5_nested_testis
Homo sapiens
Protocols: PolyA+ RNAs from a panel of seven human tissues (brain, heart, kidney, liver, lung, spleen and testis) were purchased from Clontech Laboratories, Illumina Human Body Map (HBM) RNASeq data [ArrayExpress E-MTAB-513] was used to calculate RPKMs for all GENCODE v7 lncRNAs. We selected lncRNAs that were expressed in at least one HBM experiment with an RPKM > 5 and that were lacking CAGE/PET support in ENCODE cell line experiments. The spliced RNA sequences for the top 398 lncRNAs, ranked by the mean RPKM across cell lines, were extracted and used as input for primer design. Non-specific regions within the candidate sequences were masked in order to avoid RACE off-target products. These regions were established by aligning candidate sequences against all GENCODE v7 transcript sequences using the BLAST program. Regions having >80% similarity to any other GENCODE v7 transcript from a distinct locus were masked. Only stranded overlap was considered. We then generated for each candidate transcript all possible 5' and 3' RACE primers using Primer3 (http://sourceforge.net/projects/primer3) with the following parameters: PRIMER_INTERNAL_OPT_SIZE=25, PRIMER_INTERNAL_MIN_SIZE=23, PRIMER_INTERNAL_MAX_SIZE=27, PRIMER_INTERNAL_OPT_TM=70.0, PRIMER_INTERNAL_MIN_TM=68.0, PRIMER_INTERNAL_MAX_TM=72.0, PRIMER_INTERNAL_MIN_GC=50, PRIMER_INTERNAL_MAX_GC=70, PRIMER_INTERNAL_OPT_GC_PERCENT=60. Nested and non-nested 5' and 3' RACE products were obtained by using the Clontech SMART RACE cDNA Amplification kit and the Advantage 2 Proofreading Polymerase PCR kit (Clontech Laboratories) according to the manufacturer's instructions. Double-stranded cDNA synthesis, adaptor ligations to the synthesized cDNA and 12.5 μl final volume RACE reactions were performed according to the manufacturers' instructions. Nested RACEs were performed with 0.5 μl of the initial RACEs in a final volume of 12.5 μl. cDNA RACE samples were analyzed on a DNA 7500 Chip (2100 Bioanalyzer-Agilent Technologies) to assess fragment size and sample integrity. For samples with a mean fragment size of 2 Kbp or higher, 1 μg of material was subjected to nebulization and then used to to prepare a rapid ligation (RL) genomic shotgun library using the Rapid Library Preparation Method Manual (GS FLX+ Series - XL+ May 2011) (Roche 454 Life Sciences). For RACE samples with a mean fragment size smaller than 2 Kbp the nebulization step was avoided, starting library preparation directly with 800 ng of material. A modification was introduced in the small fragment removal of this library preparation, to allow only the removal of fragments under 400 bp instead of fragments under 650 bp. Then, the quality of the RL libraries was assessed by running an aliquot of the library in a High Sensitivity Chip (2100 Bioanalyzer-Agilent Technologies). Library quantification was performed generating a RL standard curve and using a 96-well Plate fluorometer, according to the manufacturer's instructions, and using the Rapid Library Quantitation Calculator (www.454.com/my454). Samples were titrated using the emPCR amplification Method Manual Lib-L SV(GS FLX+ Series- XL+. May 2011) (Roche 454 Life Sciences) in order to know the optimal point of copies per bead (cpb) needed to obtain a 10% enriched beads. Then, a large volume emulsion PCR was performed using the emPCR amplification Method Manual Lib-L LV(GS FLX+ Series- XL+. May 2011) (Roche 454 Life Sciences).
ENA-FIRST-PUBLIC
2016-06-01T17:01:03Z
ENA-LAST-UPDATE
2018-03-09T05:06:59Z
External Id
SAMEA3556794
INSDC center name
Wellcome Trust Sanger Institute
INSDC first public
2016-06-01T17:01:03Z
INSDC last update
2018-03-09T05:06:59Z
INSDC status
public
Submitter Id
E-MTAB-3912:5_nested_testis
amplification type
nested
broker name
ArrayExpress
common name
human
organism part
testis
race orientation
5'
sample name
E-MTAB-3912:5_nested_testis
scientific_name
Homo sapiens
sex
male