title: RNAP ChIP from Retinoic Acid Stimulated HL-60 Cells, 2 hours (NCBIv34)

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10*6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10*8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10*7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against RNA Polymerase II, RNAP (Covance Research Products) overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was 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 PCR amplified, fragmented DNA from DNAse I treatment was 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 = Homo sapiens, RNAP, HL-60 Cells, 2 hours Retinoic Acid

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL-60 Cells, 2 hours


title: Brain:Amygdala, Male (MoBr.MaAm)

status: Public on Nov 01 2004

type: MPSS

city: Hayward

first: Daixing

last: Zhou

state: CA

address: 25861 Industrial Blvd

department: Bioinformatics

country: USA

institute: Solexa

description:
Strain: C57BL6
Brain:Amygdala, Male total RNA (MoBr.MaAm) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoBr.MaAm.sig21
Cell type Brain:Amygdala, Male
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoBr.MaAm_sig21.5160F.c-20	10/21/2004	332755	9202	QC Passed
MoBr.MaAm_sig21.5160W.c-20	10/11/2004	461375	12980	QC Passed
MoBr.MaAm_sig21.5160F.a-20	9/27/2004	445927	11574	QC Passed
MoBr.MaAm_sig21.5160F.b-20	9/27/2004	429447	11240	QC Passed
MoBr.MaAm_sig21.5160W.b-20	9/8/2004	392995	9886	QC Passed
Run group:
Total Beads successfully sequenced - 2062499
Processed Signatures - 16595

molecule: total RNA

organism: Mus musculus

source_name: Brain:Amygdala, Male total RNA


title: Kidney:cortex, Female (MoKi.FeCr)

description:
Strain: C57BL6
Kidney:cortex, Female total RNA (MoKi.FeCr) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoKi.FeCr.sig23
Cell type Kidney:cortex, Female
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoKi.FeCr_sig23.5127F.d-20	10/26/2004	399871	7014	QC Passed
MoKi.FeCr_sig23.5127F.b-20	10/4/2004	312111	7330	QC Passed
MoKi.FeCr_sig23.5127F.a-20	10/2/2004	326199	7481	QC Passed
MoKi.FeCr_sig23.5127W.c-20	8/16/2004	677674	11364	QC Passed
MoKi.FeCr_sig23.5127W.a-20	8/10/2004	662486	10215	QC Passed
Run group:
Total Beads successfully sequenced - 2378341
Processed Signatures - 13475

source_name: Kidney:cortex, Female total RNA


title: Liver:left lobe, Male (MoLi.MaLL)

status: Public on Oct 26 2004

description:
Strain: C57BL6
Liver:left lobe, Male total RNA (MoLi.MaLL) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoLi.MaLL.sig21
Cell type Liver:left lobe, Male
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoLi.MaLL_sig21.5135F.b-20	9/30/2004	380973	5022	QC Passed
MoLi.MaLL_sig21.5135F.a-20	9/10/2004	480677	8503	QC Passed
MoLi.MaLL_sig21.5135W.d-20	8/30/2004	549540	6494	QC Passed
MoLi.MaLL_sig21.5135W.c-20	8/24/2004	608514	7048	QC Passed
Run group:
Total Beads successfully sequenced - 2019704
Processed Signatures - 10122

source_name: Liver:left lobe, Male total RNA


title: RNAP ChIP from Retinoic Acid Stimulated HL-60 Cells, 32 hours (NCBIv34)

status: Public on Jan 31 2005

type: genomic

city: Santa Clara

first: Thomas

middle: R

last: Gingeras

address: 3380 Central Expressway

institute: Affymetrix, Inc.

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10*6 cells/ml (medium and supplemental components were purchased from Invitrogen).  Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes.  Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes.  Crosslinked cells were extensively washed resulting in nuclear pellet formation.  A nuclear pellet, equivalent to 2 x 10*8 cells, was treated with 20U  of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication.
Cellular debris was separated from solubilized chromatin by high-speed centrifugation.  Isolated chromatin, equivalent to 2.5 x 10*7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C.  This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against RNA Polymerase II, RNAP (Covance Research Products) overnight at 4C.  Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C.  Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen).  A mock IP was preformed in parallel to isolate total DNA referred to as INPUT.
Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN).  DNA was recovered using a QIAquick PCR purification kit (Qiagen).  PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles.  DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above.  Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp.
PCR amplified DNA, 5-10 ug, was 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 PCR amplified, fragmented DNA from DNAse I treatment was 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 = Homo sapiens, RNAP, HL-60 Cells, 32 hours Retinoic Acid

