Expression profiling of the moss F. hygrometrica
Expression profiling of the moss F. hygrometrica
Ten fully inbred sporophytes (strain XII5A) were surface sterilized and their spore content germinated in separate petri dishes (9 mm diameter) containing full strength Hoagland's medium (14h day/10h night, 40 mmol/m2/sec, 25  degrees C). Germinated spores (gametophyte_1) were collected after 3 days and protonemata (gametophyte_2) after 14 days of growth. RNA was extracted from four biological replicates (petri dishes) separately but were pooled in equimolar quantities prior to library preparation and RNA sequencing. After 14 days the protonematal mat of the remaining two plates were divided into 16 equal pieces and were transferred to liquid culture (full strength Hoagland's solution). After 5 days contents of the liquid cultures were emptied into small pots (5 x 5 cm) filled with soil (1:1 mixture of potting soil and sand) to initiate four gametophyte and 12 sporophyte pots. Plants were kept under these conditions until gametophores filled the pot (appr. 1.5-2 months). Plants were watered (ddH2O) from below. Gametophores (gametophyte_3) were then harvested from the four gametophyte pots separately. When the first gametangia appeared in the sporophyte pots (appr. after one month) they were watered (ddH2O) for 10 minutes at 120 minute intervals by an automatic misting system to help fertilization and sporophyte production. Samples of sporophyte_1, sporophyte_2 and sporophyte_3 were each collected from a block of four different pots. RNA was extracted separately from the four biological replicates (both gametophyte and sporophyte pots) but were pooled afterwards in equimolar quantities prior to library preparation and RNA sequencing. Samples of gametophyte_1, gametophyte_2 were collected 3 and 14 days after initiation of spore germination on medium in petri dishes. Sample gametophyte_3 was grown in the green house on soil in pots. We also collected three developmental stages of the sporophyte phase, specifically sporophytes shorter than 5mm (sporophyte_1), elongated needle-like sporophytes (sporophyte_2), and sporophytes with swollen capsules (sporophyte_3) from pots that were grown in the green house. We carried out all experiments with the strain XII5A. Total RNA was isolated separately from the tissues using the RNeasy Plant Mini Kit (Qiagen, Valencia, California, USA) following the manufacturers instructions with on-column DNA digestion. RNA was extracted from the four biological replicates separately but was pooled in equimolar quantities prior to library preparation and RNA sequencing. Single end unstranded libraries were prepared according to the protocols of Illumina
Experimental Factor: developmental stage
protonemata, gametophyte