title: GSM4042913: Chuong-387; Gallus gallus; RNA-Seq
library_construction_protocol: Please find detailed protocols for ChIP and Capture-C DNA preparaion in Methods of this publication. RNA extraction for RNA-seq: Total RNAs of indicated tissues were extracted followed by the standard Trizol extraction protocol (Invitrogen, Cat.15596026). ChIPed-DNA preparation for ChIP-seq: The protocol was adapted from Dr. Ruchi Bajpai's laboratory (Bajpai et al., 2010). 3C library preparation for NG Capture-C: The protocol was adapted from Dr. Wange Lu's laboratory (Davies et al., 2016; Davies et al., 2017). For RNA-seq library preparation: TruSeq RNA Prep Kit v2 (Illumina, RS-122-2001/-2002). For ChIP-seq library preparation: NEBNext Ultra End Repair/dA-Tailing Module (New England Biolabs, Cat. E7442S), NEBNext Ultra Ligation Module (New England Biolabs, Cat. E7445S), and KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Cat. KK2601). For NG Capture-C library preparation: 5 µg of sheared 3C-DpnII DNA was used in adaptor ligation for pre-capture library using NEBNext DNA Library Prep Master Mix Set for Illumina (NEB, Cat. E6040). Adaptors were from NEBNext Multiplex Oligos for Illumina (NEB, Cat. E7500). The indexed 3C DNA was amplified using a primer pair of P5 (5'-aatgatacggcgaccaccga) and P7 (5'-caagcagaagacggcatacgagat) and purified using 1.8X AMPure XP beads. To perform the first capture, SeqCap EZ Hybridization and Wash Kit (Roche, Cat. 05634261001) was used. For one library, the indexed sequencing library, 5 µg of a Cot-1 DNA equivalent chicken Hybloc DNA (Applied Genetics Lab, Cat. CHB), 1 µl of 1xGen Universal Blocking Oligo—TS-p5 (1 nmole/µl), and 1 µl of 1xGen Universal Blocking Oligo—TS-p7(6nt) (1 nmole/µl) were mixed well and dried completely in a low retention PCR tube (Axygen, Cat. PCR-02-L-C). The dried pellet was then resuspended in Nimblegen Hybridization Buffer and Component A, mixed with 2 µl of a capture probe pool (3 pmole in total), and incubated for hybridization at 47℃ for 72 hours. After the first hybridization, biotinylated-probe-hybridized DNA (captured DNA) was mixed with 40 µl of prepared streptavidin magnetic beads (Invitrogen, Cat. 65001) and incubated at 47℃ for 45 min. The mixture was further washed using the SeqCap Wash Kit. The captured DNA was then purified using 1.8X AMPure XP beads and eluted in final 44 µl of nuclease-free water and amplified using the P5/P7 primer pair for 12 cycles. This 1st-captured DNA was finally cleaned up with 1.8X AMPure XP beads and eluted in 25 µl of water. To perform the second capture, the 1st-captured DNA was mixed with components described in the first capture part and incubated for hybridization at 47℃ for 24 hours. The double-captured DNA was then washed, pulled down by using the streptavidin beads, purified, amplified, and cleaned up as described before.
attributes.tag: GEO Accession
attributes.value: GSM4042913