GSM8908629: ENU54288B6G35_F6_neuro2058_S30_R1_001; Mus musculus; RNA-Seq
To homogenize the tissue, RNAlater was removed, and a 5 mm stainless steel bead (Qiagen) and 1.6 mL of Qiazol (Qiagen) was added. The tissue was homogenized using a Tissue Lyser II (Qiagen) for at least 30 minutes until a homogenous solution was confirmed. 400 µL of the homogenate solution was transferred into a new tube and allowed to sit for 5 minutes before adding 100 µL of chloroform (Sigma). The homogenate-chloroform mixture was shaken vigorously, allowed to stand for 2 minutes before centrifugation for 5 minutes at 4°C and 1000 x g. The top clear layer was removed and stored at -80 °C. For RNA extraction, 120 µL was transferred into a QIAgen S-block and processed on the QIAcube using the RNeasy 96 QIAcube HT Kit. cDNA fragment libraries were constructed using the SMART-Seq® v4 3' DE Kit (Takara Bio) following manufacturer's recommendations, with minor modifications. Total RNA samples were processed in batches of 1152, distributed across twelve 96-well plates. For each sample, 4 ng total RNA was used as input for cDNA synthesis, then amplified for 8 cycles, with each sample in a row receiving a unique in-line index. Amplified cDNAs within a row were then combined into a 12-plex pool, cleaned up with Ampure XP beads (Beckman Coulter) and quantified using the Qubit HS DNA assay kit (Thermo Fisher Scientific). For each of the 96 cDNA pools generated from 12 plates, 400 pg were carried into Nextera XT library preparations (Illumina Inc.), each assigned with one of the 96 unique combinations of Forward and Reverse HT Index Primers included in the SMART-Seq® v4 3' DE kit. After Ampure XP clean up and Qubit quantification, the 96 pools were combined in an equimolar manner to generate a 1152-plex Illumina library.  This library was concentrated with Ampure XP beads, quantified with Qubit and assessed on the TapeStation with the High Sensitivity D1000 assay (Agilent Technologies).