title: GSM3142120: GS1-1; Mus musculus; RNA-Seq
library_construction_protocol: RNA extraction using the Arcturus PicoPure RNA Isolation kit was performed according to the manufacturer's protocol (Life technologies). Briefly, 100 ul of extraction buffer was added to the cells, followed by brief incubation at room temperatue. While the lysate was incubated, the purification column was wetted (Pre-condition) by adding 250 ul of condition buffer, followed by centrifugation at 16,000 × g for 1 min. 100 ul of 70% ethanol was then added to the lysate, mixed well and the whole mixture was transferred to the prepared purification columns. Purification columns were centrifuged for 2 min at 100 × g, followed by centrifuging at 16,000 × g for 30 sec to remove flow through. 100 ul of wash buffer 1 was added to the purification columns, which were then centrifuged for 1 min at 8,000 × g. DNase treatment may was performed directly within the purification column. 10 µl DNase I stock solution (Qiagen, catalog#79254) was mixed with 30 µL Buffer RDD. 40 µL DNase incubation mix was applied directly into the purification column membrane followed by Incubation at room temperature for 15 minutes. 40 ul of wash buffer 1 was added to the purification columns, which were then centrifuged for 15 seconds at 8,000 × g. After that, 100 ul of wash buffer 2 was added to the purification columns, which were then centrifuged for 1 min at 8,000 × g. Another 100 ul of wash buffer 2 was added to the purification columns. Purification columns were centrifuged at 16,000 × g for 2 min. Another spin at 16,000 × g for 1 min was performed to remove the wash residue in the columns. Purification columns were transferred to new Eppendorf tubes and 11 ul of elution buffer was carefully added onto the column membrane followed by 1 min incubation at room temperature. Purification Columns were centrifuged at 1,000 × g for 1 min, and subsequently at 16,000 × g for 1 min to elute RNA. 10 ul of RNA solution was obtained. Libraries were constructed using the Nextera XT kit as recommended by the manufacturer (Illumina, San Diego, USA). 0.5 ng of cDNA was tagmented, utilizing 5 μl of Amplicon Tagment Mix with 10 μl Tagment DNA Buffer. Tagmentation reaction was performed by incubation for 20 min at 55°C. Next, index primers were added together with Nextera PCR Master Mix. Tagmented cDNA was amplified by limited-cycle PCR (10 cycles). Libraries were purified using AMPure beads (Beckman Coulter, High Wycombe, UK) and quality validated using Agilent 2100 BioAnalyzer. Libraries were normalized and 8 nM pooled libraries were then sequenced on Illumina HiSeq2000.
attributes.tag: GEO Accession
attributes.value: GSM3142120