title: GSM3673194: 07_P42_WT1; Mus musculus; RNA-Seq
library_construction_protocol: Approximately 1000 FACS sorted microglia cells were homogenized in 170 µl Trizol reagent using Precellys CK14 ceramic beads (2 cycles of 10 seconds at 6500 rpm; 10 seconds break). After 5 minutes incubation at room temperature 34 µl of chloroform was added and the samples were again incubated at room temperature for 2 minutes. Phase separation was achieved by 15 minutes centrifugation at 12,000 x g at 4 °C. An equal amount of 70 % ethanol was added to the aqueous supernatant and the mixture was applied to RNeasy MinElute spin columns (RNeasy Micro Kit, QIAGEN, Hilden, Germany), followed by an on-column DNase digestion and several wash steps. Finally total RNA was eluted in 14 μl of nuclease free water. Purity and integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Pico LabChip reagent set (Agilent, Palo Alto, CA, USA). The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate first strand cDNA from 0.3 to 1.0 ng total-RNA. Double stranded cDNA was amplified by LD PCR (14 or 15 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). 150 pg of input cDNA were tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step 1:5 dilutions of index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA). Equimolar amounts of each library were pooled, and the pools were used for cluster generation on the cBot with the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on an HiSeq 1000 instrument using the indexed, 50 cycles single-read (SR) protocol and the TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 System User Guide.
attributes.tag: GEO Accession
attributes.value: GSM3673194