title: GSM3682883: P_GRO_rep1; Arabidopsis thaliana; OTHER
library_construction_protocol: The fine tissue powder was solubilized in 30 ml extraction buffer (2 M hexylene glycol, 20 mM PIPES pH 7.0, 10 mM MgCl2, 5 mM 2-mercaptoethanol) and filtrated through 110 μm and 60 μm nylon mesh. Add triton X-100 into the lysates to the final concentration of 1% and leave on ice for 10 minutes. Cell fractionation was performed using a 30%-80% percoll gradient solution and nuclei were collected at the 30%-80% interface. Breifly, nascent RNA was treated with T4 PNK (NEB) and followed by acidic phenol/chloroform purification. Phosphorylated nascent RNA was polyA tailed using E. coli Poly(A) Polymerase (NEB) and reverse transcribed by superscript III reverse transcriptase (Invitrogen) with RT primer (GRO-oligo2, synthesized by IDT DNA, 5'-/5Phos/GA TCG TCG GAC TGT AGA ACT CTG AAC /idSp/CC TTG GCA CCC GAG AAT TCC ATT TTT TTT TTT TTT TTT TTT VN-3'). Free RT primer was removed by Exonuclease I (Fermentas) and RNA was eliminated by alkaline hydrolysis. The resulted 1st-strand cDNA was resolved from a 10% TBE-urea polyacrylamide gel. The cDNA with size above120 nt was recovered and self-ligated using CircLigase (Epicentre). The circularized single-stranded cDNA was relinearized by APE 1 (NEB). The linearized cDNA was recovered again by a 10% TBE-urea polyacrylamide gel. The cDNA was PCR amplified by Kapa HiFi polymerase with the illumina compatible primers (Universal primer F 5'GAT CGT CGG ACT GTA GAA CTC TGA ACG TGT AGA TCT CGG TGG TCG CCG TAT CAT T 3', Reverse Index primer R 5'CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA 3', NNNNNN: index). The PCR reaction starts with a 30s denaturation at 94˚C, followed by 12-14 cycles of 10s denaturation at 94˚C, 15s annealing at 60˚C and 15s extension at 72˚C. The PCR product was resolved in a non-denaturing 8% TBE polyacrylamide gel and size region 200-330 bp was recovered. cDNA library concentration was determined by Qubit QuantIT (Invitrogen) and kapa quantitative realtime PCR according to the manual.
attributes.tag: GEO Accession
attributes.value: GSM3682883