molecule: genomic DNA

organism: Homo sapiens

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL-60 Cells, 32 hours


title: Liver:right lobe, Male (MoLi.MaRL)

status: Public on Dec 02 2004

description:
Strain: C57BL6
Liver:right lobe, Male total RNA (MoLi.MaRL) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoLi.MaRL.sig21
Cell type Liver:right lobe, Male
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoLi.MaRL_sig21.5199F.a-20	9/30/2004	358481	5035	QC Passed
MoLi.MaRL_sig21.5199F.b-20	9/27/2004	406837	4529	QC Passed
MoLi.MaRL_sig21.5199F.c-20	11/30/2004	379857	3592	QC Passed
MoLi.MaRL_sig21.5199W.a-20	10/4/2004	430697	3825	QC Passed
MoLi.MaRL_sig21.5199W.d-20	11/30/2004	402573	2983	QC Passed
Run group:
Total Beads successfully sequenced - 1978445
Processed Signatures - 6683

source_name: Liver:right lobe, Male total RNA


title: Brain:Hippocampus, Male (MoBr.MaHi)

description:
Strain: C57BL6
Brain:Hippocampus, Male total RNA (MoBr.MaHi) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoBr.MaHi.sig22
Cell type Brain:Hippocampus, Male
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoBr.MaHi_sig22.5167W.c-20	10/1/2004	742344	18761	QC Passed
MoBr.MaHi_sig22.5167F.a-20	9/13/2004	480654	14351	QC Passed
MoBr.MaHi_sig22.5167F.b-20	9/13/2004	552080	19423	QC Passed
MoBr.MaHi_sig22.5167W.a-20	9/13/2004	577239	18668	QC Passed
Run group:
Total Beads successfully sequenced - 2352317
Processed Signatures - 26747

source_name: Brain:Hippocampus, Male total RNA


title: White fat, Female (MoWF.FeAb)

description:
Strain: C57BL6
White fat, Female total RNA (MoWF.FeAb) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoWF.FeAb.sig21
Cell type White fat, Female
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoWF.FeAb_sig21.5010T.f-20	6/14/2004	333763	8128	QC Passed
MoWF.FeAb_sig21.5010T.d-20	5/24/2004	450092	9538	QC Passed
MoWF.FeAb_sig21.5010W.c-20	5/17/2004	578060	12911	QC Passed
MoWF.FeAb_sig21.5010W.b-20	5/15/2004	582620	11191	QC Passed
Run group:
Total Beads successfully sequenced - 1944535
Processed Signatures - 15917

source_name: White fat, Female total RNA


title: Brain:Midbrain, Male (MoBr.MaMb)

description:
Strain: C57BL6
Brain:Midbrain, Male total RNA (MoBr.MaMb) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoBr.MaMb.sig21
Cell type Brain:Midbrain, Male
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoBr.MaMb_sig21.5169F.c-20	9/30/2004	350976	11217	QC Passed
MoBr.MaMb_sig21.5169W.c-20	9/30/2004	619979	18100	QC Passed
MoBr.MaMb_sig21.5169W.a-20	9/14/2004	551415	14529	QC Passed
MoBr.MaMb_sig21.5169F.b-20	9/13/2004	592802	17444	QC Passed
Run group:
Total Beads successfully sequenced - 2115172
Processed Signatures - 22737

source_name: Brain:Midbrain, Male total RNA


title: Bladder, Female (MoBl.Feml)

description:
Strain: C57BL6
Bladder, Female total RNA (MoBl.Feml) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoBl.Feml.sig21
Cell type Bladder, Female
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoBl.Feml_sig21.5083F.a-20	10/27/2004	242523	4809	QC Passed
MoBl.Feml_sig21.5146F.a-20	10/1/2004	386671	8157	QC Passed
MoBl.Feml_sig21.5146W.a-20	8/26/2004	496397	9477	QC Passed
MoBl.Feml_sig21.5146W.b-20	8/26/2004	477536	8845	QC Passed
MoBl.Feml_sig21.5083W.c-20	7/17/2004	494632	12071	QC Passed
MoBl.Feml_sig21.5083W.a-20	6/29/2004	473565	11324	QC Passed
Run group:
Total Beads successfully sequenced - 2571324
Processed Signatures - 15827

