title: Subculture with downshift

city: Leeds

first: David

last: Romero A.

state: West Yorkshire

address: Woodhouse lane

country: United Kingdom

institute: University of Leeds

description: FRT-seq: amplification-free, strand-specific transcriptome sequencing

data_processing:
RNA sequences from the global analysis (FRT-seq) were processed in-house using Galaxy (Goecks et al. 2010. Genome Biol 11: R86). This involved trimming adaptor sequence and masking for low-quality sequence.
Reads were mapped to the P. acnes genome using Bowtie 2.0 (Langmead and Salzberg 2012. Nat Methods 9: 357) with custom parameter (-y -a --best --strata).
Using BEDTools, we determined or each position in the genome the number of times it was read by global RNA-seq.
The counts for each position were then used to construct bedGraphs for the forward and reverse strands of each of the two libraries
Genome_build: Reference genome was that of Propionibacterium acnes strain KPA171202 (NCBI accession number AE017283)
Supplementary_files_format_and_content: bedGraphs of the forward and reverse strands for a pair of RNA samples isolated from cells subcultured with and without potassium downshift.

organism: Cutibacterium acnes KPA171202

source_name: Batch subculture with potassium downshift

growth_protocol: Propionibacterium acnes strain KPA171202 (Bruggemann et al. 2004. Science 305: 671) was cultivated in an anaerobic workstation (MACS-MG-1000, Don Whitely Scientific) at 34°C under 80% [v/v] N2, 10% [v/v] CO2, and 10% [v/v] H2. All analyses were done using cells cultivated without shaking in 100 mL of modified Holland Synthetic Medium (HSM) (Holland et al. 1979. J Appl Bacteriol 47: 383-394). Inocula were prepared in two stages. First a single colony isolated from the surface of reinforced clostridial agar (Hirsch and Grinstead 1954. J Dairy Res 21: 101) was used to inoculate 10 mL of tryptone-yeast extract-glucose (TYG) broth (Kim and Naylor 1966. Appl Microbiol 14: 690). After growing to stationary phase, an aliquot was used to inoculate 100 mL of TYG broth to an O.D.600nm of 0.2. The culture was then incubated to an O.D.600nm of 1.0, after which cells were harvested by centrifugation (3,000 x g for 20 min) and washed by first resuspending in 10 mL of HSM (pre-warmed to 34oC) and then repeating the harvesting step. Finally, the cells were resuspended in 10 mL of pre-warmed HSM and an appropriate aliquot was used to inoculate 100 mL of pre-warmed HSM to an O.D.600nm of 0.2.

extract_protocol:
Cell pellets of P. acnes were resuspended in Kirby mix (Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich.), 100 µL per 1 O.D.600nm unit, and transferred to Lysing Matrix B tubes containing fine silica beads (MP Biomedical). Tubes were then placed in a high-speed benchtop homogenizer (Fastprep-24, MP Biomedical; set at 6.5 M/s). Cells were lysed by three cycles of homogenisation for 1 min with cooling between each cycle in an ice-water bath for 1 min. Lysates were extracted using an equal volume of acidic phenol: chloroform: isoamyl alcohol (50: 50: 1) and then chloroform: isoamyl alcohol (49: 1). Nucleic acid in the aqueous phase was precipitated by adding NaCl to 150 mM and 2.5 x volumes of 100% [v/v] ethanol, chilling at -20°C for 1 h, and then harvested by centrifugation (13,000 x g for 30 min at 4°C) . Nucleic acid pellets were washed twice with 700 µL of 70% [v/v] ethanol, air dried for 5 min and resuspended in RNase-free water. To remove contaminating DNA, samples were treated with DNase I using conditions described by the vendor (Ambion) and extracted with phenol: chloroform as described above. The concentration and integrity of RNA samples were determined using a NanoDropTM 1000 spectrophotometer (Thermo Fisher Scientific) and agarose gel electrophoresis (Kime et al. 2008. In RNA Turnover in Bacteria, Archaea and Organelles, Vol 447 (ed. LE Maquat, CM Arraiano), pp. 215), respectively. Prior to constructing libraries, samples were enriched for mRNA using MICROBExpress-Bacteria beads, as described by the manufacturer (Ambion).
Library construction was done by Dr. Lira Mamanova of the Wellcome Trust Sanger Institute using a published protocol (Mamanovaet al. 2010. Nat Methods 7: 130)

treatment_protocol: Potassium downshift (depletion from growth media) was performed by growing a 100 mL culture of P. acnes in HSM to an O.D.600nm of 1.0, after which the culture was separated into two equal halves and cells harvested by centrifugation. One half was washed using standard HSM, then used to inoculate 100 mL of fresh HSM and reincubated. The other half was processed in the same way, except the HSM lacked potassium (KH2PO4 and K2HPO4). After 1 h of reincubation, 12.5 mL of stop solution (95% [v/v] ethanol; 5% [v/v] phenol) was added to inhibit cell metabolism (Kime et al. 2008. In RNA Turnover in Bacteria, Archaea and Organelles, Vol 447 (ed. LE Maquat, CM Arraiano), pp. 215), and the cells were harvested by centrifugation. When necessary, cell pellets were stored frozen at -80C.

tag: strain

tag: sample type

value: Clinical isolate

tag: potassium downshift

value: with


title: Subculture without downshift

status: Public on May 15 2013

type: SRA

library_source: transcriptomic

molecule: total RNA

source_name: Batch subculture without potassium downshift

value: without


title: Ctrl_IP2

city: Heidelberg

first: Georg

last: Stoecklin

address: Im Neuenheimer Feld 280

country: Germany

institute: German Cancer Research Center

description: Sample 5

data_processing:
Basecalling with CASAVA v1.8.2
Mapping to the mouse genome NCBI build 37 with BWA.
Reads were summarized for gene models with the Bioconductor package easyRNASeq v1.2.5. The exon coordinates of RefSeq annotated mRNAs were obtained from the refGene table of the UCSC genome browser.
Differential abundance was tested for with the Bioconductor package DESeq v1.8.3.
Genome_build: NCBI build 37
Supplementary_files_format_and_content: The 'RoquinIP_processed.txt' file contains, for each sample, the count table obtained with easyRNASeq, the fold changes and the p-values computed with DESeq.

organism: Mus musculus

growth_protocol: RAW264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (PAA Laboratories), 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all PAN Biotech) at 37°C in 5% CO2.

