H5N1-UV_r2


title: MDM_18h_H5N1-UV_r1


title: MDM_18h_H5N1_r1


title: MDM_12h_mock_r1


title: HTBE_6h_mock_r1

growth_protocol: HTBE cells (Lonza) were maintained in PneumaCult-Ex Medium (Stemcell Technologies) according to the manufacturer’s recommendations.

treatment_protocol: 24 hours before infection, cells were plated at a density of 2 x 10^6 cells in a 10 cm dish. For infection, growth medium was replaced with virus diluted to the appropriate concentration in Pneumacult-Ex complete medium at a total volume of 2 mL. Dishes were incubated in a cell culture incubator with gentle rocking every 15 minutes. After 60 minutes, the inoculum was removed and cells were washed twice washed gently with 5 mL Pneumacult-Ex. 10 mL of Pneumacult-Ex was added and cells were incubated for the specified length of time.

value: human tracheobronchial epithelial cells (HTBE)


title: MDM_12h_H5N1_r1


title: HTBE_6h_mock_r2


title: HTBE_6h_H5N1_r1


title: HTBE_6h_H5N1-UV_r1


title: HTBE_18h_mock_r1


title: HTBE_18h_mock_r2


title: MDM_12h_H5N1-UV_r1


title: MDM_18h_mock_r1


title: HTBE_18h_H5N1-UV_r2


title: HTBE_6h_H5N1-UV_r2


title: HTBE_18h_H5N1_r2


title: HTBE_18h_H5N1-UV_r1


title: HTBE_6h_H5N1_r2


title: HTBE_12h_mock_r2


title: HTBE_12h_H5N1_r2


title: HTBE_12h_mock_r1


title: HTBE_12h_H5N1_r1


title: HTBE_18h_H5N1_r1


title: MCF7_siLAMN_rep1


title: MCF7_siHNRNPC_rep1


title: LINC00673 siRNA2 Rep3

city: New York

first: Luis

last: Arnes

state: New York

address: 1130 St Nicholas avenue

department: Biomedical Informatics

institute: Columbia University

data_processing:
RTA (Illumina) was used for base calling.
CASAVA (version 1.8.2) was used for converting BCL to fastq format, coupled with adaptor trimming.
Reads were mapped to a reference genome (Human: UCSC/hg19) using Tophat (Trapnell C et al. 2009 Bioinformatics) (version 2.0.4) with 4 mismatches (--read-mismatches = 4) and 10 maximum multiple hits (--max-multihits = 10).
Relative abundance (aka expression level) of genes and splice isoforms was determined using Cufflinks (Trapnell C et al. 2010 Nature Biotech) (version 2.0.2) with default settings.
Differentially expressed genes were calculated using DESeq2 (Love MI et al. 2014 Genome Biology).
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *_genes.csv files include FPKM values for all genes.
Supplementary_files_format_and_content: DEseq* files include DESeq analysis for all samples.

source_name: Panc1 cells, LINC00673 siRNA

extract_protocol:
Total RNA was isolated using QIAGEN RNeasy Mini kits. Libraries were prepared from total RNA (RIN>8) with the TruSeq RNA prep kit (Illumina).
RNA libraries were prepared for sequencing using the TruSeq RNA prep kit (Illumina).

