GSM2634053: RNA_total_SH_rep3; Mus musculus; RNA-Seq
Cells were rinsed in 1 ml of ice-cold PBS. Total RNA was isolated using silica column approach (NucleoSpin® RNA extraction kit, Macherey-Nagel, 740955.250). Briefly, cells were washed in ice-cold PBS and lysed in 350 of lysis buffer supplemented with 1% β-mercaptoethanol. After adding 100µl of chloroform, mouse samples were centrifuged at 10,000g at 4°C for 15 min. Upper polar phases and cell samples were then transferred to silica columns and spun 11,000g for 30 seconds. Total RNA was then isolated by following manufacturer’s instructions and eluted from columns with 50µl of RNase free water. 4-thiouridine (Sigma Aldrich, T4509) and RNA-seq were performed as previously described (Rabani et al., 2011). Briefly, 300μM 4-thiouridine was used for 30 minutes labeling in confluent scramble and ShZc3h10 myoblasts. After labelling, cells were harvested in 10ml of ice-cold PBS, of which 250μl were saved for total RNA-seq. 50μg of RNA were conjugated to biotin-HPDP (Abcam) and precipitated with 50μl of streptavidin (MyOne Steptavidin T1, Invitrogen) for 4sU-tagged RNA isolation. RNA samples were eluted in 50µl of water, quantified by UV spectrophotometry (NanoDrop 1000 Spectrophotometer, Thermo Scientific) and saved at -80°C. We then removed rRNA from total RNA samples by Ribo-Zero Magnetic Kit (Illumina, Mrzh116) following manufacturer’s instructions. SMART-seq2 protocol adapted from Picelli and colleagues (Picelli et al., 2014b) was used to generate full-length cDNA with modified random priming (5’-AAGCAGTGGTATCAACGCAGAGTACNNNNNN-3’). Purified cDNA was tagmented with Tn5 transposase produced in-house following described protocol (Picelli et al., 2014a) . cDNA fragments were enriched by PCR to create the final library and sequenced on an Illumina HiSeq2000 platform.   SMART-seq2 protocol adapted from Picelli and colleagues (Picelli et al., 2014b) was used to generate full-length cDNA with modified random priming (5’-AAGCAGTGGTATCAACGCAGAGTACNNNNNN-3’). Purified cDNA was tagmented with Tn5 transposase produced in-house following described protocol (Picelli et al., 2014a) . cDNA fragments were enriched by PCR to create the final library and sequenced on an Illumina HiSeq2000 platform.
GEO Accession
GSM2634053