extract_protocol:
Cells were mechanically disrupted using a tissue lyser. RNA was isolated before (Input) or after IP (ctrl IP or Roquin IP) with TriFast reagent.
RNA libraries were prepared for sequencing using the TruSeq RNA Sample Preparation Kit.

treatment_protocol: RAW264.7 cells were treated with LPS (100 ng/ml, Sigma, L2630, E. coli serotype O111:B4) for 2 h.

tag: cell line

tag: ip antibody/beads information

value: protein A/G beads alone

tag: molecule subtype

value: RNA purified from control IP without antibody


title: Roquin_IP2

description: Sample 2

value: rabbit polyclonal, Bethyl, 514A and 515A plus protein A/G beads

value: RNA purified from Roquin IP


title: BALBfabD1

status: Public on May 16 2013

city: Seattle

first: Wonjong

last: Moon

state: WA

address: 307 Westlake Avenue North, Suite 500

country: USA

institute: Seattle BioMed

description: BALB/cJ-fabb/f- Day1

data_processing:
Illumina Genome Analyzer II GA2 HiSeq and CASAVA 1.8.2 for base calling
Additional quality control was applied to the datasets by clipping or removing poor quality reads and N's by ea-utils toolkit (http://code.google.com/p/ea-utils/), with -p 10 and -q 7 settings, then mapped to P. yoelii YM strain reference  genome using tophat version 1.4.1 with default parameters
The differential transcript abundances between samples were measured in Fragments per kilobase of transcript per million mapped fragments (FPKM) using  Cufflinks and Cuffdiff by a protocol from Trapnell C. et al. (2012) Nature.
RNA-Seq differential analysis was visualized by CummeRbund
Transcripts with P values of less than 0.01 and at least 4-fold difference in expression (i.e., log fold > 2) were selected to identify the most differentially regulated genes.
Coverage plots were generated to examine the RNA distribution of reads across selected genes-of-interest by comparing the number of unique reads present at each nucleotide position
Genome_build: Mus musculus UCSC version mm10
Supplementary_files_format_and_content: tab-delimited text files (cuffdiff gene_exp.diff) include gene differential FPKM values for each Sample

source_name: perfused mouse liver

extract_protocol:
RNA was isolated by the Qiagen RNeasy Miniprep Kit with an added DNaseI treatment module.
Library was constructed by Expression Analysis, Inc using the Illumina TruSeq RNA sample preparation kit using standard methods.

treatment_protocol: Mice were infected intravenously with 100,000 transgenic P. yoelii fabb/f- salivary gland sporozoites or with debris from uninfected mosquito salivary glands. One day post infection mouse livers were perfused with HBSS.  Livers were then flash frozen in liquid nitrogen until extraction.

value: BALB/cJ

tag: tissue

value: mouse liver

tag: infection

value: 24 hours post infection with Py fabb/f-knock out transgenic  P. yoelii sporozoite infection


title: Input2

description: Sample 8

value: RNA purified from lysate before IP


title: WildtypeMouseRXRaChIPSeq

city: Sacramento

first: Yuqi

last: He

state: California

address: Rm3400, Research III, UC Davis Medical Center, 4645 2nd Ave

institute: UC Davis Medical Center

library_source: genomic

data_processing:
Basecalls performed using CASAVA version 1.4
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie
Peaks were called by using MACS
Genome_build: mm9
Supplementary_files_format_and_content: Bar file was generated from the bed file outputted from MACS

molecule: genomic DNA

source_name: Liver

extract_protocol:
Grand in liquid nitrogen, then fixed, sonication, pull down by antibody
Illumina True-Seq Kit

tag: gender

value: Male

tag: age

value: 3 Month

value: liver


title: FFPE_Total

city: Cambridge

first: Joshua

middle: Z

last: Levin

state: MA

address: 320 Charles Street

institute: Broad Institute of MIT & Harvard

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_FFPE_isoform.txt
PROCESSED DATA FILE NAME: GSE40705_FFPE_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where neither mate aligned to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
Genome_build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
Supplementary_files_format_and_content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the three protocols for the sample (RNaseH, RiboZero, and Total RNA)

organism: Homo sapiens

source_name: human normal kidney section (Cybrdi)

extract_protocol: Library construction method: Standard

tag: total rna input

tag: rna state


title: Pancreas_RNaseH

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_Pancreas_gene.txt
PROCESSED DATA FILE NAME: GSE40705_Pancreas_isoform.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where neither mate aligned to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
Genome_build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
Supplementary_files_format_and_content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the three protocols for the sample (RNaseH, RiboZero, and Total RNA)

source_name: human normal pancreas tissue (Zyagen)

extract_protocol: Library construction method: RNase H rRNA-depletion (Morlan et al. PLOS One, 2012)

value: partially degraded


title: FFPE_RiboZero

extract_protocol: Library construction method: Ribo-Zero (Epicentre)


title: FFPE_RNaseH


title: Pancreas_Total


title: Pancreas_RiboZero


title: miRNA from CMS (small RNA)