value: Pancreatic ductal adenocarcinoma derived cells

tag: sirna

value: LINC00673 siRNA2


title: LINC00673 siRNA2 Rep2


title: LINC00673 siRNA2 Rep1


title: LINC00673 siRNA1 Rep3

value: LINC00673 siRNA1


title: MCF7_siNC_rep1

value: control


title: LINC00673 siRNA1 Rep1


title: MDM_18h_H5N1_r2


title: LINC00673 siRNA1 Rep2


title: siRNA Control Panc1 Rep1

source_name: Panc1 cells, siRNA Control

value: siRNA Control


title: siRNA Control Panc1 Rep2


title: FAM83H-AS1 siRNA2 Rep1

source_name: Aspc1 cells, FAM83H-AS1 siRNA

value: FAM83H-AS1 siRNA2


title: FAM83H-AS1 siRNA1 Rep2

value: FAM83H-AS1 siRNA1


title: FAM83H-AS1 siRNA2 Rep2


title: siRNA Control Aspc1 Rep2

source_name: Aspc1 cells, siRNA Control


title: siRNA Control Aspc1 Rep1


title: FAM83H-AS1 siRNA1 Rep1


title: Dnm3os 2

city: Duarte

first: Rama

last: Natarajan

address: 1500 E Duarte Road

institute: City of Hope

data_processing:
Basecalls performed using Illumina’s Real time analysis (RTA).
RNA-seq reads were aligned to the mm9 genome assembly using tophat (version-2.1.1) with the mm9 Refseq gtf file, mm9 bowtie2 indexes and parameter -g 10
RNA-seq reads aligning to exons of each Refseq transcript were counted using custom scripts. DESeq2 package was used to normalize feature counts and find differentially regulated genes
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include raw counts and normalized counts for each transcript in each sample

organism: Mus musculus

source_name: RAW Macrophages

growth_protocol: Macrophage complete medium, i.e., DMEM containing 5.5 mmol/L glucose, 10% heat inactivated FBS, 2 mmol/L glutamine, penicillin/streptomycin (100 umol/L), 50mol/L beta-mercaptoethanol, and 10 mmol/L HEPES, pH 7.4.and 1% penicillin/streptomycin at 37°C in 5% CO2.

extract_protocol:
Total RNA was isolated using miRNeasy kit. PolyA RNA was isolated using TruSeq RNA Library Prep Kit following Illumina guidelines.
Sequencing library preparation and reactions were conducted at City of Hope’s Integrative Genomics Core using Illumina protocols on HiSeq 2500 platform (Illumina, San Diego, CA).

tag: passages

tag: vector


title: HTBE_12h_H5N1-UV_r2


title: HTBE_12h_H5N1-UV_r1


title: Empty Vector 2 (EV2)

value: empty


title: siRNA Control Panc1 Rep3

tag: cell line


title: Select3

status: Public on Aug 22 2018

city: Strasbourg

first: Jean-Noel

last: FREUND

address: 3 avenue Molière

country: France

institute: Inserm

data_processing:
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Aligned reads on rRNA human sequences were removed from FASTQ files before performing the analysis. The remaining reads have then been aligned onto the hg38 human genome assembly with tophat 2.0.14 and bowtie 2.1.0.0. Number of reads counted on each gene for each sample has been realized with the htseq-count script, provided with the HTSeq framework version 0.6.1, using features coming from the Ensembl database version 83.
Genome_build: hg38
Supplementary_files_format_and_content: The htseq files are in text format and contain the number of reads aligned on each gene

source_name: RNA samples extracted from colon cancer cells grown on soft vs rigid substrates.

growth_protocol: Colorectal adenocarcinoma epithelial SW480 cells (ATCC, CCL-228) were grown in RPMI-1640 medium (Invitrogen) supplemented with glutamax, 10% FBS, 100 µg/mL penicillin, 100 µg/mL streptomycin, 0.025 U/mL insulin, 50 mg/mL hydrocortisone and 1.25 mg/mL G418 maintained at 37 °C with 5% CO2.

extract_protocol:
RNA was extracted using guanidine thiocyanate and  phenol in a monophase solution (TRI reagent).
RNA-Seq libraries were generated from 200 ng of total RNA using TruSeq Stranded mRNA Sample Preparation Kit (Illumina, Part Number RS-122-2101). Briefly, following purification with poly-T oligo attached magnetic beads, the mRNA was fragmented using divalent cations at 94oC for 8 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during second strand cDNA synthesis using DNA Polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double stranded cDNA fragments, the products were purified and enriched with PCR (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC) to create the cDNA library. Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman Coulter) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.

