Physcomitrium patens
For protonema tip cell collection, 10-day-old sporelings were harvested from cellophane disks and fixed in ice-cold acetone overnight. Bud cell collection involved similar procedures, except bud-producing 18-day-old sporelings treated with NAA and BAP were harvested. After 24 hours fixation, fixed sporelings were adhered directly onto charged HistoBond slides (VWR, cat number UNIB75251) and left to dry at 37 deg C for six hours. The slides were then directly used for P.A.L.M. laser microdissection following the Zeiss manufacturing specifications. Selected protonema tip cells and tetrad stage gametophore bud cells were microdissected; at least 100,000 sq microns of plant tissue were collected for each cell type. Whole plant samples of 10-day-old sporelings, and 16-day-old NAA-treated/BAP-treated sporelings were prepared by grinding entire shoots in liquid nitrogen. RNA was extracted from the collected tissues using the PicoPure RNA Isolation Kit (Life Technologies) and in vitro amplified using a TargetAmp 2-round aRNA Amplification Kit 2.0 (Epicentre). Three biological replicates were prepared for the tip and bud cell samples; two whole plant replicates were prepared from the 10-day-old and 16-day-old plantlets. Illumina library construction and sequencing: Each amplified RNA sample was prepared for sequencing following the protocols (Kumar et al., 2012) with a modified procedure for single sample processing. The libraries were ligated to adapters with barcodes 3-nucleotides in length and pooled for 8-plex sequencing on an Illumina HiSeq 2000 at the Cornell Genomic Facility.
isolate
Gransden
dev_stage
Protonemal
geo_loc_name
United Kingdom: Cambridge
tissue
protonemal tip cells
BioSampleModel
Plant