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#    SOYBEABN    #   Procedure was performed in UCLA
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>> Harvesting Seeds

Soybean (Glycine max cv. Asgrow A3237) were grown in the UCLA Plant
Growth Center with a 16:8 light-dark cycle at 22C. A linear correlation
was observed between early developmental stages (globular to heart
stage) and the size of soybean pods. Independent pod replicates at
appropriate developmental stages were collected during the morning and
the seeds were staged by viewing fixed sections of whole mount embryos
using light microscopy. To increase precision in our measurements the
petioles were not included. Pods were placed immediately on ice and
carried to the lab after collection. 

>> Seed Fixation and Embedding

Seeds were dissected from pods no later than three hours after
collection. A small excision was made on the chalazal end to allow
fixative to penetrate seed tissues. After dissection, seeds were placed
in a chilled solution of Acetic acid-Ethanol (3:1). Fixation was
performed until the following day at 4C. Seeds were dehydrated with a
series of ethanol solutions with increasing concentration (75%, 85%,
95%, 3x 100%) for a minimum of 3 hours each. Seeds were then infiltrated
with different xylene dilutions (in ethanol) (25%, 50%, 75%, 3x100%) for
a similar period of time. Finally, seeds were incubated approximately
9-10 times with melted paraffin at 60C for a minimum of 3 hours,
embedded with paraffin in foil boats, and kept at 4C until further use.

>> Seed Sectioning

Soybean seed paraffin sections of 6 um and 8 um were generated using a
Reichert-Jung 820-II microtome. The paraffin ribbons were visualized
using a compound microscope with a 10x magnification, and only ribbons
containing visible embryos were placed on PEN-Foil slides. The slides
were kept on a slide warmer at 45C for approximately 24 hours, and then
kept in a slide holder at room temperature to minimize exposure to
humidity. A range of 5 to 8 slides per replicate was generated.

>> LCM

The PEN-Foil slides containing the paraffin sections were treated with
xylene solution in order to melt the paraffin. Two incubations of two
minutes each were performed in the fume hood, where the slides were kept
at room temperature until the following day. The slides were then placed
back in the slide holder and stored as previously mentioned in the Seed
Sectioning section. The slides were used within two months of the xylene
treatment.

We used either the Leica AS LMD or LMD6000 system to capture the
different seed regions. Only sections containing a full embryo (i.e.
embryo proper and suspensor) were captured and used for gene expression
analysis. For each slide, the sections meeting this criterion were
selected, a picture of the region to be captured was taken, the region
was captured by LCM, and a final picture was taken documenting the
captured area. Captured material was collected on a 0.5 ml eppendorf
tube cap containing 30 ul of the extraction buffer from the Arcturus
PicoPure RNA isolation kit. After collecting all samples from a
particular seed region on a slide, the tube containing the captured
material was stored at room temperature until the end of the LCM
session. Another tube was placed in the rack and a different seed region
was captured from exactly the same sections that were used previously.
At the end of the session, tubes containing the collected material were
placed at -80C until further use. As an example, one of the replicates
with five slides contained 245 sections, but only 143 contained a full
embryo from where the seed regions were captured. These 143 sections
represented 36 seeds.