GSM8291750: tcim_rep2; Danio rerio; RNA-Seq
RNA was prepared from two mm SC sections at 5 dpi in tcima/b-/- and wild-type controls for bulk RNA sequencing. 9-10 SCs were pooled for each replicate and 3 biological replicates were used. Total RNA was prepared using NucleoSpin® RNA Plus XS (Clontech, cat# 740990). Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 10ng of total RNA with a Bioanalyzer RIN score greater than 8.0. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer's protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.