GSM6635183: week16_lib2, mRNA-derived cDNA; Mus musculus; OTHER
Isolated thymi were cleaned from adipose tissue, separated into the two lobes, and subsequently subjected to three rounds of enzymatic digestion with Liberase (2.5 mg/ml, Roche) and DNaseI (10 mg/ml, Roche) diluted in PBS (Sigma) at 37°C. After filtration through a 100 μm cell strainer and resuspension in FACS buffer (PBS supplemented with 2% FBS (Sigma)), cell number was determined using a CASY cell counter (Innovatis). cDNA library was prepared using Chromium Single Cell 3ʹ v2 protocol (10x Genomics). For ADT library the following i7 primer 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3' (Illumina) was used, N-sequence stands for the sample index. The following 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-3' SI-PCR primer as i5 primer (Illumina) was used for CITE-seq library amplification. Sequencing was performed on one lane of Illumina NovaSeq 6000 instrument with a mix of 90% cDNA library and 10% ADT library resulting in 151nt-long paired-end reads.