title: Tomato Rhizosphere Metatranscriptome, biochar-amended, time 2
design: Bulk soil samples and rhizosphere soil were collected, and flash frozen in liquid nitrogen, at four time points representing key tomato plant developmental stages: (1) 25 days  emergence of third leaf, (2) 40 days  vegetative growth, pre-flowering, (3) 55 days  flowering, and (4) 70 days  fruit production (75% of fruit at red ripe stage). At 70 days, mature fruits were harvested, and above-ground biomass was collected by cutting the plant at soil level. Fruit biomass and shoot fresh weight was recorded for each treatment group.  Hexadecyltrimethylammonium bromide (CTAB) extraction buffer was prepared by mixing equal volumes of 10% (wt/vol) CTAB in 0.7 M NaCl with 240 mM potassium phosphate buffer (pH 8.0). RNA precipitation solution was prepared by dissolving 30% (wt/vol) polyethylene glycol 8000 in 1.6 M NaCl solution. 0.5 g rhizosphere sample (wet soil + roots) was weighed and transferred to 2 mL screw cap centrifuge tubes containing 0.5g (0.5 M) glass. For RNA extraction, 0.5 mL of CTAB extraction buffer and 0.5 mL phenol-chloroform-isoamyl alcohol (25:24:1) (pH 7.15) was added to the soil samples. Samples were then lysed for 3 minutes using Qiagen TissueLyser-II with machine speed set at 30 1/s. The samples were then centrifuged at 14,000 rpm at 4C for 10 minutes. The aqueous phase was then transferred to fresh 1.5 mL Eppendorf tube and equal volume of chloroform-isoamyl alcohol (24:1) was added, followed by centrifugation at 14,000 rpm at 4C for 10 min to remove the residual phenol. The aqueous layer was again transferred to fresh 1.5 mL Eppendorf tube. For RNA precipitation, 2 volumes of RNA precipitation solution were added to the extracted aqueous layer and incubated at room temperature for 2 hours. Samples were then centrifuged at 14,000 rpm at 4C for 10 minutes, and the supernatant was gently discarded. The pellets were then washed twice in ice cold 100% ethanol and air dried prior to resuspension in 50 L DEPC-treated water. Resuspended samples were treated with DNase using Ambion Turbo DNA-free kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturers guidelines. RNA was quality checked using agarose gel electrophoresis, and quantified using Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA libraries underwent library preparation and sequencing at the WSU Genomics Core on an Illumina HiSeq 2500 as 100-base single-end reads.