title: Illumina HiSeq 2000 sequencing; GRO-seq analysis U1810 cells after 8h Rapamycin treatment and DMSO control
design: GRO-seq analysis U1810 cells after 8h Rapamycin treatment and DMSO control
library_construction_protocol: 8h rapamycin (300nM) treated U1810 cells compared to DMSO treated cells. Cells cultured in RPMI medium supplemented with 10% FCS Cells were washed with cold 13 PBS buffer and swelled in swelling buffer (10mM Tris-Cl pH 7.5, 2mM MgCl2, 3mM CaCl2) for 5 min on ice and collected. Cells were lysed in lysis buffer (swelling buffer with 0.5% NP-40, 2 units per millilitre Superase In and 10% glycerol) and finally re-suspended in 100 ml of freezing buffer (50mM Tris-Cl pH 8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). For the run-on assay, re-suspended nuclei were mixed with an equal volume of reaction buffer (10mMTris-Cl pH 8.0, 5mMMgCl2, 1mMDTT, 300mM KCl, 20 units of SUPERase In, 1% sarkosyl, 500 mM ATP, GTP, and Br-UTP, 2 mM CTP) and incubated for 5 min at 30 uC. The nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol LS reagent (Invitrogen) following the manufacturer’s instructions. NRO-RNA was then subjected to base hydrolysis on ice for 40 min and followed by treatment withDNase I and antarctic phosphatase. To purify the Br-UTP-labelled nascent RNA, the NRO-RNA was immunoprecipitated with anti-BrdUargarose beads (Santa Cruz Biotech) in binding buffer (0.53 SSPE, 1mM EDTA, 0.05% Tween-20). To repair the end, the immunoprecipitated BrU-RNA was re-suspended in a 50 ml reaction (45 ml DEPC water, 5.2 ml T4 PNK buffer, 1 ml SUPERase In and 1 ml T4 PNK (NEB)) and incubated at 37 uC for 1 h. The RNA was extracted and precipitated using acidic phenol-chloroform. The complementary DNA (cDNA) synthesis was performed basically as in ref. 36 with a few minor modifications. First, RNA fragments were subjected to a poly-A tailing reaction by poly-A polymerase (NEB) for 30 min at 37 uC. Subsequently, reverse transcription was performed using oNTI223 primer (59-pGATCGTCGGACTGTAGAACTCT; CAAGCAGAAGACGGCATACGA TTTTTTTTTTTTTTTTTTTTVN). Second, tailed RNA (8.0 ml) was subjected to reverse transcription using superscript III (Invitrogen).The cDNA products were separated on a 10% polyacrylamide TBE-urea gel and the extended firststrand product (100–500 base pairs) was excised and recovered by gel extraction. After that, the first-strand cDNA was circularized by CircLigase (Epicentre) and relinearized by Ape1 (NEB). Relinearized single strand cDNA (sscDNA) was separated on a 10% polyacrylamide TBE gel and the product of needed size was excised (,120–320 base pairs) for gel extraction. Finally, single strand cDNA template was amplified by PCR using the Phusion High-Fidelity enzyme (NEB) according to the manufacturer’s instructions with two oligonucleotide primers, oNTI200 (59-CAAGCAGAAGACGGCATA) and oNTI201 (59-AATGATACGG CGACCACCGACAGGT TCAGAGTTCTACAGTCCGACG).
attributes.tag: Experimental Factor: rapamycin
attributes.value: compound
attributes.tag: Experimental Factor: 300
attributes.value: dose