city: Hangzhou

first: Jinghua

last: Yang

address: Yuhangtang 388

country: China

institute: Zhejiang University

description:
miRNA from CMS
small RNA sequencing

data_processing:
The small RNAs (sRNA) libraries of reproductive development from Brassica juncea CMS and MF were constructed by RNAs from all floral buds of one intact inflorescence. Deep sequencing generated a total number of 11845753 from MF and 3339182 from CMS raw reads (Supporting information Table S1). After removal of corrupted adapter sequences, reads with length <15 and >32 and junk reads, 7331575 and 3192676 mappable reads were finally obtained for MF and CMS libraries, respectively (Supporting information Table S1, S2). The majority of sRNA were 21-24 nucleotides (nt) for both two libraries that are within the typical size range for Dicer-derived products, in which the 21 nt sRNA was the most abundantly presented, followed by the 24 nt sRNA as the second largest percentage representing the class of endogenous small RNAs family (Fig. 1). We further compared the unique sRNAs between MF and CMS, which were 228 unique sRNAs for MF and 173 sRNAs for CMS. The number of the 21-24 nt unique sRNAs in MF was 173 and 141 in CMS, of which 21 nt unique sRNA was 98 in MF and 90 in CMS (Supporting information Table S3)
To identify the miRNAs from the reproductive development of Brassica juncea, sRNA sequences identified from Brassica juncea by deep sequencing were compared with the currently known mature plant miRNAs in miRBase 18.0. In miRbase, there are 291 Arabidopsis thaliana pre-microRNAs (pre-miRNAs) been reported corresponding to 328 mature miRNAs. Here, 99 pre-miRNAs corresponding to 94 miRNAs had been detected, besides, 63 5’ or 3’ new mature microRNAs corresponding to reported pre-miRNA in miRbase had been first time detected (supporting information Table S4, Table S5). There were 4 novel pre-miRNAs corresponding to 6 mature miRNAs originating from other pre-miRNAs except Arabidopsis thaliana. Among them, 3 5’ or 3’ new mature miRNAs corresponding to reported pre-miRNA in miRbase had been first time detected in Brassica juncea. The other 2 pre-miRNAs corresponding to 2 mature miRNAs come from 2 other plant species (Glycin max and Rehmannia glutinosa) can also been mapped to Arabidopsis thaliana genome had been first time detected here (Supporting information Table S6). 5 novel miRNAs originating from 7 pre-miRNAs that can not be mapped to Arabidopsis thaliana genome had been identified, of which these novel miRNAs were mapped to the other plant species genome and the extended sequences at the mapped positions of the genome potentially from hairpins (Supporting information Table S7). 17 novel miRNAs originating from pre-miRNAs that can not be mapped to Arabidopsis thaliana genome had been identified, of which these novel miRNAs were mapped to the other plant species genome and the extended sequences at the mapped positions of the genome, however failed in hairpin structure prediction (Supporting information Table S8). 13 other novel miRNAs which were conserved in plant species were observed, but they can not be found in Arabidopsis thaliana genome (Supporting information Table S9).
To identify new miRNAs, we removed all reads with low abundance (<5) from the sequence data. The data were used to query the non-coding RNAs sequence at database (ftp://ftp.arabidopsis.org/Sequences/ATH cDNA EST sequences FASTA/), the Rfam (ftp://ftp.sanger.ac.uk/pub/databases/Rfam/9.1/) database and repeat-repbase (http://www.girinst.org/repbase/update/index.html). Then, the consensus surrounding the genomic regions of each miRNA was retrieved and its secondary structure was formed according to public method (Zuker, 2003). All genomic loci-generating sRNAs that can be fold into the secondary structure were considered as miRNA candidate. Totally, 78 pre-miRNAs corresponding to 93 unique mature miRNAs had been first identified here, of which these candidate miRNAs were originated from predicted RNA hairpins (Supporting information Table S10). The secondary hairpin structures of the representative miRNAs were shown in Fig.2 and all the secondary hairpin structures of candidate miRNAs were last listed in Supporting Information Fig. S1. Interestingly, some new candidate miRNAs were organelle-derived non-coding sRNA, of which miRNAs, PC-5p-13, PC-3p-14, PC-3p-39, PC-5p-40, PC-3p-54, PC-3p-72, PC-5p-75, PC-5p-88 and PC-3p-90, are derived from chloroplast, and miRNAs, PC-5p-17, PC-3p-18, PC-3p-25 and PC-5p-26, are derived from mitochondria. Several pre-miRNAs sequences of new candidate miRNAs were aligned to be located in nuclear and mitochondrial genome, including pre-miRNA22/ pre-miRNA23, pre-miRNA24/ pre-miRNA25, pre-miRNA73/ pre-miRNA74 and pre-miRNA75/ pre-miRNA76 (Supporting information Table S10).  Finally, 290 miRNAs including known and new candidate were identified in reproductive development of MF and CMS Brassica juncea (Table 1).
miRNAs in MF and CMS were for differentially expression analysis using online service IDEG6 (http://telethon.bio.unipd.it/bioinfo/IDEG6_form/). The selection methods of differentially expression were Audic and Claverie, Fisher exact test and Chi-squared 2×2, and the selection threshold was 0.01 (http://telethon.bio.unipd.it/bioinfo/IDEG6_form/detail.html#AC). Finally, 47 miRNAs had been observed to be differentially expression between MF and CMS. Among these differentially expressed miRNAs, 25 miRNAs and 12 miRNAs were observed to be down- and up-regulated respectively according the sequencing reads (Table 2). The expressions of 8 selected miRNAs were tested by using quantitative real-time RT-PCR (qRT-PCR) analysis, which further validated the differentially expression data obtained from sequencing on the whole (Fig. 3). The expression pattern of 6 of 8 miRNAs was consistent with the sequencing reads. The expressions of ath-miR396b and ath-miR159_R-2 were shown no significant differences between CMS and MF, however, the expressions of these two miRNAs were detected to be down-regulated in CMS (Fig. 3).
we performed a genome-wide sequencing of miRNA-cleaved mRNA and Cleavland analysis based on recently developed high-throughput degradome sequencing technology (Addo-Quaye et al., 2008; German et al., 2008; Addo-Quaye et al., 2009).  The genome build(s)/miRBase version(s) used in the analysis of our data are TAIR10 of Arabidopsis arabidopsis genome from http://www.arabidopsis.org/ and miRbase version 19.0.

organism: Brassica juncea

source_name: floral buds

growth_protocol: when CMS and MF lines were flowering under long-day length.

extract_protocol:
Total RNA of four samples was extracted using mirVana miRNA Isolation Kit (Ambion, Austin, USA) according to the manufacturer's protocol. Briefly, 10 μg of RNA sample was size fractionated with YM-100 column (Millipore). The larger RNA fraction (>100 KDa) was removed. The smaller RNA fraction (<100KDa) was further fractioned on a 15% tris-borate-EDTA (TBE) urea polyacrylamide gel and 15-50nt fraction was obtained.
Subsequently, 5' and 3' RNA adapters were ligated to the small RNA by T4 RNA ligase (Promega), followed by reverse transcription and PCR products purification. Purified products were quantified on the TBS-380 mini-fluorometer (Turner Biosystems) using Picogreen dsDNA quantitation reagent (Invitrogen) and diluted to 10 nM and transferred for sequencing on the Illumina/Solexa G1 sequence (Illumina, San Diego, USA ).

tag: developmental stage

value: reproductive development

value: floral buds

tag: sequencing method

value: small RNA sequencing

tag: line

value: cytoplasmic male sterile (CMS)


title: SMART

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where neither mate aligned to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
Genome_build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
Supplementary_files_format_and_content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the three protocols for the sample (RNaseH, RiboZero, and Total RNA)

source_name: human CML cell line K-562 grown in tissue culture (Ambion)

extract_protocol: Library construction method: SMARTer (Clontech)

value: 1 ng

value: intact


title: microRNA from sample C constructed by the kit from NEB  [LW_CN_1]

city: Milwaukee

first: Liang

last: Wang

state: Wisconsin

address: 8701 Watertown Plank Rd.

