 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 21, 2014 |
| Title |
IVT polyA selected |
| Sample type |
SRA |
| |
|
| Source name |
Mammalian gene collection
|
| Organism |
Homo sapiens |
| Characteristics |
bc accession ids: BC000128,BC000129,BC000130,BC000131,BC000132,BC000133,BC000134,BC000135,BC000138,BC000140,BC000141,BC000142,BC000145,BC000146,BC000147,BC000148,BC000149,BC000150,BC000151,BC000152,BC000153,BC000154,BC000155,BC000157,BC000158,BC000159,BC000160,BC000161,BC000162,BC000163,BC000165,BC000166,BC000168,BC000169,BC000170,BC000171,BC000172,BC000175,BC000176,BC000177,BC000178,BC000179,BC000180,BC000181,BC000793,BC000794,BC000795,BC000797,BC000799,BC000802,BC000803,BC000804,BC000805,BC000806,BC000807,BC000808,BC000809,BC000810,BC000813,BC000814,BC000819,BC000820,BC000823,BC000824,BC000827,BC000829,BC000830,BC000832,BC000834,BC000835,BC000836,BC000837,BC000846,BC000849,BC000850,BC000851,BC000852,BC000853,BC000854,BC000855,BC000857,BC000861,BC000864,BC000866,BC000868,BC000870,BC000871,BC000872,BC000873,BC000875,BC000877,BC000878,BC000879,BC000881,BC000882,BC000884,BC000887,BC000889,BC000890,BC000891,BC000892,BC000893,BC000894,BC000895,BC000896,BC000897,BC000898,BC000899,BC000901,BC000902,BC000903,BC000904,BC000905,BC000906,BC000907,BC000908,BC000910,BC000912,BC000913,BC000914,BC000915,BC000916,BC000918,BC000921,BC000923,BC000924,BC000926,BC000927,BC000930,BC000931,BC000932,BC000933,BC000934,BC000936,BC000937,BC000938,BC000939,BC000941,BC000942,BC000944,BC000948,BC000949,BC000952,BC000953,BC000954,BC000956,BC000959,BC000961,BC000962,BC000964,BC000965,BC000966,BC000968,BC000971,BC000972,BC000973,BC000974,BC000977,BC000978,BC000979,BC000980,BC000981,BC000983,BC000985,BC000986,BC000988,BC000989,BC000990,BC000991,BC000992,BC000993,BC000994,BC000995,BC000996,BC000997,BC000998,BC001000,BC001001,BC001003,BC001226,BC001228,BC001229,BC001231,BC001232,BC001233,BC001234,BC001235,BC001236,BC001238,BC001239,BC001240,BC001241,BC001242,BC001243,BC001244,BC001245,BC001247,BC001249,BC001250,BC001251,BC001252,BC001253,BC001254,BC001255,BC001256,BC001257,BC001258,BC001259,BC001261,BC001262,BC001263,BC001264,BC001265,BC001267,BC001268,BC001269,BC001270,BC001271,BC001272,BC001273,BC001274,BC001275,BC001276,BC001277,BC001278,BC001279,BC001280,BC001281,BC001282,BC001283,BC001284,BC001285,BC001286,BC001287,BC001288,BC001289,BC001291,BC001293,BC001294,BC001295,BC001297,BC001298,BC001300,BC001301,BC001302,BC001303,BC001304,BC001305,BC001308,BC001309,BC001310,BC001311,BC001312,BC001316,BC001317,BC001318,BC001319,BC001321,BC001326,BC001327,BC001328,BC001329,BC001331,BC001333,BC001334,BC001337,BC001338,BC001340,BC001341,BC001342,BC001344,BC001345,BC001346,BC001964,BC001965,BC001966,BC001967,BC001968,BC001970,BC001971,BC001972,BC001979,BC001980,BC003352,BC003353,BC003354,BC003355,BC003356,BC003357,BC003358,BC003359,BC003360,BC003361,BC003362,BC003364,BC003365,BC003366,BC003367,BC003369,BC003370,BC003371,BC003373,BC003375,BC003376,BC003377,BC003378,BC003379,BC003381,BC003382,BC003383,BC003384,BC003385,BC003387,BC003388,BC003389,BC003390,BC003394,BC003395,BC003397,BC003398,BC003400,BC003401,BC003402,BC003403,BC003407,BC003408,BC003409,BC003410,BC003412,BC003413,BC003417,BC003418,BC003682,BC003683,BC003684,BC003685,BC003686,BC003688,BC003689,BC003690,BC003691,BC003694,BC003701,BC004815,BC004816,BC004817,BC004818,BC004819,BC004822,BC005404,BC005408,BC005700,BC006768,BC006769,BC006772,BC006781,BC006784,BC006786,BC006791,BC006793,BC006794,BC006795,BC006804,BC006807,BC006808,BC006811,BC006818,BC006819,BC006821,BC006823,BC006825,BC006831,BC006832,BC006837,BC006838,BC006839,BC006849,BC006850,BC007121,BC007122,BC007123,BC007124,BC008087,BC008090,BC008091,BC008092,BC008094,BC008095,BC008100,BC008145,BC008146,BC008149,BC008151,BC008178,BC008180,BC008182,BC008183,BC008185,BC008188,BC008191,BC008194,BC008195,BC008196,BC008197,BC008198,BC008200,BC008201,BC008202,BC008203,BC008205,BC008207,BC008211,BC008212,BC008214,BC008215,BC008219,BC008226,BC008235,BC008246,BC008250,BC008251,BC008253,BC008254,BC008505,BC008506,BC008567,BC008568,BC008569,BC008572,BC008573,BC008584,BC008585,BC008586,BC008594,BC008600,BC008602,BC008603,BC008604,BC008605,BC008607,BC008608,BC008611,BC008613,BC008618,BC008621,BC008624,BC008625,BC008628,BC008629,BC008631,BC008632,BC008634,BC008636,BC008640,BC008641,BC008650,BC008651,BC008652,BC008654,BC008656,BC008658,BC008659,BC008662,BC008663,BC008664,BC008666,BC008667,BC008668,BC008669,BC008671,BC008673,BC008674,BC008675,BC008678,BC008679,BC008680,BC008682,BC008684,BC008685,BC008686,BC008688,BC008689,BC008690,BC008691,BC008972,BC008973,BC008975,BC008976,BC008979,BC008981,BC008982,BC008983,BC008984,BC008986,BC008987,BC008988,BC008990,BC008991,BC008992,BC008993,BC009009,BC009010,BC009011,BC009012,BC009014,BC009015,BC009016,BC009017,BC009025,BC009026,BC009031,BC009032,BC009037,BC009039,BC009040,BC009041,BC009046,BC009047,BC009048,BC009049,BC009051,BC009052,BC009053,BC009054,BC009055,BC009065,BC009073,BC009074,BC009077,BC009078,BC009081,BC009084,BC009139,BC009523,BC009524,BC009529,BC009530,BC009538,BC009540,BC009545,BC009548,BC009552,BC009553,BC009561,BC009564,BC009614,BC009617,BC009618,BC009620,BC009621,BC009623,BC009624,BC009631,BC009642,BC009644,BC009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rna selection method: polyA selection (included with TruSeq library prep kit)
