GSM4975432: mRNA-liver-FE-offspring broiler3 [B1d_FOLATE_3]; Gallus gallus; RNA-Seq
Total RNA from 10 sperm samples and 10 liver samples of breeder roosters, and 40 liver samples of 1 day-old offspring broilers from folic acid-control and folic acid-excess groups were extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedures. The RNA amount and purity of each sample was quantified using NanoDrop ND-2000 (NanoDrop, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number > 7.00, and confirmed by electrophoresis with denaturing agarose gel. 4 RNA samples of four separate 1 day-old broilers' livers from same replication were mixed equally together as a pooled RNA sample according to the purity and quantity of total RNA. The mRNAs with poly (A) tails from 1 µM total RNA were isolated and purified with Dynabeads oligo (dT) 25-61005 (Thermo Fisher, CA, USA) using two rounds of purification. The mRNAs were randomly segmented into small fragments using Magnesium RNA Fragmentation Module (NEB, USA) at 94 °C for 5 min. Using random hexamer primers, fragmented mRNAs were used as templates to synthesize first-stand cDNA. Following RNase I treatment, DNA polymerase I was used to synthesize second-strand cDNA. cDNA fragments which Purified by using AMPure XP beads were then ligated with sequencing adapters according to the protocol (Illumina, San Diego, USA). Following PCR amplification, target fragments were obtained by agarose gel electrophoresis to create the cDNA library. Then we performed paired-end sequencing (2 × 150 bp) on an Illumina Novaseq™ 6000 at LC-Bio (Hangzhou, China) following the vendor's recommended protocol.