department: Pathology

institute: Medical College of Wisconsin

description: Characterization of human plasma-derived exosomal RNAs

data_processing:
Perl Scripts were developed to process data from RNA sequencing. Raw reads were firstly extracted from FASTQ files, and trimmed with sequencing quality control of Q > 13 , and then cleaned up 3’ adaptor sequences within read sequences. Those prepared sequences filtered with the length ≥16 nt were combined into a FASTA file and aligned against human miRNA sequences downloaded from miRBase (Release 19, 2043 entries) and human genome reference sequences downloaded from NCBI ftp site (Release 103) with Bowtie (version 0.12.8) , of which the parameters for alignment were -m 3 -n 1 -f -a -best --strata. Normalization of miRNA profiling was the read counts of a target miRNA by million bases versus the sum of read counts of all mappable miRNAs on human miRNA sequences.
Genome_build: miRNA from miRBase(release19)
Supplementary_files_format_and_content: Data frame of normalized miRNA transcription level  in *.txt

extract_protocol:
we selected plasma samples (samples A, B and C) from 3 anonymous blood donors and split each sample into two for technical replication.
We tested the six samples (A1 and A2, B1 and B2, C1 and C2) using two small RNA library preparation kits: NEBNext Multiplex Small RNA library Prep Set from New England Biolab (NEB) and NEXTflex Small RNA Sequencing Kit from Bioo Scientific (BS). We also tested Illumina’s TrueSeq Small RNA Sample Preparation Kit (ILMN) in sample A1 and A2. Together, we tested these plasma samples by sequencing 14 indexed libraries.
Human plasma samples were obtained from Mayo Clinic and stored at -80°C before use. Exosomes were isolated from 250 µL of plasma prepared from whole blood using the ExoQuick Exosome Precipitation Solution (SBI) according to the manufacturer’s instructions with minor modifications. Briefly, the plasma was incubated with Thromboplastin D (Thermo) for 15 min at 37oC. After centrifugation at 10,000 rpm for 5 min, the supernatant was mixed with 75 µL of ExoQuick Solution and RNase A (Sigma) at final concentration of 10 µg/mL. The mixture was kept at 4oC overnight and then further mixed with 150 units/mL of murine RNase inhibitor (NEB) before centrifugation at 1500 g for 30 min. The exosome pellet was dissolved in 25 µL 1×PBS, 2 µL of which was reserved for evaluation of exosome size and concentration using NanoSight LM 10 instrument (Particle Characterization Laboratories). The remainder was subjected to RNA extraction immediately. The usage of human biospecimen for this study was approved by Institutional Review Board of Medical College of Wisconsin and Mayo Clinic.
RNA was prepared by using miRNeasy Micro Kit (QIAGEN). Twenty-three µL of exosome suspension was mixed with 700 µL QIAzol Lysis buffer, and was processed by the standard protocol from the manufacturer. The extracted RNA was eluted with 14 µL of RNase-free water. The quantity and quality of the RNA were determined by Agilent Bioanalyzer 2100 with Small RNA Chip and RNA 6000 Pico Kit as indicated (Agilent Technologies).
The concentration and the size distribution of isolated exosomes were measured using NanoSight. Prior to sampling, the sample solutions were homogenized by vortexing, followed by serial dilution to the final dilution of 1:100,000 in 0.2 µm-filtered 1x PBS. NIST (National Institute of Standards and Technology) traceable 97 nm ± 3 nm polystyrene latex standards were added and analyzed in conjunction with the diluted exosome solution to validate the operation of the instrumentation. A blank 0.2 µm-filtered 1x PBS sample was also run as a negative control. Each sample analysis was conducted for 90 seconds. The Nanosight’s automatic analysis settings (High Sensitivity, Blue laser [405 nm, 645 mW]) were utilized for the processing of the data. All samples were evaluated in triplicates.

value: blood

tag: fraction

value: exosome


title: microRNA from sample C constructed by the kit from NEB  [LW_CN_2]


title: microRNA from sample C constructed by the kit from Bioo scientific  [LW_CB_2]


title: microRNA from sample C constructed by the kit from Bioo scientific  [LW_CB_1]


title: miRNA from MF (small RNA)

description:
miRNA from MF
small RNA sequencing

value: maintainer fertile (MF)


title: microRNA from sample B constructed by the kit from NEB  [LW_BN_1]


title: microRNA from sample B constructed by the kit from NEB  [LW_BN_2]


title: microRNA from sample B constructed by the kit from Bioo scientific  [LW_BB_2]


title: A5-post-prednisolon

city: Amsterdam

first: Ching Yong

last: Yick

address: Meibergdreef 9

department: Respiratory Medicine

country: Netherlands

institute: Academic Medical Center

data_processing:
The general quality of the sequencing run was checked using the GS Run Browser version 2.6 (Roche).
Sequence reads were mapped against the human genome (hg19; UCSC) using the GS Reference Mapper version 2.6 (Roche).
Comparison of the numbers of reads per gene between the prednisolon and placebo study group was carried out with the R package DESeq. RNA-Seq reads were assumed to follow a negative binomial distribution.
With 12 subjects in total, 4 biopsy specimens per subject, and a significance level of 0.0001, we expected to find 68 true and 2 false significant genes. This resulted in a false discovery rate of approximately 3%.
Genome_build: hg19
Supplementary_files_format_and_content: Comma-separated files contain mapped genes and the following information: number of unique matching reads, percent of  all unique matches, percent of all reads

source_name: airway smooth muscle

extract_protocol: 4 endobronchial biopsies per subject were collected during a bronchoscopy. Airway smooth muscle was collected from the biopsies by laser capture microdissection and total RNA isolated with TRIzol. cDNA was prepared using the Ovation RNA-Seq System (NuGEN). cDNA libraries of >350bp were constructed using SPRIworks Fragment Library System II (Beckman Coulter) with the addition of GS FLX Titanium Rapid Library multiplex identifier (MID) adaptors (Roche). Emulsion-based clonal amplification was performed using the GS FLX Titanium emPCR Kit (Lib-L; Roche). RNA-Seq was performed using the GS FLX+ instrument (454/Roche).