source_name: Bladder, Female total RNA


title: Kidney:contex, Male (MoKi.MaCr)

description:
Strain: C57BL6
Kidney:contex, Male total RNA (MoKi.MaCr) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoKi.MaCr.sig21
Cell type Kidney:contex, Male
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoKi.MaCr_sig21.5001T.d-20	6/3/2004	447120	11030	QC Passed
MoKi.MaCr_sig21.5001W.e-20	5/27/2004	533136	13173	QC Passed
MoKi.MaCr_sig21.5001W.d-20	5/19/2004	521427	15116	QC Passed
MoKi.MaCr_sig21.5001T.c-20	5/17/2004	397844	9288	QC Passed
MoKi.MaCr_sig21.5001T.a-20	5/17/2004	425707	8591	QC Passed
MoKi.MaCr_sig21.5001W.b-20	5/14/2004	652448	14229	QC Passed
Run group:
Total Beads successfully sequenced - 2977682
Processed Signatures - 21011

source_name: Kidney:contex, Male total RNA


title: Kidney:medulla, Male (MoKi.MaMd)

description:
Strain: C57BL6
Kidney:medulla, Male total RNA (MoKi.MaMd) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoKi.MaMd.sig21
Cell type Kidney:medulla, Male
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoKi.MaMd_sig21.5102W.b-20	7/13/2004	510493	10425	QC Passed
MoKi.MaMd_sig21.5102W.a-20	7/12/2004	580264	12251	QC Passed
MoKi.MaMd_sig21.5102T.a-20	7/12/2004	340798	7550	QC Passed
MoKi.MaMd_sig21.5102T.b-20	7/12/2004	292133	7597	QC Passed
MoKi.MaMd_sig21.5002T.e-20	6/15/2004	328502	6699	QC Passed
MoKi.MaMd_sig21.5002T.c-20	5/24/2004	300987	7809	QC Passed
MoKi.MaMd_sig21.5002W.c-20	5/19/2004	451213	9996	QC Passed
Run group:
Total Beads successfully sequenced - 2804390
Processed Signatures - 16407

source_name: Kidney:medulla, Male total RNA


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 2 hours  (NCBIv34)

type: RNA

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C in 5% CO2 to a density of 0.75 x 10*6 cells/ml (medium and supplemental components were purchased from Invitrogen). Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer?s protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
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 = Homo sapiens, PolyA+ RNA, HL-60 Cells, 2 hours Retinoic Acid

molecule: polyA RNA


title: Spinal cord:entire, Female (MoSC.FeEn)

status: Public on Nov 15 2004

description:
Strain: C57BL6
Spinal cord:entire, Female total RNA (MoSC.FeEn) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoSC.FeEn.sig21
Cell type Spinal cord:entire, Female
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoSC.FeEn_sig21.5264F.b-20	11/8/2004	263717	16920	QC Passed
MoSC.FeEn_sig21.5157W.d-20	10/5/2004	559892	32000	QC Passed
MoSC.FeEn_sig21.5157F.e-20	10/5/2004	330440	19611	QC Passed
MoSC.FeEn_sig21.5157F.a-20	10/2/2004	295758	18775	QC Passed
MoSC.FeEn_sig21.5157W.c-20	9/10/2004	317082	26012	QC Passed
MoSC.FeEn_sig21.5157W.b-20	9/8/2004	532370	28916	QC Passed
Run group:
Total Beads successfully sequenced - 2299259
Processed Signatures - 42482

source_name: Spinal cord:entire, Female total RNA


title: Brain:Thalamus, Female (MoBr.FeTh)

description:
Strain: C57BL6
Brain:Thalamus, Female total RNA (MoBr.FeTh) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoBr.FeTh.sig22
Cell type Brain:Thalamus, Female
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoBr.FeTh_sig22.5171W.c-20	10/5/2004	464501	12553	QC Passed
MoBr.FeTh_sig22.5171F.b-20	9/14/2004	561575	21893	QC Passed
MoBr.FeTh_sig22.5171F.a-20	9/13/2004	558644	20750	QC Passed
MoBr.FeTh_sig22.5171W.a-20	9/13/2004	487411	18142	QC Passed
Run group:
Total Beads successfully sequenced - 2072131
Processed Signatures - 28755

