GSM5911600: B11_blood_CTLA4_late_rep1; Mus musculus; RNA-Seq
Blood was collected with cheek bleeds into BD Microtainer capillary blood collection tubes (365974) to a volume of at least 100μL per animal. Care was taken to remove clots from samples and 100μL of each sample was transferred into microcentrifuge tubes containing 1mL TRIzol. RNA was then isolated using Qiagen RNeasy® Mini Columns (74004; Qiagen, Germantown MD) according to manufacturers recommendations and including an on-column DNase I digestion. Yield and integrity of RNA was confirmed by reading absorbance at 260, 280 and 230 nm using a NanoDrop ND-1000 (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA) and with the Agilent 2200 Tapestation (Agilent Technologies, Inc.). Library preparation and paired-end 150bp (PE150, Illumina) RNA sequencing was performed by Novogene (Novogene Corporation, Sacramento, USA). Paired end reads were aligned using STAR v2.7.9 using default options. STAR index was created using the GRCm39 primary genome FASTA and annotation files. Resulting BAM files were sorted by name using samtools v1.7 then gene counts were quantified using HTSeq-count v0.13.5. Pairwise differential expression was calculated and PCA plots were created using DESeq2 v1.32.0.