GSM1695751: Soybean Heart Stage Hilum-Biological replicate 2; Glycine max; RNA-Seq
Soybean seeds (Williams 82) were fixed in 3:1 ethanol:acetic acid for 16-20 hours at 4°C, then dehydrated with 75% ethanol, 85% ethanol, 95% ethanol, and three times with 100% ethanol, each wash in one hour intervals. Then they were infiltrated with 3:1 ethanol-xylenes, 1:1 ethanol-xylenes, 1:3 ethanol-xylenes, and three times with 100% xylenes, dilution in two hour intervals. They were then infiltrated with molten paraffin at 60 deg  C for 1-4 days, depending on the size of the tissue. Laser capture microdissection was used to isolate specific subregions of the seed after tissues were sectioned in 5-10um sections. Total RNA was isolated with Ambion RNaqueous-Micro kit (Grand Island, NY) and quantified with Quant-iT™ RiboGreen® RNA reagent (Grand Island, NY) on a Nanodrop ND-3300. Sequencing libraries were prepared using 250 nanograms of total RNA (unless otherwise noted), using the Illumina TruSeq Small RNA Sample Preparation Kit (cat no RS-200-0012 & RS-200-0024). Briefly, total RNA was ligated with 3' and 5' RNA adapter using T4 RNA ligase. Adapter-ligated small RNA was converted to complementary DNA (cDNA) and amplified by PCR for 15 cycles.  Libraries were size-selected on a polyacrylamide gel for the desired size of 147-160 bp and purified. Libraries were quantified using Quant-iT PicoGreen dsDNA Reagent (Grand Island, NY) and a Nanodrop ND-3300 instrument (Thermo Scientific, Waltham, MA).  Phi-X174 DNA was spiked in to the library by the sequencing facility before cluster formation and sequencing.
GEO Accession
GSM1695751