source_name: Brain:Thalamus, Female total RNA


title: Kidney:medulla, Female (MoKi.FeMd)

description:
Strain: C57BL6
Kidney:medulla, Female total RNA (MoKi.FeMd) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoKi.FeMd.sig21
Cell type Kidney:medulla, Female
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoKi.FeMd_sig21.5088T.b-20	7/8/2004	334137	7742	QC Passed
MoKi.FeMd_sig21.5088T.a-20	7/1/2004	360439	8237	QC Passed
MoKi.FeMd_sig21.5088W.a-20	7/1/2004	523286	11119	QC Passed
MoKi.FeMd_sig21.5088W.b-20	7/1/2004	468659	11631	QC Passed
MoKi.FeMd_sig21.5032T.c-20	6/4/2004	377661	6834	QC Passed
MoKi.FeMd_sig21.5032W.a-20	5/28/2004	578888	11639	QC Passed
MoKi.FeMd_sig21.5032T.a-20	5/27/2004	371621	6469	QC Passed
Run group:
Total Beads successfully sequenced - 3014691
Processed Signatures - 17060

source_name: Kidney:medulla, Female total RNA


title: Esophagus, Male (MoEs.Male)

description:
Strain: C57BL6
Esophagus, Male total RNA (MoEs.Male) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoEs.Male.sig21
Cell type Esophagus, Male
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoEs.Male_sig21.5004T.d-20	6/4/2004	386200	6058	QC Passed
MoEs.Male_sig21.5004W.d-20	6/2/2004	525677	9929	QC Passed
MoEs.Male_sig21.5004W.c-20	5/14/2004	487429	7199	QC Passed
MoEs.Male_sig21.5004T.a-20	5/14/2004	384784	4944	QC Passed
MoEs.Male_sig21.5004T.b-20	5/14/2004	380169	4849	QC Passed
MoEs.Male_sig21.5004W.a-20	5/14/2004	408950	5932	QC Passed
Run group:
Total Beads successfully sequenced - 2573209
Processed Signatures - 11650

source_name: Esophagus, Male total RNA


title: Bladder, Male (MoBl.Male)

description:
Strain: C57BL6
Bladder, Male total RNA (MoBl.Male) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoBl.Male.sig21
Cell type Bladder, Male
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoBl.Male_sig21.5061T.c-20	7/9/2004	433479	10982	QC Passed
MoBl.Male_sig21.5061T.a-20	6/28/2004	518425	16189	QC Passed
MoBl.Male_sig21.5061W.b-20	6/23/2004	476890	12374	QC Passed
MoBl.Male_sig21.5061W.a-20	6/22/2004	614642	16480	QC Passed
MoBl.Male_sig21.5061T.b-20	6/21/2004	408780	13030	QC Passed
Run group:
Total Beads successfully sequenced - 2452216
Processed Signatures - 22407

source_name: Bladder, Male total RNA


title: Heart:ventricles and septum, Female (MoHe.FeVS)

description:
Strain: C57BL6
Heart:ventricles and septum, Female total RNA (MoHe.FeVS) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoHe.FeVS.sig21
Cell type Heart:ventricles and septum, Female
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoHe.FeVS_sig21.5044T.d-20	7/12/2004	447604	7493	QC Passed
MoHe.FeVS_sig21.5044W.c-20	6/21/2004	525751	8421	QC Passed
MoHe.FeVS_sig21.5044W.b-20	6/18/2004	529209	8021	QC Passed
MoHe.FeVS_sig21.5044T.b-20	6/14/2004	376452	7247	QC Passed
MoHe.FeVS_sig21.5044T.a-20	6/11/2004	392258	7934	QC Passed
MoHe.FeVS_sig21.5044W.a-20	6/9/2004	530837	9781	QC Passed
Run group:
Total Beads successfully sequenced - 2802111
Processed Signatures - 14461


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 8 hours (NCBIv34)

