title: GSM2306287: RS-Mock Rep2; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Leaves from ~24 three-week-old plants (2 leaves/plant; ~1.0 g) were collected. Tissue was fast frozen and ground in liquid nitrogen. 5 ml cold polysome extraction buffer [PEB; 200 mM Tris pH 9.0, 200 mM KCl, 35 mM MgCl2, 25 mM EGTA, 5 mM DTT, 1 mM phenylmethanesulfonylfluoride (PMSF), 50 μg/ml cycloheximide, 50 μg/ml chloramphenicol, 1% (vol/vol) Brij-35, 1% (vol/vol) Igepal CA630, 1% (vol/vol) Tween 20, 1% (vol/vol) Triton X-100, 1% Sodium deoxycholate (DOC), 1% (vol/vol) polyoxyethylene 10 tridecyl ether (PTE)] was added. After thawing on ice for 10 min, lysate was centrifuged at 4 °C/16,000 g for 2 min. Supernatant was transferred to 40 μm filter falcon tube and centrifuged at 4 °C/7,000 g for 1 min. Supernatant was then transferred into a 2 ml tube and centrifuged at 4 °C/16,000 g for 15 min and this step was repeated once. 0.25 ml lysate was saved for total RNA extraction for making the RNA-seq library. Another 1 ml lysate was layered on top of 0.9 ml sucrose cushion [400 mM Tris•HCl (pH 9.0), 200 mM KCl, 35 mM MgCl2, 1.75 M sucrose, 5 mM DTT, 50 μg/ml chloramphenicol, 50 μg/ml cycloheximide] in an ultracentrifuge tube (#349623, Beckman). The samples were then centrifuged at 4 °C/70 k rpm for 4 h in a TLA100.1 rotor. The pellet was washed twice with cold water, resuspended in 300 μl RNase I digestion buffer [RNID; 20 mM Tris•HCl (pH 7.4), 140 mM KCl, 35 mM MgCl2, 50 μg/mL cycloheximide, 50 μg/ml chloramphenicol]11 and then transferred to a new tube for brief centrifugation. The supernatant was then transferred to another new tube where 10 μl RNase I (100 U/μl) was added for 60 min incubation at 25 °C/300 rpm. 15 μl SUPERase-In (20 U/μl) was then added to stop reaction. For RF library, ribosome recovery, footprint fragment purification, PNK treatment and linker ligation were performed according to the public protocol (Ingolia et al.,, 2012, Nature protocol). 2.5 μl 5’ deadenylase (NEB) was then added to the ligation system and incubated at 30 °C for 1 h. 2.5 μl RecJf exonuclease (NEB) was subsequently added for 1 h incubation at 37 °C. The enzymes were inactivated at 70 °C for 20 min and 10 μl of the samples were taken as template for reverse transcription. The rest of steps for the library construction were performedaccording to the public protocol (Ingolia et al., 2012, Nature protocol) except biotinylated oligos, rRNA1 and rRNA2, for Arabidopsis according to another reported method Juntawong et al., 2014, PNAS). For RNA-seq library, 0.75 ml TRIzol® LS (Ambion) was added to the 0.25 ml lysate saved from the Ribo-seq library construction. Total RNA was extracted, quantified and qualified using Nanodrop (Thermo Fisher Scientific Inc). 50-75 μg total RNA was used for mRNA purification with Dynabeads® Oligo (dT)25 (Invitrogen). 20 μl of the purified poly (A) mRNA was mixed with 20 μl 2× fragmentation buffer (2 mM EDTA, 10 mM Na2CO3, 90 mM NaHCO3) and incubated for 40 min at 95 °C before cooling on ice. 500 μl of cold water, 1.5 μl of GlycoBlue and 60 μl of cold 3 M sodium acetate were then added to the samples and mixed. Subsequently, 600 μl isopropanol was added before precipitation at -80 °C for at least 30 min. Samples were then centrifuged at 4°C/12,000 rpm for 30 min to remove all liquid and air dried for 10 min before resuspension in 5 μl of 10 mM Tris (pH 8). The rest of the steps were the same as Ribo-seq library preparation from footprint fragment purification.
attributes.tag: GEO Accession
attributes.value: GSM2306287