title: MIT Boissonneault-Housman Ps-n10772 feature cDNA microarray v1
status: Public on Sep 08 2013
contact.city: Cambridge
contact.first: Katie Rose
contact.last: Boissonneault
contact.email: katieroseboissonneault@gmail.com
contact.state: MA
contact.address: 77 Massachusetts Ave., 76-553
contact.department: Biology
contact.country: USA
contact.web_link: http://to be added
contact.institute: Massachusetts Institute of Technology
contact.phone: 6175019336
organism: Pseudo-nitzschia multiseries
description: Pseudo-nitzschia multiseries CL-125 (Ps-n).
  A number of the cDNAs printed on the array were sequenced more than once and in both directions (5' and 3'). All the Ps-n ESTs were submitted as separate files to the NCBI EST database. All of the replicate sequences for each cDNA clone have been included in the platform data table, with the corresponding NCBI #, to offer the reader the full dataset for each clone.
manufacture_protocol: cDNA library: Ps-n strain CL-125 cells from non-axenic cultures were harvested during the late exponential through mid-stationary growth phases, under predominantly toxin-producing conditions. Cultures were split into 250-500 mL aliquots that were centrifuged for 15 min at 1000 g. The loose pellets were pooled, and centrifuged again briefly to remove any residual culture medium. Total RNA was extracted immediately by homogenizing the cells in TRIzol® (Invitrogen Corporation, Carlsbad, CA, USA). Insoluble material was removed by low-speed centrifugation of the samples. RNA was precipitated twice with salt and ethanol, resulting in high-quality total RNA, as indicated by 260/280 O.D. ratios and formaldehyde agarose gel electrophoresis. Poly (A)+ RNA was then isolated from total RNA using a biotin-labeled oligo(dT)20-streptavidin kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) following the manufacturer’s instructions. First-strand cDNA was prepared from 5 µg poly (A)+ RNA using Superscript II (Invitrogen, Grand Island, NY, USA), NC-p7 (an RNA chaperone), and oligo pd(TZ) (an oligo-dT primer with some of the internal thymidine residues replaced with 3-nitropyrrole to minimize mispriming to internal A-rich sequences). Double-stranded cDNA was generated using RNase H, E. coli DNA polymerase I, and E. coli ligase. The ends of the cDNAs were polished with T4 DNA polymerase, and BstXI adaptors were ligated to the cDNA ends. The cDNAs were then fractionated on sucrose gradients, ligated into pMD1 (a pUC-based vector) and transformed, by electroporation, into E. coli DH10B cells [Das et al., 2001]. Following an initial library plating, 19,200 individual colonies were picked and stored at -80ºC in 15% glycerol for further analysis.(cDNA library constructed at McGill University, Pelletier lab (Boissonneault, KR, Harvey, I, Nguyen, N, and Pelletier, J)).
  cDNA preparation: 5,265 clones from the Ps-n cDNA library were grown overnight in Luria broth with carbenicillin (50 µg mL-1) at 37ºC on a shaker table. Three vector only and twelve Homo sapiens cDNA control clones (J. Pelletier) were also grown under these conditions.  Ten microliters of bacterial culture were used in 100-µL PCR reactions with primers T7 forward (TAATACGACTCACTATAGGG) and M13 reverse (CAGGAAACAGCTATGAC), which flanked the cloning site of the pMD1 vector. PCR amplification was performed using HiFI Taq polymerase (Invitrogen Corporation). An initial DNA denaturation step at 94ºC for 2 min was followed by 35 amplification cycles (0:30 melting at 94ºC, 0:30 annealing at 55ºC, 1:00 extension at 68ºC). PCR products were purified using MultiScreen size-exclusion filter plates (Millipore, Billerica, MA, USA). The DNA was then resuspended in 100 µL of nuclease-free de-ionized water and transferred to clean plates using a mechanical pipetting station. DNA quality was verified by 1% agarose gel electrophoresis for eight samples per 96-well plate; DNA concentration was determined by PicoGreen fluorescent staining [63]. Fifty µL of each PCR product was dried by vacuum centrifugation and then resuspended in 10 µL of 1.5 M Betain /3X SSC print buffer, yielding an average final concentration of 600 ng µL-1.
  Printing Array: Ps-n cDNA probes were printed onto CMT-GAPS slides (Corning, Corning, NY, USA), using a MicroGrid 610 TAS array printer (Biorobotics, Woburn, MA, USA) with quill pins. A total of 5,169 Ps-n cDNAs were printed in duplicate, and 96 were printed in quadruplicate; in addition, 3 vector only cDNAs, 12 H. sapiens control cDNAs, and 10 control cDNAs from SpotReport Alien Array Validation System (Stratagene, La Jolla, CA, USA) were printed in duplicate, resulting in a final chip that included 10,772 features. Spots were printed with a 32 print-tip head, producing a lay-out represented by 8 x 4 grids. Each grid was sub-divided into two sections, representing replicate spots. Individual features were 13 µm in diameter and were separated by 130 µm (from one spot to the next). 0.005 µL of ~600 ng µL-1 DNA (2-3 ng) was transferred to each spot. Final Ps-n arrays displayed a strong signal to noise ratio, with virtually no background. (For visual representation of microarray, see supplementary file: MIT Housman Psn 10772 cDNA v1 Figure). Experimental hybridization results demonstrated a high degree of reproducibility between replicate spots on the Ps-n chip.