GSM3473314: LungMF KO scRNASeq; Mus musculus; RNA-Seq
The CD45+CD11c+SiglecF+  lung alverolar macrophages (AMs) were sorted by FACS Aria II flow cytometer. 5,000 FACS-sorted cells were loaded into each reaction for gel bead-in-emulsion (GEM) generation and cell barcoding. Single cell sequencing libraries were generated using the 10X Genomics Chromium Single Cell 3' Reagent Kit (v2 Chemistry) and Chromium Single Cell Controller. Reverse transcription of the GEM (GEM-RT) was performed in a Thermocycler (Veriti™ 96-Well Fast Thermal Cycler, Applied Biosynthesis; 53 °C 45 min, 85 °C 5 min, 4 °C hold). cDNA amplification was performed after GEM-RT cleanup with Dynabeads MyOne Silane (Thermo Fisher Scientific) with the same Thermocycler (98 °C 3 min; 98 °C 15 sec, 67 °C 20 sec, 72 °C 1 min, repeat 12 cycles; 72 °C 1 min, 4 °C hold). Amplified cDNA is cleaned up with SPRIselect Reagent Kit (Beckman Coulter) and followed by library construction procedure, including fragmentation, end repaired, adaptor ligation, and library amplification. A Bioanalyzer (Agilent) was used for library quality control. Libraries were sequenced on an Illumina HiSeq4000 using paired-end flow cell: Read 1, 26 cycles, i7 index 8 cycles; Read 2: 98 cycles.
GEO Accession
GSM3473314