title: GSM8969654: SP16_002655_A3; Homo sapiens; RNA-Seq
library_construction_protocol: 2-4 25µM FFPE tissue sections of human TB granuloma biopsy specimens were dissociated into single cells. Nuclei and cells were isolated from formaldehyde fixed and paraffin embedded (FFPE) tissue blocks using the 10x Genomics Sample Preparation from FFPE Tissue Sections protocol. FFPE scrolls were deparaffinized then dissociated into nuclei and cells using Dissociation Enzyme mix and counted then the 10x Genomics Fixed RNA Profiling Reagent Kits protocol was followed. After the RT reaction, the GEMs are broken, full length cDNAs are cleaned with Silane Dynabeads, and then amplified via polymerase chain reaction (PCR) and purified using SPRIselect (Beckman Coulter – Brea, CA) bead size selection. Resulting cDNAs are assayed using a 4200 TapeStation System High Sensitivity D5000 ScreenTape and reagents (Agilent - Santa Clara, CA) for qualitative and quantitative analysis. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size for the sequencing library preparation in which Illumina P5 and P7 sequences, a sample index, and TruSeq read 2 primer sequence are added through End Repair, A-tailing, Adaptor Ligation PCR. The final libraries contain P5 and P7 primers used in Illumina bridge amplification. Following additional bead purification, libraries are assayed for quality using 4200 TapeStation System HSD1000 ScreenTape and reagents, then quantitated and checked for successful adapter ligation with the KAPA Library Quantification Kit (Roche - Indianapolis, IN) on the Applied Biosystems QuantStudio 5 (Thermo Fisher Scientific - Waltham, MA). Sequence is generated using paired end sequencing (one end to generate cell specific, barcoded sequence and the other to generate sequence of the expressed poly-A tailed mRNA).  These libraries were sequencing on an Illumina NovaSeq6000.