title: GSM4725740: ccl34b.1_neg_4; Danio rerio; RNA-Seq
library_construction_protocol: Whole brains were dissected and collected into 1ml ice-cold PBS, and homogenized with 27G needles. Cells were spun down (800g 4°C for 5 min) and resuspended with 0.25% Trypsin-EDTA, then incubated at 30 °C for 20-30 mins. After termination of the reaction with 10 μM CaCl2 and 10% FBS, samples were centrifuged and washed with ice-cold 1% BSA/PBS buffer, and finally pushed through a Falcon® 40 µm Cell Strainer to obtain single-cell suspensions. ccl34b.1+mpeg1+ and ccl34b.1- mpeg1+ microglia (100 cells for each sample) were directly sorted into lysis buffer (0.2% Triton X-100 solution) in a FACS Aria IIIu sorter based on the eGFP and DsRedx fluorescence. The Smart-seq2 protocol was used for whole transcriptome amplification and library preparation. Cell lysates were hybridized together with oligo-dT primer and dNTP at 72 °C for 3 min, then added with reverse transcription mix containing SuperScript™ II Reverse Transcriptase and a template-switching oligo. After reverse transcription, first-strand reaction products were PCR amplified using KAPA HiFi HotStart ReadyMix with 24 PCR cycles and purified using Ampure XP beads with a magnetic stand. cDNA quality was assessed using an Agilent Fragment Analyzer System. Sequencing libraries were prepared using TruePrep DNA Library Prep Kit V2 for Illumina® following the instructions (0.5 ng cDNA input). After tagmentation, samples were amplified with TruePrep Index Kit V2 for Illumina® (12 cycles) and purified with Ampure XP beads. Library quality was assessed using Agilent Fragment Analyzer System.