GSM5128954: Ad-CMV-GOL_resolved_spleen_CX3CR1_3; Mus musculus; RNA-Seq
Samples for RNA-seq were sorted in 1.5 mL reaction tubes including lysis buffer (2xTCL buffer (Qiagen, #1070498) + RNase-free water + beta-mercaptoethanol (1% total volume)). Library preparation for bulk 3'-sequencing of poly(A)-RNA was done as described previously in Drop-Seq pipeline. Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adapter. 5' ends of the cDNAs were extended by a template switch oligo (TSO) and after pooling of all samples full-length cDNA was amplified with primers binding to the TSO-site and the adapter. cDNA was tagmented with the Nextera XT kit (Illumina) and 3'-end-fragments finally amplified using primers with Illumina P5 and P7 overhangs. The P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 75 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.