GSM4906698: Leaf bulk RNA seq- Library 1; Arabidopsis thaliana; RNA-Seq
Seven to nine abaxial epidermis-peeled and cut leaves attached to time tape were immediately immersed in the petri dish containing 15 mL freshly prepared protoplast isolation solution (1% Cellulase Onuzuka R-10 (Duchefa, Haarlem), 0.3% macerozyme R-10 (Duchefa, Haarlem), 0.6 M Mannitol, 20 mM MES pH 5.7, 20 mM KCL, 1 mM DTT, 10 mM CaCl2, and 0.1 % BSA). Dithiothreitol (DTT) and bovine serum albumin (BSA) were added to the enzyme solution to protect the protoplasts. Leaves were shaken at 30 rpm on a platform shaker for 2 hours. After confirming the release of the protoplasts into the solution, 10 mL of wash buffer (0.6 M Mannitol, 20 mM MES pH 5.7, 20 mM KCl, 1 mM DTT, 10 mM CaCl2, and 0.1 % BSA) was slowly added to the petri dish containing the protoplasts. The solution was filtered into a round bottom 20 mL tube using a 70 um pore size filter (Corning, New York). The protoplast solution was centrifuged at 100 x g for 3 min in a swinging rotor. The protoplasts were washed four times with 20 mL washing buffer. After the final wash step, protoplasts were slowly resuspended in 1 mL wash buffer and gently filtered twice using a 40 um Flowmi cell strainer (Bel-Art SP Scienceware, New Jersey). Total RNA was extracted from leaves (not protoplasted) and leaf protoplasts isolated. For mRNA poly-A enrichment, 5 ug of total RNA was purified using the Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Massachusetts). Libraries were constructed using the ULTRA II directional library kit (New England Biolab, Massachusetts), and size selection was done using SPRI beads (New England Biolab, Massachusetts) following the manufacturer manual with the following exceptions: 7 min of fragmentation time and 26.5 and 10 uL SPRI beads pre-PCR were used to enrich ~400 bp inserts. Library amplification included 10 PCR cycles, and 0.7 volumes of (35 uL) of SPRI beads were used for post-PCR purification. QC-tested libraries (Agilent Technologies, California) were sequenced on an Illumina HiSeq 2500 lane with 150 bp paired-end.