title: New Zealand White Rabbit Heart 2.8K cDNA
status: Public on Jan 28 2009
contact.city: Boston
contact.first: James
contact.middle: D
contact.last: McCully
contact.email: James_mccully@hms.harvard.edu
contact.state: MA
contact.address: 77 Avenue Louis Pasteur
contact.department: Cardiothoracic Surgery
contact.country: USA
contact.institute: BIDMC/Harvard Medical School
contact.phone: 617-667-0725
organism: Oryctolagus cuniculus
manufacture_protocol: cDNA Library : Isolation of  total heart (atria and ventricles)RNA:  RNA was isolated  from New Zealand rabbit (15-20 weeks; 3 to 4 kg) heart in an RNase-free environment using RNase-free lab-wear, materials and equipment according to the  procedure as described by Chomczynski and Sacchi (Anal Biochem 162:156-15). In brief tissue was pulverized under liquid nitrogen using  mortar and pestle and extracted two times in acidic guanidinium isothiocyanate-phenol-chloroform and incubated for 5 min. at room temperature to permit the complete dissociation of nucleoprotein complexes. The mixture was centrifuged at 14,000x g for 15 min. at 4oC. Following centrifugation, the aqueous phase containing the RNA was carefully removed and transferred to a new tube containing 0.5 mL isopropanol and incubated for 10 min. at room temperature to precipitate the RNA. The RNA was collected by centrifugation at 14,000x g for 20 min. at 4oC. The RNA pellet washed with 1 mL of room temperature 75%  ethanol, re-centrifuged and the RNA pellet air-dried and dissolved in DEPC-treated H2O.
  POLY(A)+ RNA fractions were enriched by oligo(dT) cellulose chromatography. In brief, 1 mg of  total RNA was adjusted to 0.55 mg/mL RNA concentration in DEPC-treated H2O, and incubated for 5 min. at 650C and then chilled on ice.  The salt concentration was adjusted to 0.5M NaCl and the RNA sample mixed with 1 mL of oligo(dT) cellulose. The  suspension was  incubated for 10 min. at 37oC to allow the mRNA to bind to the oligo(dT) cellulose. The unbound RNA was eluted  and re-heated for 5 min. at 65 oC, chilled on ice and re-suspended in the oligo(dT) cellulose. The unbound RNA was washed from the column with a low salt buffer and the bound mRNA eluted from the oligo(dT) cellulose beads with 1 mL H2O (65 oC). The eluted mRNA was re-heated for 5 min. at 65 oC, chilled on ice and re-suspended in the oligo(dT) cellulose and then eluted as described above in order to further reduce any rRNA contamination. Fractions (200 uL) was collected in microfuge tubes. Positive fractions containing mRNA were determined by UV spectrophotometric analysis, pooled and the mRNA precipitated overnight, at -20oC with 50 ug/mL glycogen, 0.1 volume 7.5 M ammonium acetate and 2 volumes ethanol (-20 oC) and then collected by centrifugation  at 14,000x g for 30 min. at 4 oC. The mRNA pellet was washed twice with 75% ethanol, air-dried and dissolved in DEPC-treated H2O. The mRNA yield was determined by UV spectrophotometric analysis (260 nm) with the quality of the mRNA determined by gel electrophoresis on a 1% agarose gel containing 3% formaldehyde, 0.02 M MOPS pH 7.4 (3-[N-morpholino] propanesulfonic acid) and 0.001 M disodium EDTA (ethylenediamine tetraacetic acid) .
  cDNA LIBRARY CONSTRUCTION:  cDNA libraries were constructed  using the  SuperScript Plasmid System (GibcoBRL, cat. No.18248-013) according to the manufacturer’s instructions.  The cDNAs were ligated into the plasmid vector pSPORT 1  (4,109 bp) and then transformed into DH5 competent cells (GibcoBRL, Cat. No.10643-013) according to the manufacturer’s instructions.
  Estimation of cDNA library size was performed by titration of transformed  bacteria on LB-agar plates (1% bacto-tryptone, 0.5% bacto-yeast extract, 1% NaCl, 1.5% bacto-agar)containing IPTG/X-gal (0.02 ug/mL each)and ampicillin (75 ug/mL). Aliquots of cDNA libraries were stored in 7% DMSO at -80oC.
