GSM4223966: CALS7_4; Arabidopsis thaliana; RNA-Seq
Arabidopsis root protoplasts were isolated according to the published protocol (Clark et al., 2018). In short, sterilized seeds were plated onto the nylon mesh (57-103, Nitex) placed on the top of MS agar media. Two rows of seeds comprising approximately 100-150 seeds per row were then grown vertically for 5 days in long-day growth condition. To isolate root protoplasts, ⅓ of the root was cut and chopped on the surface of the mesh and collected in buffer containing cell wall degrading enzymes - 1.5 g Cellulase (C1794, Sigma) and 0.1 g Pectolyase (P3026, Sigma) in 100 ml protoplasting solution. After 1 hour of incubation with occasional stirring, the protoplasts were spun down (200 rcf for 6 min) in 15 ml Falcon tube and after removal of supernatant, cells were resuspended in 700 ul protoplasting solution without enzymes. After filtering with 70 um and 40 um cell strainers sequentially, FACS was performed using 70 micron nozzle at 10 psi. Fluorescence positive cells were collected in a round-bottom polystylene tube that contains 150 - 200 ul RLT buffer with beta-mercaptoethanol (10 ul / 1 ml RLT buffer, RNeasy micro kit protocol, Qiagen). Cell sorting was performed for about 15 min and collected cells were immediately frozen on dry ice and kept in -80 °C for up to one week. Frozen cells were quickly thawed by adding the same volume of EtOH (150 - 200 µl), and RNA extraction was done using RNeasy Micro Kit (Qiagen) as previously described by Clark et al., 2018. RNA integrity was measured with Agilent HS RNA TapeStation system following the manufacturer's instructions. Samples of RNA integrity value (RIN) 6.3 and above were taken to the following cDNA synthesis step. Clontech Smarter Ultra Low Input Library Kit V4 was used for cDNA synthesis and amplification (18 PCR cycles). Samples were then submitted to Novogene for library construction and RNA sequencing on an Illumina HiSeq SE50 run.  The precise name of the instrument used to produce the reads is not available, although it was a HiSeq series instrument.