The Gencode v12 annotation was used as a reference to guide assembly (-GTF-guide) additional parameters included upper-quartile normalization (-upper-quartile-norm), library type (-library-type fr-firststrand), and maximum bundle length (-max-bundle-length 7500000).Ĭuffmerge (v2.0.2) was then used to merge all the cufflinks assemblies and the reference annotation into one large set of transcript structures. Transcript structure assembly was performed with Cufflinks (v.2.0.2) on each sample for each tissue type. Using Picard Tools, the alignment files were sorted by genomic coordinates, read-group data was added, and duplicate reads were marked and removed. Raw reads were mapped to the human genome sequence using Tophat v2.0.6, allowing for four mismatches per 100bp read, a maximum of 6 edit distances per read, and one mismatch in the splice anchor region. Insert sizes were estimated using the Picard Tools (v1.79) tools SortSam.jar and CollectInsertSizeMetrics.jar on Bowtie2(v2.0.0-beta7)-generated sam files from a subset of 500,000 paired-end reads per sample (bowtie2 options -q -very-fast -phred33). Total RNA was isolated from LCLs using the PureLink RNA Mini Kit (Life Technologies). The protocol was modified to increase fragment size and to yield final libraries containing only the first strand-synthesized product by: (a) reduction of elute-prime-frag treatment time from 8 minutes to 4 minutes (to increase library insert size), (b) second strand synthesis using a mastermix containing dUTP (in place of dTTP) and RNAse H (to remove RNA), and (c) following the final adapter ligation cleanup, libraries were digested with uracil DNA deglycosylase (to remove dUTP-containing second strand product). The corresponding vendor protocol was used for almost all steps (including polyA selection) with a final PCR enrichment using 15 cycles. Immortalized lymphoblastoid cell lines (LCLs) were grown at 37☌ with 5% CO2 in RPMI 1640 media supplemented with 10% FBS, 500 U/ml penicillin/streptomycin, and 2 nmol/L GlutaMAX (Life Technologies).ĬDNA libraries were prepared using the TruSeq RNA Sample Prep Kit v2. GEO help: Mouse over screen elements for information.
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