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Sample GSM51631 Query DataSets for GSM51631
Status Public on May 18, 2005
Title Human Placenta Poly(A)+ RNA Bioligical Sample 3
Sample type RNA
 
Source name Human Placenta
Organism Homo sapiens
Extracted molecule total RNA
 
Description Triple selected polyA RNA for placenta was obtained from Ambion (Austin, TX). All RNA samples had an agilent ratio greate than 1 indicating that degradation had not occurred. All RNA samples were prepared from a pool of several different individuals.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70?C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately. Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia).
Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel. The fragmented cDNA was then end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution.
Keywords = Transcript Mapping, Human, Affymetrix, Genome Tiling Arrays
 
Submission date May 17, 2005
Last update date May 29, 2005
Contact name Joel Rozowsky
E-mail(s) rozowsky@csb.yale.edu
Phone 203-432-5405
Organization name Yale University
Department Molecular Biophysics & Biochemistry
Lab Gerstein Lab
Street address 266 Whitney Ave, Bass 437
City New Haven
State/province CT
ZIP/Postal code 06520
Country USA
 
Platform ID GPL1789
Series (1)
GSE2671 ENCODE Transcript Mapping for Human Placental Poly(A)+ RNA

Data table header descriptions
ID_REF Chromosome and Position of 25mer Oligonucleotide (NBCIv34)
VALUE Signal is an estimate of RNA abundance using a Hodges-Lehman (pseudomedian) estimator, which is the median of all pairwise averages of PM-MM is calculated for all probes pairs from all availible technical replicates within a sliding window of 101bp of genomic locale centered at each probe location. Replicate slides are quantile normalized to each other after having been median scaled.
ABS_CALL The call that indicates if at genomic location ID_REF the signal is significantly over background levels with P indicating RNA is present and A indicating absence. A genomic location ID_REF is determined to be present (P) if its intensity is within the top 90th percentile of intensities and it is within a run of probes whose maximum gap between probes above threshold is less than 50bp and the minimum run of probes is greater than 50bps. All other genomic location are determined to be absent (A).

Data table
ID_REF VALUE ABS_CALL
chr1_148374672 0 A
chr1_148374692 0.5 A
chr1_148374719 0 A
chr1_148374740 0 A
chr1_148374767 0 A
chr1_148374787 1 A
chr1_148374809 0 A
chr1_148374824 1.5 A
chr1_148374846 44 P
chr1_148374865 45.5 P
chr1_148374886 46.5 P
chr1_148374906 76.5 P
chr1_148374927 64 P
chr1_148374949 60.5 P
chr1_148374968 60.5 P
chr1_148374991 38.5 P
chr1_148375007 7 A
chr1_148375029 2 A
chr1_148375050 0 A
chr1_148375073 -0.5 A

Total number of rows: 737679

Table truncated, full table size 14560 Kbytes.




Supplementary file Size Download File type/resource
GSM51631_techrep1.CEL.gz 7.9 Mb (ftp)(http) CEL
GSM51631_techrep2.CEL.gz 7.9 Mb (ftp)(http) CEL

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