  Estimation of cDNA insert size was determined by random selection of cDNA clones from IPTG/X-gal LB-agar plates containing ampicillin. In brief, single isolated cDNA clones were selected and used to inoculate 2 mL LB liquid media (1% bacto-tryptone, 0.5% bacto-yeast extract, 1% NaCl) containing ampicillin (75 ug/mL) and  incubated overnight at 500 rpm at 37oC in a New Brunswick incubator. The recombinant bacteria werre chilled on ice and the recombinant bacterial pellet collected by brief centrifugation. Plasmid cDNA was isolated by alkaline lysis using  Qiagen columns (cat. No. 12125) and the plasmid DNA precipitated with 0.6 volumes isopropanol by centrifugation at 14,000x g for 30 min. The cDNA pellet was washed twice with 70% ethanol, air-dried and dissolved in TE buffer (10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA, pH 8.0). The purified plasmid cDNA was digested for 1 hour at 37oC in a reaction mixture containing a 3-fold excess of the restriction enzyme Mlu I (New England Biolabs, Cat. No. R0198S) which cleaves the unique restriction enzyme sites located at the termini of the cDNA insert distal to the  Not I and Sal I insertion sites. The digested cDNA was resolved on a 1% agarose gel in 40 mM Tris-acetate and 2 mM EDTA  (TAE) and the gel stained with ethidium bromide then photographed under UV illumination. DNA molecular weight markers (1 kb; New England Biolabs; 323-2S)  were used for estimation cDNA molecular weights.
  Isolation and growth:  cDNA libraries were plated at low density  on  IPTG/X-gal LB-agar plates containing ampicillin (75 ug/mL) and  incubated overnight at 37oC.  The following day cDNA clones were selected, individually and used to inoculate individual wells in 96-well culture blocks (USA Scientific, Cat. No.7556-9600) containing 1.3 mL/well liquid LB media containing ampicillin (75ug/mL) and the plates oriented and  labeled to indicate clonal position and then sealed with Breathe–Easy sealing film (USA Scientific) and incubated overnight at 500 rpm at 37oC in a New Brunswick incubator.
  Replicate plates were produced following overnight incubation by removing 100 uL samples from each well in 96-well culture blocks for the  inoculation of replicate wells in 96-well culture blocks containing 0.9 mL/well liquid LB media containing ampicillin (75 ug/mL). The replicate plates were oriented, labeled in the same manner as above, sealed with Breathe–Easy sealing film (USA Scientific) and incubated overnight at 500 rpm at 37oC in a New Brunswick incubator. Following overnight incubation replicate plates were cooled on ice and glycerol added to a  final concentration of 20 % /well and the plates sealed with  Thermowell sealers (Costar 6524) and stored at –80oC.
  5’ Sequencing:  Plasmid cDNA were isolated by Qiagen Bio Robot 8000 automated nucleic acid purification and liquid handling workstation available for use by our laboratory through core facilities. Purified cDNAs were sequenced using the Applied Biosystems Taq DyeDeoxy Terminator cycle sequencing kit which utilizes a fluorescently-labeled dideoxy-nucleotide chain termination method. After cycle sequencing and clean up the DNA samples were resolved by capillary electrophoresis on an PE BIOSYS 3700 automated sequencer which translates the fluorescent signals into their corresponding base pair sequence.
  Sequence  analysis of all cDNAs was performed against the non-redundant GEN Bank/ EMBL/ DDBJ nucleotide, the non-redundant Gen Bank CDS translation/ PDB/ Swiss  Prot/ PIR/ PRF peptide and the dbEST databases using the BLAST algorithm on a UNIX platform. Assignment of putative identity for cDNAs exhibiting matches to known genes required a maximum value of E=1e-25 (minimum p=10-10)and nucleotide sequence identity >95%. Prior to analysis of sequence data, sequence quality were evaluated using the accuracy assessment program  phred of the phred/phrap/consed assembly package. All sequence traces from the  ABI model sequencer werebe saved in fasta output format.
  Sequence submission and storage: Sequence quality was evaluated using the accuracy assessment program  phred of the phred/phrap/consed assembly package. All sequence traces were saved in fasta output format. Sequence analysis was performed against the non-redundant GEN Bank/ EMBL/ DDBJ nucleotide, the non-redundant Gen Bank CDS translation/ PDB/ Swiss  Prot/ PIR/ PRF peptide and the dbEST databases using the BLAST algorithm on a UNIX platform . All cDNA’s were 5’ sequenced and are available under accession numbers EC618425-EC626095 and  dbEST40150263-40157933 and at www.mccull