alias: GSM2808408
title: GSM2808408: CD11b_healthy_2; Mus musculus; RNA-Seq
library_construction_protocol: For RNA sequencing kidney single cells suspensions were prepared. In brief, Kidneys were cut into small pieces and then stirred for 30 min at 37 °C in 5 ml of complete RPMI (Thermo Fisher Scientific) with 30 U/ml DNase I and 250 μg/ml collagenase B (Sigma-Aldrich). For removal of debris, cells were passed through a 100 μm strainer before red blood cells were lysed with ammonium chloride–potassium bicarbonate lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 3 min at room temperature and then passed through a 70 μm cell strainer. Immune cells were enriched with anti-mouse CD45-beads and LS columns (Miltenyi Biotec, San Diego, CA) according to the manufacturer's instructions, before proceeding to surface. Cell sorting was performed on a FACS Aria II flow cytometer (BD) and sorted directly into the lysis buffer of the RNAqueous micro isolation kit (ThermoFisher). Lysates were immediately frozen on dry ice and stored at -80°C until RNA preparation and DNase treatment according to the manufacturer's standard protocol. RNA integrity was determined using an Agilent bioanalyzer. Library preparation was performed with total RNA after integrity was determined using an Agilent bioanalyzer.  Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara Bio USA, Mountain View, CA) per the manufacturer's protocol. cDNA was fragmented using a Covaris E210 sonicator using duty cycle 10, intensity 5, cycles/burst 200, time 180 seconds. cDNA was blunt ended, had an adenine added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-2500 (Illumina, San Diego, CA) using single reads extending 50 bases.
identifiers.namespace: GEO
identifiers.id: GSM2808408