title: NextSeq 500 sequencing; EVOREPRO: Physcomitrella
library_construction_protocol: The Gransden wild-type strain from Physcomitrella patens Bruch & Schimp (Ashton and Cove, 1977) was used for this study. To initiate plant growth and culture, 3 Mature sporophytes were sterilized using a 5% commercial bleach solution for 5 minutes and rinsed twice in molecular grade water. Sterilized sporophytes were then broken using a pipette tip and diluted into 5mL molecular grade water. Spore containing solution was then distributed into 4 sterile peat pellets (Jiffy-7, Jiffy Products International) and two 9cm Petri dishes containing KNOPS medium (Reski and Abel, 1985) supplemented with 0.5 g/l ammonium tartrate dibasic (Sigma-Aldrich Co). Petri dishes were kept at 25°C, 50% humidity, and 16 h light (light intensity 80 μlum/m/s). Protonema samples were collected 10 days after spore germination. Plants in Phytatray™ II (Sigma-Aldrich Co) containing 4 sterile peat pellets (Jiffy-7, Jiffy Products International) were grown for 6-8 weeks at 25°C, 50% humidity, and 16 h light (light intensity 80 μlum/m/s). Water was supplied to the bottom of each box. Leave samples were collected after 6 weeks, prior to induction of gametangia development. For gametangia and sporophyte development, water was again supplied to the bottom of each box containing four pellets and were transferred to 17°C, 8 h light, and 50% humidity (light intensity 50 μlum/m/s) to induce the development of reproductive structures (Hohe et al., 2002). Gametangia samples (archegonia, paraphysis and sperm cell packages) were collected 15 days after reproductive induction. Antheridia samples were collected at several time points during their development. Further development of the sporophyte was conducted under these conditions and sporophyte samples were collected at different time points during sporophyte development. S1 sporophytes were collected 7 days after sperm cell (SC) release, S2 sporophytes 15 days after SC release, S3 sporophytes 20 days after SC release (green spore capsules) and SM samples 28 days after SC release (brown spore capsules).  The Gransden wild-type strain from Physcomitrella patens Bruch & Schimp (Ashton and Cove, 1977) was used for this study. To initiate plant growth and culture, 3 Mature sporophytes were sterilized using a 5% commercial bleach solution for 5 minutes and rinsed twice in molecular grade water. Leaves, Protonema and Sporophytes were collected under the Stereoscope using tweezers, placed in 2.5 uL of RLT+ buffer (Qiagen), and shock frozen in liquid nitrogen. Before RNA-seq library preparation, these samples were mechanically disrupted using sterile pellet pestles (Z359947, Sigma-Aldrich Co). Antheridia, Archegonia, Paraphysis and Sperm Cell packages were collected using a Yokogawa CSU-W Spinning Disk confocal with 10x 0.25NA objective, using the brightfield channel and an Andor Zyla 4.2 sCMOS camera. For each of these samples the plants were prepared under a stereoscope, isolating the whole gametangia for ca. 10 shoots. They were placed in 20 uL of molecular grade water on a glass slide. Using a cover slip the gametangia were disrupted into individual antheridia by applying slight pressure. Slides were placed under a microscope and specific organs were identified and collected, using an Eppendorf CellTram® Air/Oil/vario micromanipulator with glass capillaries (borosilicate glass with fire polished ends, without filament GB100-9P) pulled with a Narishige PC-10 puller. Then they were transferred to another clean slide, and subsequently excessive liquid containing possible contaminations, such as cell debris, was removed. For paraphysis samples 8-15 individual paraphysis were collected directly into 2 uL of RLT+ buffer and flash frozen in liquid nitrogen. For antheridia samples 5 to 20 individual antheridia of each specific stage (9 to 15 days after induction, distinguished by size) were collected and then burst under a microscope by applying pressure on a cover slip applied to the samples on the slide The slide was washed with 4 uL of RLT+ buffer and the buffer transferred into a PCR tube, subsequently flash frozen.  Archegonia samples were prepared from 3-5 archegonia following the same procedure. Released sperm cell packages (2-5 per sample) were collected from gametangia preparations (as described above; antheridia 15 days after induction) without clean up, transferred into a tube with 2 uL of RLT+ buffer, flash frozen in liquid nitrogen and subsequently used for RNA-seq library preparation. RNA-seq library preparation for all samples was performed as described in (Misra et al., 2019), with the addition of mixing the PCR tubes on a Thermomixer C (Eppendorf) every 15 minutes at 200 rpm for 1 min during the RT step.