tag: subject

tag: treatment

value: post-prednisolon

value: airway smooth muscle layer from airway wall

tag: health status

value: Atopic asthma


title: A6-post-prednisolon


title: microRNA from sample A constructed by the kit from NEB  [LW_AN_2]


title: microRNA from sample A constructed by the kit from Bioo scientific  [LW_AB_2]


title: microRNA from sample B constructed by the kit from Bioo scientific  [LW_BB_1]


title: NuGEN 1f

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999537_NuGEN_1f.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999537_NuGEN_1f.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.

extract_protocol: Library construction method: Ovation RNA-Seq (NuGEN)

tag: cell type

value: CML

value: fragmented


title: microRNA from sample A constructed by the kit from Bioo scientific  [LW_AB_1]


title: microRNA from sample A constructed by the kit from NEB  [LW_AN_1]


title: A4-post-prednisolon


title: NuGEN 1i

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999536_NuGEN_1i.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999536_NuGEN_1i.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.

tag: cell line


title: TruSeq

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999535_TruSeq.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999535_TruSeq.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.

extract_protocol: Library construction method: TruSeq RNA-Seq (Illumina)


title: Total

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999534_Total.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999534_Total.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.


title: PolyA

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999533_PolyA.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999533_PolyA.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.

extract_protocol: Library construction method: Oligo (dT) Dynabeads (Invitrogen)

value: 1000 ng (10 ng polyA+)


title: A3-post-prednisolon


title: Ribo-Zero

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999531_Ribo-Zero.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999531_Ribo-Zero.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.


title: A2-post-prednisolon


title: A1-post-prednisolon


title: NuGEN 100f

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999532_NuGEN_100f.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999532_NuGEN_100f.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.


title: A6-pre-prednisolon

value: pre-prednisolon


title: A5-pre-prednisolon


title: RNase H NS

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999530_RNase_H_NS.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999530_RNase_H_NS.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.


title: A4-pre-prednisolon


title: RNase H

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999529_RNase_H.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999529_RNase_H.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.


title: microRNA from sample A constructed by the kit from Illumina [LW_AI_2]


title: A3-pre-prednisolon


title: microRNA from sample A constructed by the kit from Illumina [LW_AI_1]


title: A2-pre-prednisolon


title: DSN-lite PCR

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999528_DSN-lite_PCR.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999528_DSN-lite_PCR.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.

extract_protocol: Library construction method: Standard followed by DSN treatment


title: DSN-lite

data_processing:
**********************************************************************

PROCESSED DATA FILE NAME: GSM999527_DSN-lite.genes.results.txt.gz
PROCESSED DATA FILE NAME: GSM999527_DSN-lite.isoforms.results.txt.gz
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
Reads where neither mate aligned to rRNA were extracted using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the USCS transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the USCS transcriptome using bowtie version 0.12.7 allowing for one multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quantified using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: rsem sample_name.genes.results and sample_name.isoforms.results documentation on these file formats can be found at http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_K562_Isoform.txt
PROCESSED DATA FILE NAME: GSE40705_K562_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing.  To do this, reads where at least one mate did not align to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice.  The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome.  This was done using bowtie version 0.12.7 allowing for one hit per read.  The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read.  This was the second, final alignment.  The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
genome build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
processed data files format and content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM.  Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file.  Each file contains TPM values calculated for each of the protocols listed.

extract_protocol: Library construction method: Standard without PCR followed by DSN treatment


title: A1-pre-prednisolon


title: A5-post-placebo

value: post-placebo


title: LPD2

city: New Haven

first: Gang

last: Fang

state: CT

address: 266 Whitney Ave

institute: Yale University

description: Sample 14

data_processing:
Raw color space data from SOLiD sequencing was mapped to the C. crescentus NA1000 chromosome (NC_011916) (plus and minus strands, separately) using SOCS software with a mismatch cutoff of 5 nucleotides (see PMID:18842598 for SOCS reference ). We assigned weights of 1, 0.95, 0.9, 0.85, 0.8 and 0.75 to reads with 0 to 5 mismatches, respectively, when summing them together to get the RNA raw coverage.
We employed a dynamic programming segmentation algorithm from the tillingArray (R package) to divide the CV curve into segments. We removed segments with CV >1 before quantifying gene expression. We then calculated the weighted mean coverage in the remaining segments that fell within annotated CDS or RNA coordinates as gene expression value.
Normalization: in each of the 15 samples, genes with ≤1000x follow power-law distributions with very similar parameter (alpha is about 1.75), and a few extreme highly expressed genes (>1000x) do not follow the major power-law distribution in either of the sample. Genes were thus divided into two groups (≤1000x and >1000x), and normalized by the sum of each group separately.
Genome_build: Caulobacter crescentus NA1000; GenBank ID CP001340.1
Supplementary_files_format_and_content: CSV file with gene expression in 15 samples; Scores represent gene (CDS and non-coding RNA) expression value in number of RNA coverage; Genome annotation is from GenBank CP001340.1; Gene ID is taken from GenBank annotation

organism: Caulobacter vibrioides

source_name: Caulobacter crescentus late predivisional cells

growth_protocol: C. crescentus NA1000 (also known as CB15N) was grown at 30ºC in M2G until the exponentially growing culture reached an OD660 of about 0.3. Cell synchronization, which includes a centrifugation in a density gradient of silica (Ludox) at 4°C using 1 L of culture. After synchronization, the purified swarmer cell population was resuspended in pre-warmed M2G medium. A total of 5 synchronies were done to obtain 3 time points such that, in total, we obtained three replicates (50 ml aliquots of cells) at 0, 30, 60, 90 and 120 min following synchronization.

extract_protocol:
Total bacterial RNA was isolated using phenol-chloroform extraction.  The quality of the extracted RNA was assessed by agarose electrophoresis; rRNA bands appeared intact and no RNA smear was apparent. RNA samples were immediately frozen and stored at -80oC. RNA samples were later enriched for mRNA using the Invitrogen Ribominus Transcriptome Isolation Kit (Yeast and Bacteria) to remove ribosomal RNA per the manufacturer’s protocols except for the use of custom-made nucleic acid probes (Invitrogen) designed against C. crescentus ribosomal sequences.  All RNA samples were tested for integrity on a BioRad Experion capillary electrophoresis system. Possible residual DNA was removed by addition of Ambion Turbo DNase.
Fifteen sequencing libraries for Applied Biosystems SOLiD system sequencing were created using the Applied Biosystems Whole Transcriptome Library Preparation for SOLiD Sequencing, and individual samples were barcoded using Applied Biosystems Small RNA Expression Kit (SREK) barcodes (per the manufacturer’s protocols).  Transcriptome library preparation was performed for labeling in a strand-specific manner.

