GSM7234119: Mono_Prf1_Ruxo_3 2511432_15; Mus musculus; RNA-Seq
RNA was extracted from sorted cell populations using the RNeasy Micro Kit (Qiagen, Germantown, Maryland). The quality and quantity of the initial RNA was determined by using the Agilent 2100 Bioanalyzer with the Eukaryote Total RNA Pico kit. For each good quality sample, 2.5-25 nanograms of total RNA was used in the Tecan Ovation RNA sequencing system v2 protocol as written to create SPIA (single primer isothermal amplification)-cDNA. Once the cDNA was purified, five hundred nanograms of each sample was sheared using the Covaris LE220 focused ultra sonicator. The shearing plate used was the 96 microtube-50 AFA fiber plate and the target base pair size was 300. The settings for the shearing were the following: Peak incident Power (W) of 450, duty factor of 15%, cycles per burst 1000 with a 100 second treatment time. Once the cDNA was sheared, libraries were created using the KAPA Hyper-Prep kit from Roche. Based on the starting cDNA input amount, the Kapa Hyper protocol recommended four pcr cycles for the amplification step while the rest of the protocol was followed as described. The indexes used were the UDI DNA indexes from Illumina. Eukaryote Total RNA Pico kit (catalog: 5067-1513) Tecan (formally NuGen) Ovation RNA sequencing system v2 (catalog: 7102-32) Covaris 96 tube plate (catalog:520168) KAPA Hyper-Prep (catalog: KK8504) UDI DNA Indexes from Illumina (catalog: 20022370)