title: GSM4191818: PLT2_Est_2; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Reporter line pCALS7::H2B-RFP was crossed with pNAC86(XVE)::PLT2-3YFP. For the induction of PLT2, 5-day-old seedlings grown on the standard ½ media were transferred onto 10 uM 17-b-estradiol containing ½ MS media for 6 hours. Arabidopsis root protoplasts were isolated according to the published protocol (Clark et al., 2018).  In short, roots were cut and collected in the protoplasting solution containing the same volume of estradiol and DMSO respectively, and incubated for 12 min with vigorous swirling with a forcep. After additional 50 min of incubation in the same solution, protoplasts were precipitated and resuspended for the following cell sorting.  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 ul), 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.
attributes.tag: GEO Accession
attributes.value: GSM4191818