tag: strain

value: late predivisional cells

tag: time point

value: 120 min following synchronization


title: LPD3

description: Sample 15


title: A6-post-placebo

tag: tissue


title: LPD1

description: Sample 13


title: A4-post-placebo


title: PD2

description: Sample 11

source_name: Caulobacter crescentus predivisional cells

value: predivisional cells

value: 90 min following synchronization


title: PD3

description: Sample 12


title: A3-post-placebo


title: PD1

description: Sample 10


title: EPD3

description: Sample 9

source_name: Caulobacter crescentus early predivisional cells

value: early predivisional cells

value: 60 min following synchronization


title: A2-post-placebo


title: A1-post-placebo


title: EPD1

description: Sample 7


title: ST3

description: Sample 6

source_name: Caulobacter crescentus stalked cells

value: stalked cells

value: 30 min following synchronization


title: A4-pre-placebo

value: pre-placebo


title: A5-pre-placebo


title: EPD2

description: Sample 8


title: A6-pre-placebo


title: A3-pre-placebo


title: A2-pre-placebo


title: ST1

description: Sample 4


title: SW3

description: Sample 3

source_name: Caulobacter crescentus swarmer cells

value: swarmer cells

value: 0min following synchronization


title: SW2

description: Sample 2


title: ST2

description: Sample 5


title: Magnaporthe infected rice leaf 96 hpi

status: Public on May 14 2013

city: Newark

first: Vidhyavathi

last: Raman

state: DE

address: 531 S college Ave

department: Plant and Soil Sciences

institute: University of Delaware

data_processing:
Adapters were removed from the sequencing reads and matched to the M. oryzae genome (version 6 assembly) from the Broad Institute
Reads with sequencing errors were filtered out using PERL scripts
tRNA, rRNA, snRNA or snoRNAs -matched reads were removed from the total reads, and genome-matched reads were obtained using the program Bowtie, accepting only perfect matches
From the total genome matched reads, distinct genome matched reads were calculated and the abundance of each read was normalized to 5 million units of transcripts per 5 million (TP5M)
Genome_build: Magnaporthe oryzae genome assembly 6 from Broad Institute
Supplementary_files_format_and_content: tab-delimited text file including small RNA count

organism: Pyricularia oryzae

source_name: Infected rice leaf

extract_protocol:
Total RNA was isolated using a standard TRIZOL-based method
Small RNA libraries were generated as described by Lu et al. (Methods 2007, 43:110) with modifications using standard Illumina sRNA adapters, followed by sequencing with Illumina's SBS method.

value: in planta tissues

value: Magnaporthe infected rice leaf 96 hpi


title: Magnaporthe infected rice leaf 72 hpi

value: Magnaporthe infected rice leaf 72 hpi


title: A1-pre-placebo

value: airway smooth muscle layer from airway wall

value: Atopic asthma


title: Mycelium grown in paraquat treated complete media for 24 h

source_name: Mycelium

value: Mycelium


title: Mycelium grown in nitrogen starved media for 16 h


title: Mycelium grown in minimal media for 16 h


title: Mycelium grown in carbon starved media for 16 h


title: Mycelium grown in complete media for 16 h


title: Sham1

city: Charlottesville

first: Elizabeth

last: Ames

state: VA

address: Box 800759, Health System

department: Biomedical Engineering

institute: University of Virginia

data_processing:
Illumina Casava1.7 software used for basecalling for Samples1-4, 7 and 8 (and then converted to maq ill2sanger), Casava1.8 used for Samples 5, 6, and 9.
Sequenced reads were mapped to rn4 whole genome using tophat v1.3.1 with parameters with butterfly search option (--butterfly) and individual insert sizes specified (-r). Insert size was estimated from highest peak on Agilent DNA High Sensitivity electropherogram
Transript models were built for each sample using Cufflinks v1.0.3 with multiread correction (-u), upper quartile normalization (-N),  cDNA fragment bias correction (-b with ensembl rn4 fasta reference), and the reference transcript annotation was used to guide assembly (-g). Transcript models were merged using cuffcompare with genomic sequence (-s) with refernce GTF (-r) and transcripts were reduced with (-R) by only keeping transcripts that overlapped with input loci. Differential expression between the three groups (TAC, sham, and fetal) and statistical significance was calculated with cuffdiff using upper quartile normalization (-N).
Genome_build: rn4
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample

organism: Rattus norvegicus

source_name: heart (left ventricle)

extract_protocol:
Hearts were removed, stored in RNAlater, and RNA was harvested using either Trizol reagent or Qiagen Rneasy Fibrous Tissue Kit (as noted in Samples section). Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 5 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: heart (left ventricle)

value: adult

value: Sprague-Dawley

tag: rna isolation method

value: Qiagen Rneasy Fibrous Tissue Kit

tag: surgical procedure

value: Sham (identical to TAC procedure without constriction)


title: Litter2

source_name: heart (ventricle)

value: heart (ventricle)

value: embryonic day 18

value: Trizol


title: Sham3


title: Sham2


title: Litter1


title: Litter3


title: TAC1

value: TAC (transverse aortic constriction)


title: TAC3


title: TAC2


title: RNA_C2C12_24hDAPT_repl1

status: Public on May 13 2013

city: Nijmegen

first: Arjen

last: Brinkman

address: NCMLS #274, Geert Grooteplein Zuid 30

department: Molecular Biology

institute: Radboud University, Nijmegen Center for Molecular Life Sciences

description:
Mouse myoblast cell line (established from thigh muscle from an adult C3H strain female).
Ribozero processed and fragmented RNA.

data_processing:
Strand-specific RNA-seq: Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequences were aligned to the mouse (mm9) reference genome using the Illumina Analysis Pipeline, allowing one mismatch. Only the tags uniquely aligning to the genome were considered for further analysis. Further analysis was performed using the 36 bp sequence reads. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All sequence analyses were conducted based on the Mus musculus mm9 genome assembly.
Genome_build: mm9

source_name: C2C12, DAPT 24h

growth_protocol: C2C12 cells were cultured in DMEM medium supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen) at 37 °C in 5% CO2 atmosphere.

