Zr small rna ladder

4 un-opened flower buds (stage 12 and younger) from individual
mutant plants as well as 3 individual wild-type (WT) controls were
collected, frozen in liquid nitrogen and kept at −80°C until
use. The total RNA extraction and small RNA enrichment were performed as
previously described^{68 (link)}with the following minor modifications: (1) for the small RNA enrichment
step an equal volume of 20% polyethylene glycol 8000/2M NaCl was added to
each total RNA sample and (2) the ZR-small RNA ladder (Zymo Research, Cat#
R1090) was used to determine the gel region corresponding to the
17–29 nucleotide (nt) size range. The resulting small RNAs were then
used for library preparation with the NEBNext Multiplex Small RNA Library
Prep Set for Illumina (New England Biolabs, Cat# E7300) following the
user’s manual. The final library products were further purified using
an 8% polyacrylamide gel to excise 130–160nt products relative to the
pBR322 DNA-MspI Digest ladder (New England Biolabs, Cat# E7323AA). The
libraries were pooled and sequenced (single end 50bp, SE50) on a HiSeq 2500
machine (Illumina).

Denature the half of the total RNA set aside at the beginning of step 2.2b, the 10 μl of oxidation-elimination treated RNA, and 10 μl of ZR small-RNA ladder (Zymo Research R1090) in RNA Loading Dye (NEB B0363S) at 70°C for 5 minutes. Run the samples and ladder on a 15% TBE urea PAGE gel until the dye front comes close to the end of the gel (i.e. 180V, 75 minutes). Stain the gel with SYBR Gold Nucleic Acid Gel Stain (Thermo S11494) for 5 min at room temperature according to the manufacturer’s instructions, and cut out the small RNA (17-29 nt) portion of the gel, using the ladder as a reference (Fig. 3A). To recover piRNAs, cut the gel band between 17 to just over 30 nt. Recover the small RNAs using ZR small-RNA PAGE Recovery Kit (Zymo Research R1070), eluting with 6 μl of nuclease-free water.