7 protocols
nucleic acid sequencing protocol
Hiseq2000, 100bp Illumina paired end sequencing
growth protocol
Arsenate tolerant and non-tolerant Holcus lanatus L. Genovars were selected randomly from a screened population, the arsenic uncontaminated Cruickshank Botanic Garden (CBG) and tillers of the plants were grown hydroponically for 2 weeks in 50ml individual centrifuge tubes with base of the tubes covered in tin-foil to block out light, in heated glasshouse at around 18C under supplemental sodium lamps.
treatment protocol
Plant tillers from arsenic tolerant (T) and non-tolerant (N) plants were grown hydroponically for 2 weeks in 50ml complete Hogland solution (plusP treatment) or 50ml Hogland solution minus Phosphate (minusP treatment). The experiment therefore investigated 4 treatments (TplusP, TminusP, NplusP, NminusP) and was repeated 2 time. Experiment1 was conducted on 4 plants (n=1), Experiment2 was conducted on 16 plants (n=4).
nucleic acid extraction protocol
dry roots and shoots for each replicate were ground under liquid nitrogen and total RNA was extrected using the Plant Rneasy kit (Qiagen), with additional on-column Dnase treatment. Extracted RNA of each sample was stored at -80C until shipping on dry ice for sequencing
nucleic acid library construction protocol
Illumina TrueSeq RNA sample preparation
nucleic acid sequencing protocol
454 GS FLX TITANIUM, single end. Here is the output from Newbler for each file for the number of reads and number of bases before and after trimming: 315.A_Holcus_lanatus_NT-P_region2_run113_RL1_2012_05_29.sff: numberOfReads = 582952, 582888; numberOfBases = 261031336, 260606804. 315.B_Holcus_lanatus_NTplusP_region1_run113_RL2_2012_05_29.sff: numberOfReads = 501283, 501218; numberOfBases = 210242405, 209894594. “315.B_Holcus_lanatus_NTplusP_region1_run112_RL2_2012_05_23.sff: numberOfReads = 4477, 4477; NumberOfBases = 1844258, 1841681. “315.A_Holcus_lanatus_NT-P_region1_run112_RL1_2012_05_23.sff: NumberOfReads = 2364, 2364; numberOfBases = 990167, 988746.
nucleic acid library construction protocol
454-TITANIUM, normalised total RNA