GSM5600622: GF_104w_F_3; Mus musculus; RNA-Seq
Total RNA was extracted from FACS sorted CD11b+CD45lowLin- microglia cells using The ARCTURUS PicoPure RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. The SMARTer v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories) was used to generate first strand cDNA from 500 to 750 pg total-RNA. Double stranded cDNA was amplified by LD PCR (11 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina). 150 pg of input cDNA were tagmented (tagged and fragmented) by the Nextera XT transposase. 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). 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.