treatment_protocol: 1) Preparation of the culture substrata: PLL (Poly-L-Lysine, MW = 5.7 x 104 Da) and HA (Hyaluronic Acid, MW = 4.0 x 105 Da) were used for buildup (PLL/HA)24 films, and PSS (Poly Sodium 4-Styrenesulfonate, MW = 7.0 x 104 Da) and PAH (Poly Allylamine Hydrochloride, MW = 7.0 x 104 Da) for (PSS/PAH) capping films which were deposited on top of (PLL/HA)24 strata. PLL, HA, PSS, and PAH were dissolved at 1 mg/mL in a buffer solution containing 150 mM NaCl and 20 mM of tris(hydroxymethyl)-aminomethan (TRIS) at pH 7.4, and all rinsing steps were performed in the same buffer. (PLL/HA)24 strata and (PSS/PAH) capping films were prepared using a dipping machine, on Glass slides. The apparent moduli of the (PLL/HA)24-(PSS/PAH) film is about 20 kPa. For this film, we shall use the short hand notation E20. 2) SW480 cells were seeded on E20-coated coverslips placed in 24-well plate at 1.105 per cm2. The E20 substrate with the plated SW480 cells was moved for 24h into a new 24-well plate in order to avoid collection of cells that would in contact with the plastic surface of the well. The surviving cells were transferred by trypsin addition and amplified for two weeks on Glass. Then, the seeding procedure on E20 for 24h and amplification on glass was repeated 3 times. After the amplification step following the 4th seeding on E20, cells were collected for RNA extraction. For control experiments, SW380 cells were seeded on glass coverslips instead of E20-coated coverslips.

tag: substrate structure

value: soft

value: colon cancer


title: Empty Vector 1 (EV1)


title: CTRL1

value: rigid


title: CTRL2


title: Select1


title: Select2


title: Dnm3os 1


title: WG17015_H12-W08

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 1

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

data_processing:
Reads flagged as invalid by the Illumina HiSeq control software were discarded. In the remaining reads, any 3′ bases with a quality score of B were removed. If any of the six UMI bases in the 5' end had a Phred score <17 the read was discarded, else the UMI was cut away and saved. The following three bases had to be G for the read to be kept. These, as well as any additional up to totally nine (presumably template-switch derived) G:s were cut away. Reads were discarded if the remaining transcript-derived sequence ended in a poly(A) sequence leaving <25 bases, or if the remaining sequence consisted of fewer than six non-A bases or a dinucleotide repeat with fewer than six other bases at either end. The reads were sorted by the well, as defined by the combined two index read barcodes.
Alignment was performed using the Bowtie aligner, allowing for up to three mismatches and up to 24 alternative mappings. Reads with no alignments were realigned against an artificial chromosome, containing all possible splice junctions arising from the exons defined by the UCSC transcript models. The coordinates of aligned splice junctions were translated back to the corresponding genomic positions.
For expression level calculation, the exons of each locus with several transcript variants were merged to a combined model representing total expression of the locus. To account for incomplete cap site knowledge, the 5′ ends of all models were extended by 100 bases, but not beyond the 3′ end of any upstream nearby exon of another gene of the same orientation.
The annotation step was performed separately for each well. For each genomic position and strand combination the number of reads in each UMI was counted. Any multiread that mapped  to some repeat outside exons was assigned randomly as one of these repeats and did not contribute to the transcript corresponding to the exon. Else, if the multiread mapped to some exon, and not to any repeat outside exons, it was assigned to the exon where it was closest to the transcript model 5′ end. If it had no exon mapping, it was assigned randomly at one of the mappings. The total number of molecules at each mapping position was determined by the number of distinct UMIs observed. Any UMI represented by only a single read was excluded, in order to reduce false molecules due to PCR and sequencing errors. The raw UMI count was corrected for the UMI collision probability. For the human samples all reads mapping anywhere within the whole locus, defined as the region (including actual introns) from the start of the 100 base 5'end extension of the first exon up to the end of the last exon, were counted as exon-derived.
Genome_build: UCSC mm10
Supplementary_files_format_and_content: Tab-delimited table of total number of detected mRNA molecules from each gene in each cell

source_name: striatum

growth_protocol: Mice were housed on a standard 12+12 light/dark cycle with 2-5 mice per cage