mouse strain: NA
mouse tissue: NA
plasmid backbone: NA
ivt rna input quantity: 1000 ng
mouse rna input quantity: 0 ng
|
| Growth protocol |
cDNAs from the Mammalian Gene Collection, cloned into pCMV-Sprot 6 plasmid, were collected from three 384-well plates (total of human 1062 transcripts) as follows: 10 µl sterile dH2O was added to each well and incubated at 37oC for 10 min to resuspend plasmid DNA in water. Plasmid DNAs were collected and combined in 1.5 mL tube with aid of multichannel pipette and concentrated by ethanol precipitation. To amplify the library 10 ng of plasmid library was transferred into E.coli DH5α (Invitrogen catalog no. 18258-012) with heat shock method. Cells were incubated with plasmid library for 5 min on ice and were subjected to 42oC for 30 sec. Then cells were transferred back to ice and incubated for 2 min. Next, 0.95 mL SOC medium was added to the cells and incubated at 37 oC for 1 h by shaking at 225 rpm. Cells were plated on LB-agar plates containing 100 µg/ml ampicilin. Plates were incubated for 16h at 37oC to grow the colonies and 3500 (approx 3-fold of library size) colonies were collected with liquid LB. Cells were transferred into 100 mL liquid LB and incubated at 37oC for 2 h. Plasmids were purified using Qiagen maxiperep kit (catalog no. 12163), according to the manufacturer's protocol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For IVT RNA: Plasmids were linearized by NotI-HF enzyme so that the SP6 polymerase promoter site will be upstream of the sequences to be transcribed. Reactions consists of 5 U NotI-HF (NEB catalog no. R3189L), 5 µg library plasmid DNA, 1 X NEBuffer 4 (supplied with enzyme) and 90 µl of dH2O. Reaction was incubated at 37oC for 2 h to achieve complete digestion. The complete digestion of plasmid DNA was assessed by DNA gel electrophoresis. To eliminate NotI-HF and possible RNase in reaction mixture, samples was subjected to Proteinase K treatment. SDS and Proteinase K were added to the reaction mixture to a final concentration of 0.5% and 100 µg/mL, respectively. Sample was incubated at 37oC for 30 min. After Proteinase K treatment, sample was subjected to the phenol/chlororform extraction, followed by ethanol precipitation. Pellet was dissolved in 50 µl of RNase-free water. Next in vitro transcription was carried out using MAXIscript® SP6 Kit (Ambion catalog no: AM1308). Reaction composed of 1 µg of library plasmid, 1X transcription buffer, 0.5 mM of NTPs (GTP,ATP, CTP, and UTP), 40 U of SP6 RNA polymerase and 10 µl of RNase-free water. Reaction was incubated at 37oC for 30 min. Next, samples were treated with TURBO DNase to remove the plasmid templates. Briefly, 10 U of TURBO DNase (included with MAXIscript SP6 kit) were added to reaction mixture and incubated at 37oC for 15 min. To stop the reaction 1 μL of 0.5 M EDTA was added. To remove unincorporated NTPs and other impurities sample was precipitated with ammonium acetate/ethanol. The following reagents were added to the DNase -treated reaction mixture: 30 μL RNase-free water to bring the volume to 50 μL, 5 μL 5 M Ammonium Acetate, and 3 volumes 100% ethanol. Sample was chilled at -20oC for 30 min and then centrifuged at maximum speed in a 4oC table-top microfuge. The supernatant was discarded and pellet was washed with ice-cold, 70% ethanol. Pellet was dissolved in 50 μL RNase-free water and quality of RNA was assessed by agrose gel electrophoresis. In addition, PCR was carried out with in vitro transcribed RNA to confirm total depletion of plasmid DNA as well. For mouse RNA: WT 6-week old male C57/BL6 mice were acquired from Jackson Labs. Mice were sacrificed and liver samples were quickly dissected and snap-frozen in liquid nitrogen. RNA was isolated from frozen mouse liver samples by TRIzol reagent according to manufacturer’s protocol (Invitrogen catalog no. 15596-026). All animal experiments were performed in accordance with the approval of the Institutional Animal Care and Use Committee.
