title: GSM7777735: LN thoracic replicate 1; Homo sapiens; RNA-Seq
library_construction_protocol: Freshly isolated human LN, spleen were flushed with ice-cold PBS. Fat tissues were removed from the samples. 0.5 g of the tissue was cut it into small pieces of 2-4 mm in length and transferred into the GentleMACS C Tube containing the enzyme mix and RPMI-1640 medium on ice. GentleMACS Program for the tissue type of interest (i.e., 37° C 'multi.B' preset program for human spleen) was selected.BM samples were collected in anticoagulated tubes and processed on the same day. Density gradient separation of BM samples was performed using Lymphoprep, StemCell. Cells were sorted using FACSAria III or FACSMelody (BD Biosciences, San Jose, CA). Library construction was performed using the 10X Genomic Chromium Single Cell 3' Reagent Kit per manufacturer's protocols. Briefly, cells were resuspend in master mix and loaded into the chip with partitioning oil and gel beads to generate GEM. The mRNA will concvert to cDNA and cleaned up with Silane DynaBeads. After amplification, the size selection was doen using SPRIselect beads for library construction. In the library construction part, the adaptors for sequencing and index were added for sequencing.