GSM1940311: JCLIA002A; Saccharomyces cerevisiae; RNA-Seq
(1) Extraction of ribosome-protected mRNA: Frozen cell pellets were lysed by low-frequency cryogenic mixer milling.  Lysate was clarified of cell debris and 25 A260 units of extract were treated with 450 U of RNase I (Ambion) for 1 h at room temperature with gentle rotation; digestion was stopped by addition of 120 U of Superase-IN.  Ribosomes were pelleted using high-speed centrifugation through a 1 M sucrose cushion.  miRNeasy Mini kit (Qiagen) was used to purifiy ribosome-protected mRNA fragments following the manufacturer’s instructions. (2) Extraction of total RNA: Total RNA was extracted using the RiboPure Yeast kit (Ambion, Austin, TX, USA), according to manufacturer's instructions, except cells were disrupted by bead-beating 3 times for 30 sec, with a 30 sec pause between runs. (1) Ribosome profiling library: Preparation of ribosome profiling libraries followed similar protocols as those in (Ingolia et al., 2012; Ingolia et al., 2009). (2) RNA-seq library: 4 µg of total RNA were used to prepare the multiplexing libraries with barcodes following the standard instructions of the Illumina TruSeq RNA Sample Prep Kit.
GEO
GSM1940311
GEO Accession
GSM1940311