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C in 5% CO2 to a density of 0.75 x 10*6 cells/ml (medium and supplemental components were purchased from Invitrogen). Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer?s protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
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 = Homo sapiens, PolyA+ RNA, HL-60 Cells, 8 hours Retinoic Acid


title: Brain:Cerebellum, Male (MoBr.MaCb)

description:
Strain: C57BL6
Brain:Cerebellum, Male total RNA (MoBr.MaCb) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoBr.MaCb.sig21
Cell type Brain:Cerebellum, Male
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoBr.MaCb_sig21.5166W.d-20	10/5/2004	474748	15446	QC Passed
MoBr.MaCb_sig21.5166W.c-20	10/2/2004	348203	12931	QC Passed
MoBr.MaCb_sig21.5166F.c-20	10/1/2004	273331	10446	QC Passed
MoBr.MaCb_sig21.5166F.a-20	9/17/2004	517363	23043	QC Passed
MoBr.MaCb_sig21.5166F.b-20	9/17/2004	298009	12544	QC Passed
MoBr.MaCb_sig21.5166W.b-20	9/16/2004	310871	12182	QC Passed
Run group:
Total Beads successfully sequenced - 2222525
Processed Signatures - 27979

source_name: Brain:Cerebellum, Male total RNA


title: Adrenal, Female (MoAd.Feml)

description:
Strain: C57BL6
Adrenal, Female total RNA (MoAd.Feml) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoAd.Feml.sig21
Cell type Adrenal, Female
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoAd.Feml_sig21.5224W.a-20	10/21/2004	669953	14346	QC Passed
MoAd.Feml_sig21.5224W.b-20	10/21/2004	432288	10863	QC Passed
MoAd.Feml_sig21.5224F.a-20	10/19/2004	473815	12018	QC Passed
MoAd.Feml_sig21.5224F.b-20	10/19/2004	476627	8956	QC Passed
Run group:
Total Beads successfully sequenced - 2052683
Processed Signatures - 17813

source_name: Adrenal, Female total RNA


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 0 hours  (NCBIv34)

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C in 5% CO2 to a density of 0.75 x 10*6 cells/ml (medium and supplemental components were purchased from Invitrogen). Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer?s protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
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 = Homo sapiens, PolyA+ RNA, HL-60 Cells, 0 hours Retinoic Acid


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 32 hours (NCBIv34)

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C in 5% CO2 to a density of 0.75 x 10*6 cells/ml (medium and supplemental components were purchased from Invitrogen). Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer?s protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
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 = Homo sapiens, PolyA+ RNA, HL-60 Cells, 32 hours Retinoic Acid


title: Uterus, Female (MoUt.Feml)

description:
Strain: C57BL6
Uterus, Female total RNA (MoUt.Feml) received from National Human Genome Research Institute was analyzed through Bioanalyzer and passed our quality control test.  The mRNA was processed according to the MPSS protocol as outlined in the previous publications (Brenner, S., et al. (2000) Proc Natl Acad Sci U S A 97(4): 1665-1670 and Brenner, S. et al., Nat. Biotechnol 18(6): 630-634).  Briefly, the mRNA was reverse transcribed and the cDNA was digested with Dpn II. The 20 bases adjacent to the 3? most Dpn II site was cloned into a Megaclone vector.  The resulting library was amplified and loaded onto microbeads. About 1.6 million microbeads were loaded into each flow cell and the signature sequences were determined by a series of enzymatic reactions as outlined in the above publications. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparisons between samples.
Cells/tissue:
Library MoUt.Feml.sig21
Cell type Uterus, Female
Source NHGRI
RNA isolation LYNX mRNA QC passed
cDNA library:
Library 	DpnII restriction - (signature cloning using MmeI)
Sequence length	20 bp
MPSS:
runs
MoUt.Feml_sig21.5159F.e-20	11/4/2004	434208	7256	QC Passed
MoUt.Feml_sig21.5159F.d-20	10/29/2004	446420	7479	QC Passed
MoUt.Feml_sig21.5159W.c-20	10/21/2004	352898	7078	QC Passed
MoUt.Feml_sig21.5159W.a-20	9/3/2004	525703	9063	QC Passed
MoUt.Feml_sig21.5159W.b-20	9/3/2004	410704	7416	QC Passed
Run group:
Total Beads successfully sequenced - 2169933
Processed Signatures - 12269

source_name: Uterus, Female total RNA