extract_protocol:
Dorso-lateral striatum (bregma, AP: 1.42 to -0.58 mm) from Lhx6Cre:RFP, Pvcre:TdT and Htr3aEGFP mice was dissociated into a single cell suspension as described in Zeisel et al., Science vol. 347  p.1138 (2015) . Dissociated cells were FACS sorted based on fluorescence (RFP+ and EGFP+) and positive cells were sorted using BD FACSAria II SORP into a 9600 multiwell array (2400 cells/plate) prefilled with 50 nl lysis buffer (500nM STRT-P1-T31, 4.5nM dNTP, 2% Triton-X-100, 20mM DTT, 1.5U/μl TaKaRa RNase Inhibitor), followed by 3min lysis at 72°C.
85nl reverse transcription (RT) mix (2.1X SuperScript II First-Strand Buffer, 12.6mM MgCl2, 1.79M betaine, 14.7U/μl SuperScript II, 1.58U/μl TaKaRa RNase Inhibitor, 10.5μM P1B-UMI-RNA-TSO) were dispensed and RT carried out 42°C for 90 minutes. 96 Transposome stocks were assembled (6.25μM barcoded adapter (STRT-Tn5-Idx[1-96]), 6.25μM Tn5 transposase, 40% glycerol), 37°C 1h.  Tn5 reactions were assembled with 3μl transposome and 2μl amplified cDNA, in a total 20μl 1x CutSmart buffer (NEB), and incubated at 55°C 20min.

tag: strain

tag: Sex

value: male

tag: postnatal days

value: Pthlh


title: WG17015_H11-W23


title: WG17015_H12-W02


title: WG17015_H12-W32


title: WG17015_H12-W04


title: WG17015_H11-W17


title: WG17015_H12-W26


title: WG17015_H11-W14


title: WG17015_H11-W27

value: Npy+Sst+


title: WG17015_H12-W10


title: WG17015_H11-W16


title: WG17015_H12-W20

value: Th


title: WG17015_H12-W12


title: WG17015_H12-W28


title: WG17015_H11-W25


title: WG17015_H12-W22


title: CTRL3


title: WG17015_H12-W18


title: WG17015_H11-W11

value: Chat


title: WG17015_H11-W12


title: WG17015_H11-W10


title: WG17015_H11-W08


title: WG17015_H11-W28


title: WG17015_H11-W06


title: WG17015_H11-W05


title: WG17015_H11-W20


title: WG17015_H11-W04


title: WG17015_H11-W13


title: HCC70_shRNA_NFIB_2

status: Public on Aug 25 2018

city: Jerusalem

first: Ittai

last: Ben-Porath

address: Ein-Kerem

department: Department of Developmental Biology and Cancer Research

country: Israel

institute: The Hebrew University Faculty of Medicine

data_processing:
Read data demultiplxing & mapping was done using the CELseq pipeline script https://github.com/yanailab/CEL-Seq-pipeline
UMI correction was done using the formula transcript # = -(4^umi_length)*ln(1-(umi_count/umi_length) to convert UMI into transcript
Transcript data was grouped from two sequencing runs to form a single matrix
Data was normalized into transcripts per million (tpm) by dividing the number of each transcripts for each gene with the total number of transcripts for the sample and multiply by 500K
Genome_build: Ensembl GRCh37
Supplementary_files_format_and_content: Tab-delimited text files include RPKM values for each sample

source_name: HCC70  breast cancer cell line

growth_protocol: Cell lines were obtained from ATCC. HCC70 cells were grown in 96 well plates with RPMI medium containing 10% FBSand supplemented with penicillin and streptomycin.

extract_protocol:
RNA was extracted using the Single Cell RNA Purification Kit (Norgen) and 3’ cDNA was synthesized and barcoded in batches of 12 samples, followed by RNA synthesis and amplification by in vitro transcription based on the CELseq protocol DOI: 10.1186/s13059-016-0938-8. Some samples were ran a second time due to low representation in the first run.
Libraries were prepared and libraries were prepared and sequenced using Illumina’s RNA sequencing protocol.

treatment_protocol: Cells were infected with the indicated shRNA or treated as indicated

tag: shRNA id/treatment


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