IVT RNA (2500 ng, 150ng, 75ng, 15 ng, and 0 ng) was pooled with mouse liver RNA (0 ng, 2350 ng, 2425 ng, 2485 ng, and 2500 ng respectively) to a final quantity of 2.5 µg. Each pool was split into two replicate samples of 1 µg each. RNA pools were treated with Ribo-Zero Gold kit (Epicentre catalog no. RZHM11106) and converted into Illumina RNA-seq libraries with the TruSeq RNA sample prep kit (Ilumina catalog no. FC-122-1001). Briefly, rRNA was removed from 1 ug of pooled RNA using Ribo-Zero Gold kit and purified via ethanol/sodium acetate precipitation according to manufacturer’s protocol. After drying, the RNA pellet was dissolved in 18 μL of Elute, Prime, Fragment mix (provided with TruSeq RNA sample prep kit). RNA was fragmented for 8 minutes and 17 uL of this fragmented RNA was used to make the RNA-seq library according to Illumina TruSeq RNA sample prep kit protocol. After fragmentation/ priming, first strand cDNA synthesis with SuperScript II (Invitrogen catalog no. 18064014), second-strand synthesis, end-repair, a-tailing, and adapter ligation, the library fragments were enriched with 15 cycles of PCR. Quality and size of library was assessed using Agilent 2100 BioAnalyzer. The five libraries from each replicate were pooled together and sequenced using a single lane from an Illumina HiSeq 2000 (paired 100 bp reads).
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| |
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
polyA RNA
As with the other RNA-seq libraries, these libraries were prepared using the TruSeq RNA sample prep kit (Illumina catalog no. FC-122-1001). For the polyA sample, 1 µg of IVT RNA was treated with polyA selection reagents included with the TruSeq RNA sample prep kit according to manufacturer's protocol. RNA was fragmented for 8 minutes and 17 uL of this fragmented RNA was used to make the RNA-seq library according to Illumina TruSeq RNA sample prep kit protocol. After fragmentation/ priming, first strand cDNA synthesis with SuperScript II (Invitrogen catalog no. 18064014), second-strand synthesis, end-repair, a-tailing, and adapter ligation, the library fragments were enriched with 15 cycles of PCR. Quality and size of library was assessed using Agilent 2100 BioAnalyzer. The five libraries from each replicate were pooled together and sequenced using a single lane from an Illumina HiSeq 2500 (paired 100 bp reads).
|
| Data processing |
Raw reads from all sequencing samples were aligned to the human genome (GRCh37/hg19) using the RNA-seq Unified Mapper (v2.0.4) with default parameters. RUM also generated coverage bedgraph files and calculated RPKM values using the annotations from the MGC genes track in the UCSC genome browser.
Genome_build: hg19
Supplementary_files_format_and_content: txt files contain RPKM values for each exon, intron, and transcript contained in the IVT sequences
Supplementary_files_format_and_content: bigwig files contain coverage plots for uniquely-aligned reads.
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| |
|
| Submission date |
Aug 29, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicholas Lahens |
| Organization name |
University of Pennsylvania
|
| Department |
ITMAT
|
| Street address |
Smilow Center for Translational Research 10-110 3400 Civic Center Blvd, Bldg 421
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE50445 |
IVT-seq reveals extreme bias in RNA-sequencing |
|
| Relations |
| BioSample |
SAMN02339360 |
| SRA |
SRX341400 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1219408_polyA_Unique.bw |
5.7 Mb |
(ftp)(http) |
BW |
| GSM1219408_polyA_feature_quant.txt.gz |
298.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
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