extract_protocol: Strand-specific RNA-seq using RiboZero: Total RNA was extracted using the RNeasy Mini and Micro kit (Qiagen) following the manufacturer's instructions. Ribosomal RNA was removed from 200 ng total RNA using the Ribo-Zero rRNA Removal Kit Low Input for Human/Mouse/Rat (Epicentre Biotechnologies) according to the manufacturer’s protocol. RNA was subsequently fragmented in fragmentation buffer (40 mM Tris-Ac pH8.2, 100 mM KAc, 30 mM MgAc) for 90 s at 95 ºC and purified by ethanol purification. cDNA was synthesized using random hexamers by Superscript III Reverse Transcriptase (Invitrogen) in the presence of 6 ng/µl ActinomycinD. cDNA was purified (MinElute Reaction Cleanup Kit, QIAGEN) and subjected to second strand synthesis by E. coli DNA Polymerase I (Invitrogen) and E. coli DNA Ligase (New England Biolabs) in the presence of RNaseH (Ambion). For second strand synthesis, dUNTPs were used, containing dUTP instead of dTTP. Ds-cDNA was purified (MinElute Reaction Cleanup Kit, QIAGEN) and prepared for Illumina sequencing according to standard procedures using the DNA sample Prep Kit (Illumina, cat# IP-102–1001) and starting from the A-tailing step. After ligation of adapters, a 200 bp band is excised using the E-Gel system (Invitrogen). Before final PCR amplification, uracil-containing second strand DNA was removed by USER enzyme (New England Biolabs) for 15 min at 37 ºC followed by 10 min at 95 ºC in 1x Phusion Buffer (Finnzymes). PCR amplification was performed with 14 cycles.

treatment_protocol: DAPT treatments: DAPT dissolved in DMSO (Calbiochem) was added to Fc-only dishes at a final concentration of 20µM for 6h or 24h.

value: DAPT 24h

tag: technique

value: cDNA-sequencing


title: SW1

description: Sample 1

value: swarmer cells

value: 0min following synchronization


title: RNA_C2C12_6hDAPT_repl2

source_name: C2C12, DAPT 6h

value: DAPT 6h


title: RNA_C2C12_24hDAPT_repl2


title: RNA_C2C12_6hDelta_repl2

source_name: C2C12, Delta 6h

treatment_protocol: Delta treatments: For ligand-dependent activation of Notch signaling, cells were grown in the presence of the extracellular domain of Delta-like 1 fused to the Fc fragment of human IgG (Hicks et al., 2002) (kindly provided by N. Gupta upon permission from G. Weinmaster). Culture dishes were first coated with 10 µg/ml anti-Fc antibody for 1h at RT. Dishes were then incubated with conditioned medium from transfected 293T cells expressing the fusion proteins, for 1h at RT. C2C12 cells were plated in fresh medium and grown for 6h or 24h on the immobilized-ligand containing dishes.

value: Delta 6h


title: RNA_C2C12_6hDAPT_repl1


title: RNA_C2C12_24hDelta_repl1

source_name: C2C12, Delta 24h

value: Delta 24h


title: RNA_C2C12_6hDelta_repl1


title: ChIP_NICD_GFPAb_C2C12_NICDGFP

description: C2C12 cells expressing GFP-tagged human NICD (NICD-EGFP). C2C12 cells were transduced with a lentivirus Ubiquitin C-rTTA3 and a lenti tetO-hNICD-EGFP. The lentiviral backbones are the same and derive from pTRIP-∆U3 lentivirus (for reference, see PMID 11319904). Only promoters and transgene were modified accordingly. To induce NICDGFP expression, cells were induced for 8h with 2ug/ml doxycycline.

data_processing:
ChIP-seq: Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequences were aligned to the mouse (mm9) reference genome using the Illumina Analysis Pipeline, allowing one mismatch. Only the tags uniquely aligning to the genome were considered for further analysis. Duplicated reads were discarded to obtain a non-redundant set. Further analysis was performed using the 36 bp sequence reads. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All sequence analyses were conducted based on the Mus musculus mm9 genome assembly.
Genome_build: mm9

source_name: C2C12_NICDGFP, GFP ChIP

extract_protocol:
Cells were trypsinized according to standard procedures. A cell suspension with cell density of 5 million/ml was made in cell medium. Cells were subsequently cross-linked for 30 min at RT in medium plus 1% formaldehyde and 1/14 volume of buffer A (0.1M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 50mM HEPES pH7.6), quenched with 0.125 M glycine and washed at 4°C with the following buffers: (i) PBS, (ii) buffer B: 0.25% Triton-X-100, 10mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6, (iii) buffer C: 0.15M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 50 mM HEPES pH7.6. During each washing step, cells were rotated for 10 min at 4°C and subsequently centrifuged for 7 min at 500g 4°C. Cells were then suspended in ChIP incubation buffer (0.15% SDS, 1% Triton-X-100, 0.15M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6, 1x protease inhibitor cocktail complete (Roche)) at 20 million/ml and sheared using a Bioruptor (Diagenode) with the following settings: high power, 30s on/off, 18 cycles. Sonicated chromatin was centrifuged for 5 min at 13000 rpm in eppendorf tubes, supernatant was snap-freezed in liquid nitrogen and stored at -80 °C or directly used in ChIPs. For one ChIP, 100 µl chromatin was incubated with 2 µg antibody, 20 µl 50% protein A/G bead (Santa Cruz), in 1x ChIP incubation buffer with 0.1% BSA in a total volume of 300 µl o/n at 4°C. Beads were then washed with the following buffers: twice with buffer1: 0.1% SDS, 0.1% NaDOC, 1% Triton-X-100, 0.15M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6, once with buffer2: 0.1% SDS, 0.1% NaDOC, 1% Triton-X-100, 0.5M NaCl, 1mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6, once with buffer3: 0.25M LiCl, 0.5% NaDOC, 0.5% NP-40, 1mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6, and twice with buffer4: 1mM EDTA pH8, 0.5mM EGTA pH8, 20mM HEPES pH7.6. During each washing step, beads were rotated for 5 min at 4°C and subsequently centrifuged for 2 min at 4000 rpm 4°C. Precipitated chromatin was then eluted from beads in 200 µl elution buffer (1% SDS, 0.1M NaHCO3) for 40 min at RT. Materials (precipitated chromatin or input chromatin in elution buffer) were decross-linked for 5h at 65 °C with addition of 200 mM NaCl. DNA was purified on QIAquick spin columns according to QIAGEN protocols. For sequencing, 6-8 ChIPs were pooled.
1 to 20 ng was used for library preparation using the ChIP-seq DNA sample Prep Kit (Illumina, cat# IP-102–1001) using the recommended protocol. After ligation of adapters, a 300 bp band is excised using the E-Gel system (Invitrogen), which correspond to a 234 bp fragment of genomic DNA. PCR enrichment of adapter-ligated DNA was done using 14 cycles of PCR.

