/ml, respectively made up to 200 l using Tris-EDTA buffer. The suspensions were vortexed for 10 s and incubated at room temperature for 10 min with 2 min mixing interval. RNA extraction of the treated cells was buy TAPI-2 performed using RNeasy Plus Mini kit (Qiagen, Germany) according to manufacturer’s guidelines and eluted in DEPC water (Bioline, UK). A total of three biological replicates were included for each treatment group and an untreated control group for comparison analysis. RNA-Seq library preparation and analysis.Quality of RNA was verified using Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and NanoDrop spectrometer. Samples that passes quality control (minimum RNA integrity number (RIN) of 7, absorbance ratios A260/280 in the range 2.0?.2 and A260/230 above 1.8), a non-normalized cDNA library was constructed. Barcoded libraries were multiplexed by 12 in each lane and sequenced on an Illumina HiSeq 2000 system using the single-end mode. The length of the reads was around 100 bp. Quality control of the RNA-Seq data was performed using FastQC and detailed information about the quality of reads in each replicate is provided in Additional file (xx_). Sequence reads have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA308880 (www.ncbi.nlm.gov/bioproject/ PRJNA308880).MethodsQuality check (QC) with FastQC. Adapters from the fastQ file were removed using Cutadapt (https:// code.google.com/p/cutadapt/). Removal of reads with phred score below 20 were performed using fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Mapping and Expression analysis. Raw reads in fastq format from illumina sequencing were used to map against streptococcus pneumoniae TIGR4 genome (NC_003028) by TopHat v2.0.10 program47. To compare expression analysis among samples output bam file from TopHat and GFF file from gene prediction were used as input to cuffdiff v2.1.1 program48 with classic method of normalization with FPKM to identify the differentially expressed genes between all the samples. Gene clustering and Heat map.Differentially expressed genes were clustered using PD173074 web K-means clustering algorithm using ComplexHeatmap41 package from Bioconductor in R. Clusters generated by K-means were submitted to DAVID 6.7 web server40 for gene enrichment studies. Annotations from various databases such as KEGG pathways, gene ontology (GO), and swissprot were also retrieved from DAVID 6.7 server40.
www.nature.com/scientificreportsOPENReceived: 23 November 2015 accepted: 03 May 2016 Published: 01 JuneMicrosomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkersRavindra Varma Polisetty1,*,, Poonam Gautam1,*,, Manoj Kumar Gupta1,2,3, Rakesh Sharma2, Harsha Gowda2, Durairaj Renu4, Bhadravathi Marigowda Shivakumar5, Akhila Lakshmikantha6, Kiran Mariswamappa6, Praveen Ankathi7, Aniruddh K. Purohit7, Megha S. Uppin7, Challa Sundaram7 Ravi Sirdeshmukh1,2,Diffuse astrocytoma (DA; WHO grade II) is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on 2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tu./ml, respectively made up to 200 l using Tris-EDTA buffer. The suspensions were vortexed for 10 s and incubated at room temperature for 10 min with 2 min mixing interval. RNA extraction of the treated cells was performed using RNeasy Plus Mini kit (Qiagen, Germany) according to manufacturer’s guidelines and eluted in DEPC water (Bioline, UK). A total of three biological replicates were included for each treatment group and an untreated control group for comparison analysis. RNA-Seq library preparation and analysis.Quality of RNA was verified using Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and NanoDrop spectrometer. Samples that passes quality control (minimum RNA integrity number (RIN) of 7, absorbance ratios A260/280 in the range 2.0?.2 and A260/230 above 1.8), a non-normalized cDNA library was constructed. Barcoded libraries were multiplexed by 12 in each lane and sequenced on an Illumina HiSeq 2000 system using the single-end mode. The length of the reads was around 100 bp. Quality control of the RNA-Seq data was performed using FastQC and detailed information about the quality of reads in each replicate is provided in Additional file (xx_). Sequence reads have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA308880 (www.ncbi.nlm.gov/bioproject/ PRJNA308880).MethodsQuality check (QC) with FastQC. Adapters from the fastQ file were removed using Cutadapt (https:// code.google.com/p/cutadapt/). Removal of reads with phred score below 20 were performed using fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Mapping and Expression analysis. Raw reads in fastq format from illumina sequencing were used to map against streptococcus pneumoniae TIGR4 genome (NC_003028) by TopHat v2.0.10 program47. To compare expression analysis among samples output bam file from TopHat and GFF file from gene prediction were used as input to cuffdiff v2.1.1 program48 with classic method of normalization with FPKM to identify the differentially expressed genes between all the samples. Gene clustering and Heat map.Differentially expressed genes were clustered using K-means clustering algorithm using ComplexHeatmap41 package from Bioconductor in R. Clusters generated by K-means were submitted to DAVID 6.7 web server40 for gene enrichment studies. Annotations from various databases such as KEGG pathways, gene ontology (GO), and swissprot were also retrieved from DAVID 6.7 server40.
www.nature.com/scientificreportsOPENReceived: 23 November 2015 accepted: 03 May 2016 Published: 01 JuneMicrosomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkersRavindra Varma Polisetty1,*,, Poonam Gautam1,*,, Manoj Kumar Gupta1,2,3, Rakesh Sharma2, Harsha Gowda2, Durairaj Renu4, Bhadravathi Marigowda Shivakumar5, Akhila Lakshmikantha6, Kiran Mariswamappa6, Praveen Ankathi7, Aniruddh K. Purohit7, Megha S. Uppin7, Challa Sundaram7 Ravi Sirdeshmukh1,2,Diffuse astrocytoma (DA; WHO grade II) is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on 2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tu.
