GSM8517677: 32A_coeliac; Homo sapiens; RNA-Seq
Cryopreserved biopsies were thawed by immersing in a 37°C water bath for ~ 2 min with periodic shaking. RPMI with 10% FCS was added to each biopsy, dropwise in a 50 ml tube. The biopsies were washed in serum free RPMI over a cell strainer. Single cell suspensions were made by placing biopsies in a mixture of Liberase TM (SIGMA – 5401127001) and DNase (SIGMA - D5025-15 KU) while slowly rotating at 37°C (Smilie et al.,). Dissocicated biopsies were washed with 0.1% BSA over a cell strainer. Dead cells were removed using EasySep™ Dead Cell Removal (Annexin V) Kit (Stemcell Technologies -  17899). Cells counts were determined using AOPI staining solution and a Nexcelom fluorescent cell counter. scRNA libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Kit v3.1 (10X Genomics) according to manufacturer's instruction on the Chromium Controller (10X Genomics). Sequence-ready libraries were validated and quantitated using the Agilent High Sensitivity D1000 ScreenTape Kit on the TapeStation 4200 System (Agilent Technologies). Individual libraries were normalized to 3nM and quantified by qPCR using the KAPA Library Quantification Kit – Illumina/ROX Low (Roche) on the QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems) according to manufacturer's instructions. The libraries were then pooled to 3nM and quantified again by qPCR as described above. The quantified library pool was denatured and diluted according to the NovaSeq 6000 Denature and Dilute Libraries Guide and loaded onto a NovaSeq 6000 System (Illumina) for paired end sequencing. BCL conversion and demultiplexing was performed using the bcl2fastq2 script (v2.20, Illumina).