treatment_protocol: GFP or NICDGFP protein induction: the cells were induced for 8h with 2ug/ml doxycycline.

value: doxycycline

value: ChIP-sequencing

tag: chip antibody

value: anti-GFP goat polyclonal

tag: antibody vendor/provider

value: M. Vermeulen


title: ChIP_control_GFPAb_C2C12_GFPonly

description: C2C12 cells expressing EGFP. C2C12 cells were transduced with a lentivirus Ubiquitin C-rTTA3 and a lenti tetO-EGFP. The lentiviral backbones are the same and derive from pTRIP-∆U3 lentivirus (for reference, see PMID 11319904). Only promoters and transgene were modified accordingly. To induce GFP expression, cells were induced for 8h with 2ug/ml doxycycline.

source_name: C2C12_GFP, GFP ChIP


title: Mock inoculated rice leaf

organism: Oryza sativa

source_name: Rice leaf

value: control

value: Mock inoculated rice leaf


title: ChIP_RBPj_1F1_C2C12_NICDGFP

source_name: C2C12_NICDGFP, RBP-J 1F1 ChIP

value: anti-RBP-J 1F1 rat monoclonal

value: Ascenion


title: RNA_C2C12_24hDelta_repl2


title: ChIP_p300_C2C12_24hDelta_repl1

description: Mouse myoblast cell line (established from thigh muscle from an adult C3H strain female).

source_name: C2C12, Delta 24h, p300 ChIP

value: anti-p300 rabbit polyclonal

value: santa cruz

tag: antibody catalog #

tag: lot#


title: ChIP_p300_C2C12_6hDAPT_repl1

source_name: C2C12, DAPT 6h, p300 ChIP


title: ChIP_RBPj_1F1_C2C12_24hDAPT_repl1

source_name: C2C12, DAPT 24h, RBP-J 1F1 ChIP

value: DAPT 24h


title: ChIP_RBPj_1F1_C2C12_24hDAPT_repl2


title: ChIP_RBPj_rabbitAb_C2C12_6hDelta_repl1

source_name: C2C12, Delta 6h, RBP-J ChIP

value: anti-RBP-J rabbit polyclonal

value: E. Kieff


title: ChIP_RBPj_1F1_C2C12_6hDelta_repl2

source_name: C2C12, Delta 6h, RBP-J 1F1 ChIP


title: ChIP_RBPj_1F1_C2C12_24hDelta_repl2

source_name: C2C12, Delta 24h, RBP-J 1F1 ChIP


title: ChIP_p300_C2C12_24hDAPT_repl1

source_name: C2C12, DAPT 24h, p300 ChIP


title: ChIP_p300_C2C12_6hDelta_repl1

source_name: C2C12, Delta 6h, p300 ChIP


title: ChIP_RBPj_1F1_C2C12_24hDelta_repl1


title: ChIP_RBPj_1F1_C2C12_6hDAPT_repl1

source_name: C2C12, DAPT 6h, RBP-J 1F1 ChIP


title: Input_C2C12_6hDelta_repl1

description:
Mouse myoblast cell line (established from thigh muscle from an adult C3H strain female).
Input DNA purified from chromatin.

source_name: C2C12, Delta 6h, ChIP input

value: input-sequencing


title: ChIP_RBPj_rabbitAb_C2C12_6hDAPT_repl1

source_name: C2C12, DAPT 6h, RBP-J ChIP

value: DAPT 6h


title: ChIP_RBPj_1F1_C2C12_6hDelta_repl1

value: Delta 6h


title: ChIP_RBPj_1F1_C2C12_6hDAPT_repl2


title: Pfeiffer EZH2

city: New York

first: Matt

last: Teater

state: NY

address: 445 E 69th St

institute: Weill Cornell Medical College

data_processing:
genome build: hg19
Illumina Casava1.8 software used for basecalling.
RNA-seq data was aligned to transcripts using TopHat with default parameters. ChIP-seq data was aligned to the appropriate genome using ELAND.
FPKM were obtained using CuffLinks with upper-quartile and GC normalization
H3K4me3 ChIP-seq reads were called into peaks using the ChIPseeqer framework (p<10-15 and fold-change over input threshold 2) (Giannopoulou and Elemento, 2011) and H3K27me3 and EZH2 ChIP-seq reads were quantified in 1kb bins genome-wide, identifying regions of enrichment as consecutive bins with read counts greater than one standard deviation of the genome-wide mean.
Supplementary_files_format_and_content: tab-delimited FPKM values for each Refseq transcript; WIG files were generatedand peak files were called using ChIPseeqer framework (http://physiology.med.cornell.edu/faculty/elemento/lab/chipseq.shtml)

source_name: Pfeiffer cell line

extract_protocol:
ChIP was performed as previously described (Ci et al., Blood, 2009). Briefly, 108 cells were fixed with 1% formaldehyde, lysed, and sonicated (Branson Sonicator; Branson) leading to a DNA average size of 200 bp. Five µg of antibodies were added to the precleared sample and incubated overnight at 4°C. The complexes were purified using protein-A beads (Roche) followed by elution from the beads and decrosslinking. DNA was purified using PCR purification columns (QIAGEN).
ChIP-seq and RNA-seq libraries were prepared using the Illumina ChIP-Seq and TruSeq RNA sample kits, respectively, according to the manufacturer. Libraries were validated using the Agilent Technologies 2100 Bioanalyzer and Quant-iT™ dsDNA HS Assay (Life Technologies)

value: Pfeiffer cell line

value: anti-EZH2 (Millipore, 07-689, lot DAM1834941)


title: ChIP_RBPj_1F1_C2C12_GFPonly

source_name: C2C12_GFP, RBP-J 1F1 ChIP

value: doxycycline

value: ChIP-sequencing


title: WSUDLCL2 EZH2

source_name: WSUDLCL2 cell line

value: WSUDLCL2 cell line


title: SUDHL6 EZH2

source_name: SUDHL6 cell line

value: SUDHL6 cell line


title: SUDHL5 EZH2

source_name: SUDHL5 cell line

value: SUDHL5 cell line


title: Farage EZH2

source_name: Farage cell line

value: Farage cell line


title: OCI-Ly7 EZH2

source_name: OCI-Ly7 cell line

value: OCI-Ly7 cell line